The Role of Glutamic Acid-producing Microorganisms in Rumen Microbial Ecosystems

로벨리아,이상석 한국생명과학회 2021 생명과학회지 Vol.31 No.5

Microbial protein is one of the sources of protein in the rumen and can also be the source of glutamate production. Glutamic acid is used as fuel in the metabolic reaction in the body and the synthesis of all proteins for muscle and other cell components, and it is essential for proper immune function. Moreover, it is used as a surfactant, buffer, chelating agent, flavor enhancer, and culture medium, as well as in agriculture for such things as growth supplements. Glutamic acid is a substrate in the bioproduction of gamma-aminobutyric acid (GABA). This review provides insights into the role of glutamic acid and glutamic acid-producing microorganisms that contain the glutamate decarboxylase gene. These glutamic acid-producing microorganisms could be used in producing GABA, which has been known to regulate body temperature, increase DM intake and milk production, and improve milk composition. Most of these glutamic acid and GABA-producing microorganisms are lactic acid-producing bacteria (LAB), such as the Lactococcus, Lactobacillus, Enterococcus, and Streptococcus species. Through GABA synthesis, succinate can be produced. With the help of succinate dehydrogenase, propionate, and other metabolites can be produced from succinate. Furthermore, clostridia, such as Clostridium tetanomorphum and anaerobic micrococci, ferment glutamate and form acetate and butyrate during fermentation. Propionate and other metabolites can provide energy through conversion to blood glucose in the liver that is needed for the mammary system to produce lactose and live weight gain. Hence, health status and growth rates in ruminants can be improved through the use of these glutamic acid and/or GABA-producing microorganisms.

김치 발효 숙성 중의 γ-aminobutyric acid (GABA) 및 아미노산 함량의 변화

이혜현 ( Hye Hyun Lee ),김건희 ( Gun Hee Kim ) 한국식품조리과학회(구 한국조리과학회) 2013 한국식품조리과학회지 Vol.29 No.6

본 연구에서는 배추김치의 저장기간별 아미노산 및 GABA의 함량을 분석하고, L-glutamic acid과 GABA의 상관성을 조사하였다. pH의 변화는 초기 5.24에서 저장 2주 후 4.40으로 감소한 뒤 7주까지 비슷한 수준으로 유지되었고, 산도는 저장 초기 0.36%에서 8주에 1.26%로 증가하는 경향을 보였다. 유리 아미노산함량은 0주부터 7주 저장기간 동안 유의적인 차이가 있는 것으로 나타났다(p<0.05). 유리 아미노산 조성은 L-valine > L-glutamic acid > glycine 순으로 함량이 많았으며 저장기간 동안 증감을 반복하며 점차적으로 증가하는 추세를 보였다. GABA는 김치제조 후 초기 72.43 μM/100 g fw의 함량을 보였으며 말기에는 229.06 μM/100 g fw를 나타내어 숙성 기간이 경과 함에 따라 증가하는 경향을 나타내었다. 초기의 빠른 GABA 생성은 GAD의 활성으로 인해 L-glutamic acid로 부터 생성 된 것으로 보여지나 0-7주간 L-glutamic acid와 GABA와의 함량에서 유의적인 양의 상관관계는 확인되지 않았다. The objective of this study was to examine the levels of free amino acids and identify the correlation between γ-aminobutyric acid (GABA)and L-glutamic acid contents in Kimchi during fermentation. During 2 weeks of fermentation, the acidity of Kinchi increased, i.e., the pH level decreased from 5.24 to 4.40. The content of amino acids determined using HPLC differed significantly (p < 0.05) during 7 weeks of fermentation. Over the 7 weeks of fermentation, the content of most free amino acids increased in the order L-valine > L-glutamic acid > L-glycine, except L-methionine decreased. Initially, the GABA content was found to be 72.43 μM/100 g fresh weight (fw), and it increased to 229.06 μM/100 g fw after 7 weeks. This rapid increase in the GABA content in the initial stage is considered to be due to L-glutamic acid. However, during the period of 0~7 weeks, no correlations were found between the L-glutamic acid and GABA contents.

Stimulation of γ-Aminobutyric Acid Synthesis Activity in Brown Rice by a Chitosan/Glutamic Acid Germination Solution and Calcium/Calmodulin

( Suk Heung Oh ) 생화학분자생물학회 2003 BMB Reports Vol.36 No.3

Changes in the concentrations of r-aminobutyric acid (GABA), soluble calcium ions, glutamic acid, and the activity of glutamate decarboxylase (GAD) were investigated in non-germinated vs. germinated brown rice. Brown rice was germinated for 72h by applying each of the following solutions :(1) distilled water, (2) 5 mM lactic acid, (3) 50 ppm chitosan in 5mM lactic acid, (4) 5mM glutamic acid, and (5) 50ppm chitosan in 5mM glutamic acid. GABA concentrations were enhanced in all of the germinated brown rice when compared to the non-germinated brown rice. The GABA concentration was highest in the chitosan/glutamic acide that germinated brown rice at 2,011 nmol/g fresh weight. The concentrations of glutamic acid were significantly decreased in all of the germinated rice, regardless of the germination solution. Soluble calcium and GAD were higher in the germinated brown rice with the chitosan/glutamic acid solution when compared to the rice that was germinated in the other solutions. GAD that was partially purified from germinated brown rice was stimulated about 3.6-fold by the addition of calmodulin in the presence of calcium. These data show that the germination of brown rice in an chitosan/glutamic acid solution can significantly increase GABA synthesis activity and the concentration of GABA.

Rhodobacter sphaeroides에 의한 ${\delta}-aminolevulinic{\;}acid$생산에 있어서 glutamic acid 및 감마 유도체의 영향

최경민,임왕진,황세영,Choi, Kyung-Min,Lim, Wang-Jin,Hwang, Se-Young 한국응용생명화학회 1993 Applied Biological Chemistry (Appl Biol Chem) Vol.36 No.3

The effect of ${\delta}-aminolevulinic\;acid$ (ALA) biosynthetic precursors and related compounds on the ALA productivity from a strain of Rhodobacter sphaeroides has been examined in vivo and in vitro systems. The relative ratios of ALA productivities by $C_{4}$- pathway to that by $C_{5}$-pathway in vivo and in vitro systems were 0.78 and 1.37, respectively. Although the expression rates of $C_{4}-$ and $C_{5}-pathways$ in cell-free systems prepared after precursors supplemented cultivations were increased 1.35 and 1.52 folds, respectively, the rate increase of $C_{4}-pathway$ was accompanied by the rate decrease of the $C_{5}-pathways$, and vice versa, as that the rates of both $C_{4}-$ and $C_{5}-pathways$ were lowered to be 0.91, 0.83, respectively. The order of cellular uptake rates of ${\gamma}-glutamyl$ derivatives relative to that found with L-glutamic acid were shown to be D-glutamic acid, 0.55: D-glutamine, 0.5: L-glutamine, 0.4: ${\gamma}-L-glutamyl$ ethylester, 0.3: GSH and Glu-pNA, 0. L and D configurations of glutamine were indicated as better substrates in vivo for ALA yields than those of glutamic acid, respectively. Rhodobacter sphaeroides 균주가 생산하는 ${\delta}-aminolevulinic\;acid(ALA)$의 생산성에 관하여 in vivo, in vitro 상에서 기질 및 관련 화합물을 이용하여 검토하였다. $C_5\;ALA$ 생합성계에 의한 ALA yield 대비의 $C_4$ 생합성계에 의한 비율은 in vive 상에서 0.78인 반면, in vitro 상에서의 비율은 1.37이었다.$C_4\;C_5$ 각 계의 기질 첨가 배양에 의한 cell-free system의 $C_4,\;C_5$ 계의 발현도는 미첨가 배양에 의한 system과 비교하여 각각 1.35, 1.52로 증가하였으나, 증가한 계에 대한 상대계의 발현도가 억제되어, $C_4$ 와 $C_5$ 계가 각각 0.91, 0.83으로 나타났다. ${\gamma}-Glutamyl\;derivatives$의 세포내 uptake rates는 L-glutamic acid를 기준으로 비교해서 D-glutamic acid, 0.55: D-glutamine, 0.5: L-glutamine, 0.4: ${\gamma}-L-glutamyl\;ethylester$, 0.3: GSH 및 Glu-pNA, 0의 순서를 보였다. Uptake rate와 관계없이 in vivo 상에서 L-과 D-glutamine이 L-, D-glutamic acid보다 균체 외 ALA의 생산에 있어서 각각 높은 yield의 효과를 보였다.

Structural Analysis of 5-aminosalicyl-L-glutamic Acid, a Colon-specific Prodrug of 5-aminosalicylic Acid, for Colon-specific Deconjugation

Kim, Ji-Hye,Kim, Jung-Yoon,Lee, Yong-Hyun,Kim, Young-Mi,Jung, Yun-Jin The Korean Society of Pharmaceutical Sciences and 2010 Journal of Pharmaceutical Investigation Vol.40 No.4

In a previous paper, we showed that 5-aminosalicyl-L-aspartic acid (5-ASA-Asp) has much greater deconjugation efficiency in the cecal contents than does 5-aminosalicyl-L-glutamic acid (5-ASA-Glu). To explore a reason for ineffective deconjugation of 5-ASA-Glu, structural analysis of the conjugate was performed. Aromatic acyl-L-glutamic acid derivatives, N-benzoyl-glumatic acid (BA-Glu), N-(2-hydroxybenzoyl)-glutamic acid (SA-Glu), N-(3-aminobenzoyl)-glutamic acid (3-ABA-Glu) and N-(4-aminobenzoyl)-glutamic acid (4-ABA-Glu), were prepared and incubated in the cecal contents. The deconjugation rates were compared with that of 5-ASA-Glu. The order of the rates was BA-Glu $\approx$ 4-ABA-Glu $\approx$ 3-ABA-Glu $\gg$ SA-Glu $\approx$ 5-ASA-Glu. The deconjugation of the aromatic acyl-L-glutamic acid derivatives was carried out by enzyme(s) in the cecal contents since the deconjugation did not occur in the autoclaved cecal contents and on incubation with N-benzoyl-D-glutamic acid. Our data suggest that the 2-hydroxyl group in 5-ASA is ascribed to the poor deconjugation of 5-ASA-Glu in the cecal contents.

Production of siderophore from L-glutamic acid as both carbon and nitrogen sole sources in Acinetobacter sp. B-W

김경자,장주호,양용준,Kim, Kyoung-Ja,Jang, Ju-Ho,Yang, Yong-Joon The Microbiological Society of Korea 2017 미생물학회지 Vol.53 No.2

포도당과 글루탐산을 함유한 배지에서 시드로포어인 2, 3-dihydroxybenzoic acid (DHB)를 생산하는 Acinetobacter sp. B-W 균주를 글루탐산을 유일한 탄소원과 질소원으로 함유한 배지에 배양한 결과, 상등액에서 2, 3-DHB가 아닌 카테콜 형의 시드로포어를 생산하는 것으로 조사되었다. 글루탐산의 농도는 3%에서 시드로포어 생산이 최고로 나타났으며, 3% 보다 높은 농도에서는 감소하는 것으로 조사되었다. 글루탐산을 유일한 탄소원과 질소원으로 함유한 배지에서 자란 균주 B-W는 배양 온도 $28^{\circ}C$에서는 시드로포어를 생산하지만 $36^{\circ}C$에서는 생산하지 않았다. 또한 $10{\mu}M\;FeCl_3$를첨가한 배지에서는 시드로포어 생산이 완전히 억제되었다. 글루탐산 배지에서 생산된 균주 B-W의 시드로포어는 TLC 전개 용매 butanol: acetic acid: water =12:3:5에서 Rf치가 0.32로 나타나 Rf치가 0.82인 2, 3-DHB와는 다른 것으로 조사되었으며, 또한 항산화 활성도 없는 것으로 나타나 항산화 활성을 가진 2, 3-DHB와는 다른 시드로포어인 것으로 추정되었다. Catechol type siderophore different from 2, 3-dihydroxybenzoic acid (DHB) was produced from Acinetobacter sp. B-W grown in medium containing L-glutamic acid as both carbon and nitrogen sole sources at $28^{\circ}C$. Optimal concentration of glutamic acid for siderophore production was 3% and production of siderophore was decreased above 3% glutamic acid. In previous report, siderophore, 2, 3-DHB was produced from strain B-W grown in medium containing glucose as carbon source and glutamic acid as nitrogen source. Rf value of siderophore produced from strain B-W grown in medium glutamic acid as both carbon and nitrogen sole sources at $28^{\circ}C$ was 0.32, while 2, 3-DHB was 0.84 in butanol-acetic acid-water (12:3:5) as developing solvent. Antioxidative activity of 2, 3-DHB was not detected in that siderophore produced from glutamic acid. Catechol nature of siderophore was detected by Arnow test. Although in iron-limited media optimal cell growth was identified at $36^{\circ}C$, significant quantities of siderophore were produced only at $28^{\circ}C$. Biosynthesis of siderophore was strongly inhibited by growth at $36^{\circ}C$. Production of siderophore was completely inhibited by $10{\mu}M\;FeCl_3$.

Stimulation of γ-Aminobutyric Acid Synthesis Activity in Brown Rice by a Chitosan/Glutamic Acid Germination Solution and Calcium/Calmodulin

Oh, Suk-Heung Korean Society for Biochemistry and Molecular Biol 2003 Journal of biochemistry and molecular biology Vol.36 No.3

Changes in the concentrations of $\gamma$-aminobutyric acid (GABA), soluble calcium ions, glutamic acid, and the activity of glutamate decarboxylase (GAD) were investigated in non-germinated vs. germinated brown rice. Brown rice was germinated for 72 h by applying each of the following solutions: (1) distilled water, (2) 5 mM lactic acid, (3) 50 ppm chitosan in 5 mM lactic acid, (4) 5 mM glutamic acid, and (5) 50 ppm chitosan in 5 mM glutamic acid. GABA concentrations were enhanced in all of the germinated brown rice when compared to the non-germinated brown rice. The GABA concentration was highest in the chitosan/glutamic acid that germinated brown rice at 2,011 nmol/g fresh weight, which was 13 times higher than the GABA concentration in the non-germinated brown rice at 154 nmol/g fresh weight. The concentrations of glutamic acid were significantly decreased in all of the germinated rice, regardless of the germination solution. Soluble calcium and GAD were higher in the germinated brown rice with the chitosan/glutamic acid solution when compared to the rice that was germinated in the other solutions. GAD that was partially purified from germinated brown rice was stimulated about 3.6-fold by the addition of calmodulin in the presence of calcium. These data show that the germination of brown rice in a chitosan/glutamic acid solution can significantly increase GABA synthesis activity and the concentration of GABA.

Effect of plasmid curing on the production of siderophore from glutamic acid as both carbon and nitrogen sole sources in Acinetobacter sp. B-W

김경자,이재림,양용준,Kim, Kyoung-Ja,Lee, Jae-Rim,Yang, Yong-Joon The Microbiological Society of Korea 2018 미생물학회지 Vol.54 No.3

Effect of plasmid curing of Acinetobacter sp. B-W on the production of siderophore from glutamic acid as both carbon and nitrogen sole sources was investigated. Plasmid cured mutant of strain B-W lost the ability to produce siderophore from glutamic acid at $28^{\circ}C$. Transformant E. coli $DH5{\alpha}$ harboring 20 kb plasmid, that was isolated from wild type of strain B-W produced siderophore from glutamic acid as both carbon and nitrogen sole sources at $28^{\circ}C$, but, not at $36^{\circ}C$. Production of siderophore from glutamic acid by transformant E. coli $DH5{\alpha}$ was completely inhibited by $10{\mu}M\;FeCl_3$. In previous report, catechol nature of siderophore produced from glutamic acid by strain B-W was detected by Arnow test. The siderophore produced from glutamic acid by transformant E. coli $DH5{\alpha}$ was also catechol type. Rf value of siderophore produced from transformant E. coli $DH5{\alpha}$ grown in medium glutamic acid as both carbon and nitrogen sole sources at $28^{\circ}C$ was 0.32 in butanol-acetic acid-water (12:3:5) as developing solvent. Rf value of the siderophore was the same with that of wild type of strain B-W. Thus a single plasmid of 20 kb seemed to be involved in the production of siderophore from glutamic acid. 플라스미드가 제거된 Acinetobacter sp. B-W 균주의 돌연변이체를 글루탐산을 유일한 탄소 원과 질소원으로 함유한 배지에 $28^{\circ}C$에서 배양한 결과 글루탐산으로부터의 시드로포어 생산이 억제되었다. B-W 원 균주의 20 kb 플라스미드를 가진 형질 전환체 대장균 $DH5{\alpha}$는 같은 조건의 배지에서 시드로포어를 생산하는 것으로 조사되었다. 그러나 $36^{\circ}C$에서는 형질 전환체 대장균 $DH5{\alpha}$의 시드로포어 생산이 강하게 억제되었으며, 돌연변이체 B-W 균주는 $28^{\circ}C$에서와 마찬가지로 $36^{\circ}C$에서도 시드로포어를 생산하지 못하였다. 형질 전환체로부터 생산된 시드로포어의 종류는 원 균주 B-W와 마찬가지로 Arnow 시험 결과 카테콜 형으로 조사되었으며, $10{\mu}M\;FeCl_3$를 첨가한 배지에서는 시드로포어 생산이 완전히 억제되었다. 형질전환체로부터 생산된 시드로포어의 TLC상에서의 Rf값은 butanol-acetic acid-water (12:3:5) 용매상에서 0.32로 원 균주 B-W에서 생산된 시드로포어와 같았다. 위와 같은 실험 결과로 글루탐산으로부터 생산된 시드로포어의 생합성에 관여하는 유전자들이 20 kb 플라스미드 상에 있는 것으로 추정되었다.

위합성용액에서 과일주스에 노출한 Non-O157 Shiga Toxin-Producing Escherichia coli의 산 저항성 평가

김광희(Gwang-Hee Kim),오덕환(Deog-Hwan Oh) 한국식품영양과학회 2016 한국식품영양과학회지 Vol.45 No.4

다양한 환경에서 분리된 시가독소 생산성 대장균(Shiga toxin-producing E. coli, STEC, n=18)을 초산혼합용액(AAS; 400 mM, pH 3.2, 30°C)에 노출한 후 산 저항성을 측정하였다. 또한, 선정된 4종류의 non-O157:H7 STEC균을 사과주스, 파인애플주스, 오렌지주스, 딸기주스(pH 3.8)에 정봉하여 4°C와 20°C에서 24시간 산 적응시킨 후 위합성용액(SGF, pH 1.5)에서 2시간 동안 생존능력을 평가하였다. Non-O157:H7 STEC를 AAS에 노출했을 때 O111 혈청형의 STEC는 평균 0.12 log CFU/mL 감소하여 다른 혈청형에 비하여 가장 강한 산 저항성을 나타냈고 O157:H7 STEC와 유의적 차이가 없었으며(P>0.05), O26 혈청형의 STEC는 가장 민감한 것으로 나타났다. 반면, AAS에 glutamic acid를 첨가하였을 경우 모든 STEC는 혈청형과 관계없이 초산에 매우 강한 저항성을 나타내었다(P>0.05). SGF에서 생존능력을 측정한 결과, 06E0218(O157:H7)은 다른 non-O157:H7 STEC 균들보다 생존능력이 낮았고 03-4669(O145:NM)가 가장 강한 생존능력을 나타내었다. 한편, 과일주스 중에서는 파인애플주스에 산 적응된 STEC가 SGF에 가장 강한 생존능력을 나타내었다. 4°C의 과일주스에 STEC를 산 적응시켰을 경우 20°C보다 SGF에 대한 생존능력이 현저하게 높았다(P<0.05). 따라서 과일주스에 의한 non-O157:H7 STEC의 산 적응력 증가는 위장관 내 생존율 및 식중독 발생을 높일 수 있으므로 이에 대한 적절한 연구와 안전관리 옵션을 제공할 필요가 있을 것으로 판단된다. The objectives of this study were Ⅰ) to compare the acid resistance (AR) of seven non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups, including O26, O45, O103, O111, O121, O145, and O157:H7 STEC isolated from various sources, in 400 mM acetic acid solution (AAS) at pH 3.2 and 30°C for 25 min with or without glutamic acid and Ⅱ) to determine strain survival upon exposure to simulated gastric fluid (SGF, pH 1.5) at 37°C for 2 h after acid adaptation in apple, pineapple, orange, and strawberry juices at pH 3.8, 4°C and 20°C. Results show that the O111 serogroup strains had the strongest AR (0.12 log reduction CFU/mL) which was very similar to that of O157:H7 STEC (P>0.05), compared to other serogroups in AAS without glutamic acid, whereas O26 serogroup strains showed the most sensitive AR. However, there was no significant (P>0.05) difference of AR among seven serogroups in AAS with glutamic acid. In the SGF study, 05-6545 (O45:H2), 08023 (O121:H19), and 03-4669 (O145:NM) strains adapted in fruit juices at 4°C and 20°C displayed enhanced survival with exposure to SGF for 60 min compared to 06E0218 (O157:H7) strains (P<0.05). In addition, 4 STEC strains adapted in pineapple juice at 4°C showed enhanced survival with exposure to SGF for 60 min compared to those strains acid-adapted in the other fruit juices. Generally, adaptation at 4°C in fruit juices resulted in significantly enhanced survival levels compared to acid-adapted at 20°C and non-adapted conditions. The AR caused by adaptation in fruit juices at low temperature may thus increase survival of non-O157 STEC strain in acidic environments such as the gastrointestinal tract. These results suggest that more careful strategies should be provided to protect against risk of foodborne illness by non-O157 STEC.

아미노산 첨가가 꽃송이버섯 균사체 성장 및 베타글루칸, GABA 함량 변화에 미치는 영향

조한교,신현재 한국버섯학회 2017 한국버섯학회지 Vol.15 No.1

Sparassis latifolia (formerly S. crispa) is used in food and nutraceuticals or dietary supplements, as rich in flavor compounds and β-glucan. Some previous studies have reported the effects of mushroom on brain function, including its neuroprotective effect. Thus, for this mushroom to be used as an effective nutraceutical for brain function, it would be desirable for it to contain other compounds such as γ-aminobutyric acid (GABA) in addition to β-glucan. In this study, the enhancement of growth and GABA production in the mycelium of medicinal and edible mushroom S. latifolia was investigated. Amino acids were added externally as the main source of nutrition, and the effects of amino acids were investigated using liquid medium, specifically amino acid-free potato dextrose broth (PDB). The amino acids added were L-glutamic acid (named PDBG medium) and L-ornithine (named PDBO medium). The growth of mycelia was determined to be 0.9 ± 0.00 g/L, 2.2 ± 0.16 g/L, and 1.93 ± 0.34 g/L PDBG respectively. The GABA content was 21.3 ± 0.9 mg/100 g in PDB medium, and it in PDBG 1.4% medium, at 115.4 ± 30.2 mg/100 g. However, the PDBO medium was not effective in increasing the GABA content of mycelia. Amino acids had little effect on the β-glucan content of mycelia. The β-glucan content was 39.7 ± 1.4 mg/100 mg, 34.4 ± 0.2 mg/100 mg, and 35.2 ± 9.2 mg/100 mg in PDB, PDBG 1.8% and PDBO 1.4% media, respectively. Addition of glutamic acid and ornithine positively affected the growth of S. latifolia mycelia, and glutamic acid positively affected GABA production; no degradation of GABA was observed with addition of glutamic acid.