전통발효식품 유래 Enterococcus faecium JK29에 의한 γ-aminobutyric acid의 생산 최적화 Optimization of γ-Aminobutyric Acid Production by Enterococcus faecium JK29 Isolated from a Traditional Fermented Foods원문보기
${\gamma}$-Aminobutyric acid(GABA)를 생산하는 희귀 젖산균을 분리하기 위하여 전통발효식품으로부터 총 147개의 젖산균을 확보한 후 1% 글루탐산 나트륨(L-monosodium glutamate, MSG)를 사용하여 GABA를 생산하는 23개의 균주를 1차 분리하였다. 2차 분리를 위하여 글루탐산탈탄산효소(glutamate decarboxylase)와 16S rRNA 유전자의 염기서열 분석을 통해 기본 MRS 배지에서 48시간 배양 후 1.56 mM의 GABA를 생산하는 Enterococcus faecium JK29를 최종 분리하였다. E. faecium JK29에 의한 GABA의 생산을 향상시키기 위하여 배양 조건을 최적화하였으며 그 결과 0.5% 자당(sucrose), 2% 효모 추출물(yeast extract), 0.5% 글루탐산 나트륨이 포함된 최적화 MRS 배지를 개발하였다. 최적화 MRS 배지를 활용하여 $30^{\circ}C$, pH 7.5에서 48시간 배양을 한 결과 E. faecium JK29이 14.86 mM의 GABA를 생산하는 것을 확인하였다.
${\gamma}$-Aminobutyric acid(GABA)를 생산하는 희귀 젖산균을 분리하기 위하여 전통발효식품으로부터 총 147개의 젖산균을 확보한 후 1% 글루탐산 나트륨(L-monosodium glutamate, MSG)를 사용하여 GABA를 생산하는 23개의 균주를 1차 분리하였다. 2차 분리를 위하여 글루탐산 탈탄산효소(glutamate decarboxylase)와 16S rRNA 유전자의 염기서열 분석을 통해 기본 MRS 배지에서 48시간 배양 후 1.56 mM의 GABA를 생산하는 Enterococcus faecium JK29를 최종 분리하였다. E. faecium JK29에 의한 GABA의 생산을 향상시키기 위하여 배양 조건을 최적화하였으며 그 결과 0.5% 자당(sucrose), 2% 효모 추출물(yeast extract), 0.5% 글루탐산 나트륨이 포함된 최적화 MRS 배지를 개발하였다. 최적화 MRS 배지를 활용하여 $30^{\circ}C$, pH 7.5에서 48시간 배양을 한 결과 E. faecium JK29이 14.86 mM의 GABA를 생산하는 것을 확인하였다.
Dominant lactic acid bacteria (LAB) strains were isolated from traditional fermented foods to obtain rare ${\gamma}$-aminobutyric acid (GABA)-producing LAB. Out of 147 isolates, 23 strains that could produce GABA with 1% (w/v) L-monosodium glutamate (MSG) were first isolated. After furthe...
Dominant lactic acid bacteria (LAB) strains were isolated from traditional fermented foods to obtain rare ${\gamma}$-aminobutyric acid (GABA)-producing LAB. Out of 147 isolates, 23 strains that could produce GABA with 1% (w/v) L-monosodium glutamate (MSG) were first isolated. After further screening of these rare GABA-producing LAB by analysis of the glutamate decarboxylase and 16S rRNA gene sequences, Enterococcus faecium JK29 was isolated, and 1.56 mM of GABA was produced after 48 h cultivation in basic de Man, Rogosa, and Sharpe (MRS) medium. To enhance GABA production by E. faecium JK29, the culture conditions were optimized. When E. faecium JK29 was cultivated in optimized MRS medium containing 0.5% (w/v) sucrose and 2% (w/v) yeast extract with 0.5% (w/v) MSG, GABA production reached 14.86 mM after 48 h cultivation at initial conditions of pH 7.5 and $30^{\circ}C$.
Dominant lactic acid bacteria (LAB) strains were isolated from traditional fermented foods to obtain rare ${\gamma}$-aminobutyric acid (GABA)-producing LAB. Out of 147 isolates, 23 strains that could produce GABA with 1% (w/v) L-monosodium glutamate (MSG) were first isolated. After further screening of these rare GABA-producing LAB by analysis of the glutamate decarboxylase and 16S rRNA gene sequences, Enterococcus faecium JK29 was isolated, and 1.56 mM of GABA was produced after 48 h cultivation in basic de Man, Rogosa, and Sharpe (MRS) medium. To enhance GABA production by E. faecium JK29, the culture conditions were optimized. When E. faecium JK29 was cultivated in optimized MRS medium containing 0.5% (w/v) sucrose and 2% (w/v) yeast extract with 0.5% (w/v) MSG, GABA production reached 14.86 mM after 48 h cultivation at initial conditions of pH 7.5 and $30^{\circ}C$.
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제안 방법
GABA production is generally affected by nutritional factors for GABA-producer and culture conditions, and these factors should be thus taken into account in the design of optimal GABA production for the application of industrial fields [13]. Accordingly, this study also describes the culture optimization of E. faecium JK29 for high GABA production by determining the optimal carbon and nitrogen sources, initial pH and exogenous L-monosodium glutamate (MSG) concentration.
After isolating E. faecium strains, the full-length gad gene from genomic DNA of E. faecium was amplified by using the following primer sets designed for the previously reported E. faecium gad gene sequences: sets MME-61 (5'-ATGTTATACGGAAAAGATAATCAAGAAG-3') and MME-62 (5'-TTAGTGAGTAAAGCCGTACGT-3').
Each strain was then identified by polymerase chain reaction (PCR) amplification of the 16S rRNA genes from each genomic DNA using the universal primer sets 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-CGGTTACCTTGTTACGACTT-3').
대상 데이터
0% (data not shown). Since this study was focused on rare LABs producing GABA, we could select 11 LABs excluding Lactobacillus strains. The quantitative analysis of GABA concentration produced from 11 LABs showed that Enterococcus-genus strains produced more GABA with compared to Leuconostoc strains (data not shown).
성능/효과
A multiple alignment of the amino acid sequence of GAD from E. faecium JK29 with various other GAD revealed that GAD from JK29 displayed the highest sequence identity (99%) with those from the already-known E. faecium DO and E. faecium 70-8-2 NODE_29 (Fig. 2).
4 kbp). After sequencing these PCR products, we finally selected one strain GABA-producing JK29 (Fig. 1) of which GAD gene was taxonomically characterized to be close to that of E. faecium TX1337RF (accession number WP_002309699) with 99% identity, because this strain only showed the difference of GAD sequence with the previously-reported GADs and a high level of GABA production (1.56 mM) compared to the other Enterococcus strains isolated in this study. A multiple alignment of the amino acid sequence of GAD from E.
In conclusion, GABA has the great potential as a bioactive component in pharmaceutical and functional food industries and LABs are regarded to be GRAS bacteria with being used as probiotics. In this study, E.
In this study, E. faecium JK29 isolated from traditional fermented foods produced less GABA (14.86 mM) with utilizing 0.5% MSG compared to major group of LABs producing GABA such as Lactobacillus- and Lactococcus-genus strains, even though the culture conditions were partially optimized for GABA production. In the previous studies, L.
In conclusion, GABA has the great potential as a bioactive component in pharmaceutical and functional food industries and LABs are regarded to be GRAS bacteria with being used as probiotics. In this study, E. faecium JK29 was isolated from traditional fermented foods as rare LAB producing GABA and its culture conditions were optimized to enhance the GABA production. The cultivation of E.
5% MSG. The addition of 0.5% MSG rather yielded the higher GABA conversion yield (49.5%) of GABA compared to that of 1% MSG (25.0%). Therefore, the MSG concentration of 0.
3A). The investigation of sucrose concentration for GABA production resulted that the GABA production significantly increased to 8.56 mM with adding 0.5% sucrose with slightly decrease over 0.5% sucrose up to 5% (Fig. 3B). The optimal carbon sources and concentrations for GABA production varied according to the GABA-producing LAB strains: 4% maltose for L.
Since this study was focused on rare LABs producing GABA, we could select 11 LABs excluding Lactobacillus strains. The quantitative analysis of GABA concentration produced from 11 LABs showed that Enterococcus-genus strains produced more GABA with compared to Leuconostoc strains (data not shown). Thereafter, 11 Enterococcus strains was PCR-amplified with the developed PCR primer sets (MME-61 and MME-62) to screen novel GAD gene-harboring strain, resulting an amplified DNA fragment with the expected size (about 1.
후속연구
86 mM GABA after 48 h of cultivation. The further studies on the detail optimization and large-scale fermentation of E. faecium JK29 as a rare GABA-producer will be needed to determine the applicability of E. faecium JK29 as functional LAB starter strain for industrialization.
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