Enhanced Production of Gamma-Aminobutyric Acid by Optimizing Culture Conditions of Lactobacillus brevis HYE1 Isolated from Kimchi, a Korean Fermented Food원문보기
Lim, Hee Seon
(Department of Life Sciences, Graduate School of Incheon National University)
,
Cha, In-Tae
(Division of Bioengineering, Incheon National University)
,
Roh, Seong Woon
(Biological Disaster Analysis Group, Korea Basic Science Institute)
,
Shin, Hae-Hun
(Division of Foodservice Industry, Baekseok Culture University)
,
Seo, Myung-Ji
(Department of Life Sciences, Graduate School of Incheon National University)
This study evaluated the effects of culture conditions, including carbon and nitrogen sources, L-monosodium glutamate (MSG), and initial pH, on gamma-aminobutyric acid (GABA) production by Lactobacillus brevis HYE1 isolated from kimchi, a Korean traditional fermented food. L. brevis HYE1 was screene...
This study evaluated the effects of culture conditions, including carbon and nitrogen sources, L-monosodium glutamate (MSG), and initial pH, on gamma-aminobutyric acid (GABA) production by Lactobacillus brevis HYE1 isolated from kimchi, a Korean traditional fermented food. L. brevis HYE1 was screened by the production analysis of GABA and genetic analysis of the glutamate decarboxylase gene, resulting in 14.64 mM GABA after 48 h of cultivation in MRS medium containing 1% (w/v) MSG. In order to increase GABA production by L. brevis HYE1, the effects of carbon and nitrogen sources on GABA production were preliminarily investigated via one-factor-at-a-time optimization strategy. As the results, 2% maltose and 3% tryptone were determined to produce 17.93 mM GABA in modified MRS medium with 1% (w/v) MSG. In addition, the optimal MSG concentration and initial pH were determined to be 1% and 5.0, respectively, resulting in production of 18.97 mM GABA. Thereafter, response surface methodology (RSM) was applied to determine the optimal conditions of the above four factors. The results indicate that pH was the most significant factor for GABA production. The optimal culture conditions for maximum GABA production were also determined to be 2.14% (w/v) maltose, 4.01% (w/v) tryptone, 2.38% (w/v) MSG, and an initial pH of 4.74. In these conditions, GABA production by L. brevis HYE1 was predicted to be 21.44 mM using the RSM model. The experiment was performed under these optimized conditions, resulting in GABA production of 18.76 mM. These results show that the predicted and experimental values of GABA production are in good agreement.
This study evaluated the effects of culture conditions, including carbon and nitrogen sources, L-monosodium glutamate (MSG), and initial pH, on gamma-aminobutyric acid (GABA) production by Lactobacillus brevis HYE1 isolated from kimchi, a Korean traditional fermented food. L. brevis HYE1 was screened by the production analysis of GABA and genetic analysis of the glutamate decarboxylase gene, resulting in 14.64 mM GABA after 48 h of cultivation in MRS medium containing 1% (w/v) MSG. In order to increase GABA production by L. brevis HYE1, the effects of carbon and nitrogen sources on GABA production were preliminarily investigated via one-factor-at-a-time optimization strategy. As the results, 2% maltose and 3% tryptone were determined to produce 17.93 mM GABA in modified MRS medium with 1% (w/v) MSG. In addition, the optimal MSG concentration and initial pH were determined to be 1% and 5.0, respectively, resulting in production of 18.97 mM GABA. Thereafter, response surface methodology (RSM) was applied to determine the optimal conditions of the above four factors. The results indicate that pH was the most significant factor for GABA production. The optimal culture conditions for maximum GABA production were also determined to be 2.14% (w/v) maltose, 4.01% (w/v) tryptone, 2.38% (w/v) MSG, and an initial pH of 4.74. In these conditions, GABA production by L. brevis HYE1 was predicted to be 21.44 mM using the RSM model. The experiment was performed under these optimized conditions, resulting in GABA production of 18.76 mM. These results show that the predicted and experimental values of GABA production are in good agreement.
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제안 방법
The full-length gad gene from genomic DNA was also amplified by using the designed primer sets based on previously reported gad sequences of several L. brevis strains: MME-63 (5’- ATGGCTATGTTGTATGGA-3’) and MME-64 (5’-TTAGTGCGT GAACCCGTA-3’).
The optimal level of each variable and the effect of their interactions on GABA production as a function of two variables were analyzed by plotting three-dimensional response surface curves (Fig. 5). The response surface representing GABA production is a function of the values of two variables with other variables at an optimal level.
brevis HYE1 were also investigated via the conventional OFAT method to determine carbon and nitrogen sources and identify fermentation factors with significant effects on GABA production. Thereafter, the optimal concentration of each fermentation factor, including the selected carbon and nitrogen sources, exogenous L-monosodium glutamate (MSG), and initial pH, were further determined to enhance GABA production by L. brevis HYE1 using statistical RSM while employing a three-level, four-variable BBD.
Optimization of GABA production by the RSM model could be dependent on GABA-producing microorganisms, in particular species, and variables used in the model. Therefore, it was meaningful to optimize GABA production by L. brevis HYE1 via the RSM model with four variables, including the concentrations of maltose, tryptone, and initial MSG and the initial pH, in this study. Further studies are needed on culture optimization using medium additives such as pyridoxal-5’-phosphate as a coenzyme of GAD, Tween-80 as a growth-stimulating factor for LAB, and sulfate ions for enhancing GAD activity [20, 40].
데이터처리
Various assemblies used in this design are shown in Table 1. Analysis of variance (ANOVA) and regression analysis were performed with the SAS software package (SAS Institute Inc., USA).
The significance of the regression coefficient was demonstrated by ANOVA analysis (Table 2). A value of “Model Prob > F” less than 0.
이론/모형
RSM with the BBD model using three coded levels was employed for determining the optimal culture conditions of the four variables monitored in the OFAT method. The behavior of the system was explained by the following quadratic equation.
Recently, culture conditions of Lc. lactis strain B isolated from kimchi and L. plantarum Taj-Apis362 from honeybees were optimized for GABA production by the RSM method based on the Box-Behnken design (BBD) and central composite design (CCD), respectively [23, 24].
성능/효과
1). Additionally, the sequence showed 83%, 82%, and 71% amino acid sequence identities with those from L. herbarum, L. plantarum 16, and Enterococcus avium, respectively. Based on the result of 16S rRNA gene sequence analysis, the isolate HYE1 was identified as L.
In order to isolate the GABA-producing microorganism, over 100 bacterial isolates obtained from traditional fermented foods were screened for GABA production by incubating the bacterial culture with 1% MSG. After TLC and spectrophotometry analyses using GABase enzyme, over 10 isolates showing high GABA production were identified by 16S rRNA gene sequence analysis, resulting in Lactobacillus genus strains with taxonomical similarities from 99.6% to 100.0% (data not shown). Sequence analysis of the GAD gene was subsequently conducted to select the novel GAD gene-harboring strain.
plantarum 16, and Enterococcus avium, respectively. Based on the result of 16S rRNA gene sequence analysis, the isolate HYE1 was identified as L. brevis based on 99% sequence similarity to those of L. brevis ATCC 367 (GenBank Accession No. CP000416) and L. brevis KB290 (AP012167). The 16S rRNA gene and GAD gene sequences from L.
38%. Finally, the optimum conditions for maximum GABA production using the quadratic regression model equation and response surface curves were 2.14% maltose, 4.01% tryptone, 2.38% initial MSG, and an initial pH of 4.74. The maximum value of GABA production predicted from this model was determined to be 21.
0. In agreement with our results, the pH conditions for most Lactobacillus-genus strains producing GABA have been optimized within the range from 4.5 to 5.5, including the following studies reporting pH 5.0 for L. buchneri MS [12], pH 5.25 for L. brevis K203 [20], and pH 5.31 for L. plantarum Taj-Apis362 [24]. The enhancement of GABA production under acidic culture conditions is closely associated with the characteristics of GAD, which shows increased activity and stability in the presence of hydrogen ions [34, 35].
4 kbp per the expected size (data not shown). The isolate HYE1 was finally selected based on the GABA production level and sequence analysis of the GAD gene, and exhibited the highest level of GABA production (14.64 mM) compared with the other GABA-producing LAB strains isolated in this study (data not shown). The amino acid sequence of GAD from the isolate HYE1 showed 99% identity to those of L.
후속연구
sakei B2-16 and Pediococcus pentosaceus HN8 were optimized using rice bran extracts and mature coconut water, which are byproducts from industrial agricultural and food productions, respectively [21, 41]. Therefore, further study will be needed regarding the commercial production of GABA by L. brevis HYE1 and its culture optimization using a costeffective medium instead of modified MRS medium. In particular, the fact that studies on culture optimization to directly control the cost-effective medium are limited will encourage the follow-up study regarding the RSM-based culture optimization for the industrial production of GABA.
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