GABA-producing Lactic Acid Bacteria Research Articles

High γ-aminobutyric acid production from lactic acid bacteria: Emphasis on Lactobacillus brevis as a functional dairy starter

ABSTRACTγ-Aminobutyric acid (GABA) and GABA-rich foods have shown anti-hypertensive and anti-depressant activities as the major functions in humans and animals. Hence, high GABA-producing lactic acid bacteria (LAB) could be used as functional starters for manufacturing novel fermented dairy foods. Glutamic acid decarboxylases (GADs) from LAB are highly conserved at the species level based on the phylogenetic tree of GADs from LAB. Moreover, two functionally distinct GADs and one intact gad operon were observed in all the completely sequenced Lactobacillus brevis strains suggesting its common capability to synthesize GABA. Difficulties and strategies for the manufacture of GABA-rich fermented dairy foods have been discussed and proposed, respectively. In addition, a genetic survey on the sequenced LAB strains demonstrated the absence of cell envelope proteinases in the majority of LAB including Lb. brevis, which diminishes their cell viabilities in milk environments due to their non-proteolytic nature. Thus, several strategies have been proposed to overcome the non-proteolytic nature of Lb. brevis in order to produce GABA-rich dairy foods.

Common Distribution of gad Operon in Lactobacillus brevis and its GadA Contributes to Efficient GABA Synthesis toward Cytosolic Near-Neutral pH.

Many strains of lactic acid bacteria (LAB) and bifidobacteria have exhibited strain-specific capacity to produce γ-aminobutyric acid (GABA) via their glutamic acid decarboxylase (GAD) system, which is one of amino acid-dependent acid resistance (AR) systems in bacteria. However, the linkage between bacterial AR and GABA production capacity has not been well established. Meanwhile, limited evidence has been provided to the global diversity of GABA-producing LAB and bifidobacteria, and their mechanisms of efficient GABA synthesis. In this study, genomic survey identified common distribution of gad operon-encoded GAD system in Lactobacillus brevis for its GABA production among varying species of LAB and bifidobacteria. Importantly, among four commonly distributed amino acid-dependent AR systems in Lb. brevis, its GAD system was a major contributor to maintain cytosolic pH homeostasis by consuming protons via GABA synthesis. This highlights that Lb. brevis applies GAD system as the main strategy against extracellular and intracellular acidification demonstrating its high capacity of GABA production. In addition, the abundant GadA retained its activity toward near-neutral pH (pH 5.5–6.5) of cytosolic acidity thus contributing to efficient GABA synthesis in Lb. brevis. This is the first global report illustrating species-specific characteristic and mechanism of efficient GABA synthesis in Lb. brevis.

Physiology-Oriented Engineering Strategy to Improve Gamma-Aminobutyrate Production in Lactobacillus brevis.

Gamma-aminobutyrate (GABA) is an important chemical in the pharmaceutical field. GABA-producing lactic acid bacteria (LAB) offer the opportunity of developing this health-oriented product. In this study, the gadA, gadB, gadC, gadCB, and gadCA gene segments of Lactobacillus brevis were cloned into pMG36e, and strain Lb. brevis/pMG36e-gadA was selected for thorough characterization in terms of GABA production after analysis of GAD activities. Subsequently, a physiology-oriented engineering strategy was adopted to construct an FoF1-ATPase deficient strain NRA6 with higher GAD activity. As expected, strain NRA6 could produce GABA at a concentration of 43.65 g/L with a 98.42% GABA conversion rate in GYP fermentation medium, which is 1.22-fold higher than that obtained by the wild-type strain in the same condition. This work demonstrates how the acid stress response mechanisms of LAB can be employed to develop cell factories with improved production efficiency and contributes to research into the development of the physiology-oriented engineering.

Application of Baechu-Kimchi Powder and GABA-Producing Lactic Acid Bacteria for the Production of Functional Fermented Sausages.

This study was carried out to determine the physicochemical, microbiological, and quality characteristics of a new type of fermented sausage manufactured by incorporating Baechu-kimchi powder and gamma-aminobutyric acid (GABA)-producing lactic acid bacteria (LAB). The LAB count was at the maximum level by day nine of ripening in inoculated sausages, accompanied by a rapid decrease in the pH. The addition of kimchi powder decreased the lightness (L*) and increased the redness (a*) and, yellowness (b*) values, while also significantly increasing the hardness and chewiness of the sausage (p<0.05). Moreover, although the thiobarbituric acid reactive substances values increased in all samples during the study period, this increase was lower in the kimchi-treated samples, indicating a reduction in lipid oxidation. Overall, our results show that the addition of Baechu-kimchi powder to sausages reduced the off-flavor properties and improved the taste profile of the fermented sausage in sensory evaluations. The GABA content of all fermented sausages increased from 17.42-25.14 mg/kg on the third day of fermentation to 60.95-61.47 mg/kg on the thirtieth day. These results demonstrate that Baechu-kimchi powder and GABA-producing LAB could be functional materials in fermented sausage to improve quality characteristics.

Characterization of the Production of Biogenic Amines and Gamma-Aminobutyric Acid in the Soybean Pastes Fermented by Aspergillus oryzae and Lactobacillus brevis.

The production of gamma-aminobutyric acid (GABA) using GABA-producing lactic acid bacteria (LAB) has been considered to be an attractive strategy. However, some LAB may produce biogenic amines (BA), which may be of concern from the safety viewpoint. The aim of the present study was to characterize the production of GABA and BA in the soybean pastes fermented by Aspergillus oryzae (A. oryzae) FMB S46471 and GABA-producing Lactobacillus brevis (L. brevis) GABA 100. After a ripening period of 90 days, the levels of BA (putrescine, cadaverine, histamine, and tyramine) and GABA in the fermented soybean were assessed by highperformance liquid chromatography. The soybean pastes fermented by A. oryzae and L. brevis showed a range of 7,130-11,592 mg/kg for GABA, 178-305 mg/kg for tyramine, 139-163 mg/kg for putrescine, 7.4-10.8 mg/kg for histamine, and 7.1-7.9 mg/kg for cadaverine, whereas the soybean pastes fermented by A. oryzae only showed a range of 30-1,671 mg/kg for GABA, 0.8-189 mg/kg for tyramine, 1.3-85 mg/kg for putrescine, up to 3.6 mg/kg for histamine, and 0.2-2.4 mg/kg for cadaverine. The results showed that the production of GABA was accompanied by the increase in the production of BA, even though the production levels of histamine and cadaverine were very low. This is the first study to simultaneously characterize the production of BA and GABA in GABA-enriched fermented soybean pastes, and warrants further study to minimize the production of BA while optimizing the production of GABA.

Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production.

Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products.

Isolation and Identification of γ-Aminobutyric acid (GABA)-producing lactic acid bacteria from Kimchi

Presumptive lactic acid bacteria (LAB) were isolated from 20 kimchi samples (total of 230 isolates) and screened for their capacity to synthesize γ-aminobutyric acid (GABA). Only 68 isolates (ca. 30%) showed this activity and were identified by a polyphasic approach consisting of morphological characteristics, catalase and biochemical tests, and species-specific polymerase chain reaction and 16S rRNA gene sequence analyses. Five species were found, including Lactobacillus plantarum (55 isolates), Lactobacillus brevis (six), Leuconostoc mesenteroides (four), Leuconostoc lactis (one), and Weissella viridescens (two). The 68 GABA-producing LAB isolates were isolated from only 11 among 20 kimchi samples indicating that they were not evenly distributed. This is the first report on the isolation of two species of Leuconostoc (Le. mesenteroides and Le. lactis) and one species of Weissella (Ws. viridescens) from kimchi with the capacity to synthesize GABA under in vitro conditions. Additionally, in previous screening results, Le. lactis and Ws. viridescens with the capacity to synthesize GABA isolated and identified from fermented food source were not observed.

유산균 발효에 의한 GABA 함유 토마토 페이스트의 생산

Abstract For the purpose of production of GABA-rich tomato paste, this study was carried out to investigate GABA producing lactic acid bacteria from Korean traditional fermented food, Kimchi and optimize the culture conditions. As a result of fermentation, Lactobacillus brevis B3-20 among lactic acid bacteria isolated at the pre-experiments was the best producer of GABA at the tomato paste medium with 50%(wet-base) levels of dionized water. At the result of fermentation on the tomato paste medium with 0.5%(w/w) yeast extract, as a source of nitrogen, 3%(w/w) MSG(monosodium glutamate) and dionized water(the ratio of tomato paste and water was 2:8), Lb. brevis B3-20 produced the maximum GABA concentration, 143.38 mM. GABA-rich tomato paste showed the activity of free radical scavenging. Because GABA-rich tomato paste have functional ingredients such as ascorbic acid, lycopene, carotenoid, as well as GABA by lactic acid bacteria fermentation, GABA-rich tomato paste can be considered high functional materials. Key words: GABA, tomato paste, Lactobacillus sp., free radical scavenging

Isolation, characterization, and utilization of γ-aminobutyric acid (GABA)-producing lactic acid bacteria from Myanmar fishery products fermented with boiled rice

γ-Aminobutyric acid (GABA)-producing lactic acid bacteria (LAB) were isolated from four types of Myanmar traditional fermented fishery products with boiled rice. All of them belonged to the genus Lactobacillus, and comparison of the effects of these representatives on GABA accumulation in fermented fishery products with boiled rice revealed that Lactobacillus farciminis D323 is the most effective strain as a starter culture. These results may contribute to the development of traditional fermented fishery products with functional properties. In addition, this study is the first to show in detail the distribution of GABA-producing LAB in Southeast Asian fermented fishery products.

Antioxidant activity and γ-aminobutyric acid (GABA) content in sea tangle fermented by Lactobacillus brevis BJ20 isolated from traditional fermented foods

GABA-producing lactic acid bacteria were isolated from kimchi and salt-fermented Jot-gal, which are traditional Korean fermented foods. The strain, BJ-20, isolated from salt-fermented Jot-gal (cod gut), possessed the highest GABA-producing ability in MRS broth with 1% monosodium glutamate (MSG), as determined by thin layer chromatography. The BJ-20 strain was identified as Lactobacillus brevis and designated as L. brevis BJ20. A sea tangle solution was fermented over 5 days to produce GABA using L. brevis BJ20. During fermentation, the GABA concentration dramatically increased, while the glutamic acid concentration decreased. This result indicates that the glutamic acid was converted to GABA by L. brevis BJ20 in the fermented sea tangle solution. Furthermore, the fermented solution exhibited strong antioxidant activities, such as DPPH scavenging, superoxide scavenging, and xanthine oxidase inhibition, which were higher than those of BHA as a positive control.

Microbial and chemical properties of aji-no-susu, a traditional fermented fish with rice product in the Noto Peninsula, Japan

Aji-no-susu is a Japanese fermented fish product prepared from salted horse mackerel Trachurus japonicus, and cooked rice. We studied the organic acid and free amino acid contents and microflora in 12 aji-no-susu products to clarify their features as a lactic-acid-fermented food. Salinity of the samples was approximately 7.0% (rice portion) and 6.0% (fish portion) (w/w). Water activity was approximately 0.9, and pH was approximately 4.4 and lower. In the rice portions, lactic acid content was very high (57 mg/g sample). The predominant amino acids were alanine (2.3 mg/g rice portion) and lysine (2.1 mg/g). In the case of long-fermented (4 and 12 months) aji-no-susu, a high content of gamma-aminobutyric acid (GABA, 1.5 and 1.4 mg/g) was detected. Total viable counts in rice and fish portions were 7.7 and 7.4 log colony-forming units (cfu)/g, respectively. The number of lactobacilli in the rice and fish portions was 7.3 and 7.1 log cfu/g, respectively. Yeasts were detected in eight samples. Furthermore, acid-tolerant lactic acid bacteria (LAB) (Lactobacillus plantarum), GABA-producing LAB (Lactobacillus sp.), and halophilic or halo-tolerant yeast (Debaryomyces hansenii) were isolated and identified. Results in this study indicate that aji-no-susu is a typical traditional lactic-acid-fermented fish product.

Enzyme-based microtiter plate assay for γ-aminobutyric acid: Application to the screening of γ-aminobutyric acid-producing lactic acid bacteria

An enzyme-based microtiter plate assay for γ-aminobutyric acid (GABA) was developed. GABA was quantified using γ-aminobutyrate glutamate aminotransferase and succinic semialdehyde dehydrogenase in the presence of NADP + and α-ketoglutarate. The NADPH produced by the series of enzymatic reactions was measured spectrophotometrically at 340 nm. A linear relationship between absorbance and the concentration of GABA was obtained in the ranges from 5.0 × 10 −4 to 1.0 × 10 −2 M. The relative standard deviation for 10 successive measurements was 0.9% at the 10 mM GABA level. This analytical method was applied to the screening of GABA-producing lactic acid bacteria in de Man–Rogosa–Sharpe (MRS) medium. The proposed method enables one to assay 96 samples for an hour without the pre-treatment of samples. The method is by far superior to the traditional HPLC method from the point of view of rapidity and simplicity.