Optimization of c-amino butyric acid production in a ... - Springer Link

Biotechnol Lett (2014) 36:93–98 DOI 10.1007/s10529-013-1326-z

ORIGINAL RESEARCH PAPER

Optimization of c-amino butyric acid production in a newly isolated Lactobacillus brevis Tran Thi Thanh Binh • Wan-Taek Ju Woo-Jin Jung • Ro-Dong Park

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Received: 9 May 2013 / Accepted: 13 August 2013 / Published online: 28 September 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract An isolate from kimchi, identified as Lactobacillus brevis, accumulated c-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in the culture medium. Optimal culture conditions for growth of L. brevis and production of GABA were 6 % (w/v) L-glutamic acid, 4 % (w/v) maltose, 2 % (w/v) yeast extract, 1 % (w/v) NaCl, 1 % (w/v) CaCl2, 2 g Tween 80/l, and 0.02 mM pyridoxal 50 -phosphate at initial pH 5.25 and 37 °C. GABA reached 44.4 g/l after 72 h cultivation with a conversion rate 99.7 %, based on the amount (6 %) of L-glutamic acid added.

Electronic supplementary material The online version of this article (doi:10.1007/s10529-013-1326-z) contains supplementary material, which is available to authorized users. T. T. T. Binh W.-T. Ju W.-J. Jung R.-D. Park Glucosamine Saccharide Materials-National Research Laboratory (GSM-NRL), Division of Applied Bioscience and Biotechnology, Institute of Agricultural Science and Technology, Chonnam National University, Kwangju 500-757, Korea Present Address: T. T. T. Binh Faculty of Agriculture and Forestry, Tay Nguyen University, Buon Ma Thuot, Dak Lak Province, Viet Nam R.-D. Park (&) Division of Applied Bioscience and Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Kwangju 500-757, Korea e-mail: [email protected]

GABA was purified using ion exchange column chromatography with 70 % recovery and 97 % purity. Keywords c-Aminobutyric acid GABA Glutamic acid Kimchi Lactic acid bacterium Lactobacillus brevis

Introduction c-Aminobutyric acid (GABA), a major inhibitory neurotransmitter, is a non-protein amino acid that is widely distributed in microorganisms, plants and animals. It has several physiological functions, such as induction of hypotension, a diuretic effect, and a tranquilizer effect, particularly with regard to sleeplessness, depression, and autonomic disorders observed during menopausal periods (Jakobs et al. 1993). GABA therefore has the potential as a bioactive component in foods and pharmaceuticals. Some GABA-containing foods, such as tea (Abe et al. 1995), soy products (Tsai et al. 2006) and rice germ (Zhang et al. 2006), have been developed. The consumption of GABA-enriched foods can depress the systolic blood pressure in spontaneously hypertensive rats (SHRs) (Hayakawa et al. 2004) and mildly hypertensive humans (Inoue et al. 2003). Lactic acid bacteria (LAB) are generally regarded as safe (GRAS) and have been extensively used in dairy products, bread, fermented vegetables, meats and fish (Lee et al. 2006; Leroy and De Vuyst 2004).

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Also, LAB have been used as probiotics due to their properties such as immunomodulation, inhibition of pathogenic bacteria, control of intestinal homeostasis, resistance to gastric acidity, bile acid resistance, and antiallergic activity (Hwanhlem et al. 2010; Tannock 2004). Thus, GABA produced by LAB in appropriate foods can make full use of the health-promoting properties of GABA and LAB themselves. Some fermented products, such as dairy products (Inoue et al. 2003), black raspberry juice (Kim et al. 2009), soymilk (Tsai et al. 2006) and kimchi (Seok et al. 2008), have been enriched in GABA using GABA-producing LAB as starter cultures. Some GABA-producing LAB strains have shown potential in large-scale fermentation for the production of GABA (Komatsuzaki et al. 2005; Cho et al. 2007; Yang et al. 2008). In this study, a GABA-producing LAB, Lactobacillus brevis K203, was isolated and characterized from kimchi. Culture conditions were then optimized in order to enhance GABA productivity.

Materials and methods Isolation and identification of GABA-producing LAB Lactobacillus brevis was isolated from kimchi and identified by standard protocols (see Supplementary data 1). It was given the strain designation of L. brevis K203.

Biotechnol Lett (2014) 36:93–98

beef extract and 0.5 % (w/v) yeast extract were removed from the MRS medium and then 1 % nitrogen sources of casein peptone, meat extract, yeast extract, malt extract, skim milk, corn milk and soybean powder were added (Kook et al. 2010). The effects of NaCl, CaCl2, Tween 80 and pyridoxal 50 phosphate on GABA productivity were also tested. Purification of GABA from the fermented broth GABA was purified from the fermented culture broth (200 ml) by centrifugation, decolorization using active carbon, ethanol precipitation and Dowex 50WX4-200 ion exchange chromatography according to Li et al. (2011) with modification. Analysis of GABA GABA contents were determined qualitatively by prestained TLC cellulose glass plates (Qiu et al. 2010) and quantitatively according to Li et al. (2009) with modifications. After development in n-butanol/acetic acid/water (5:3:2, by vol.) containing 1.2 % (w/v) ninhydrin, the plate was directly dried for color yield. GABA spots were scratched out from the plate and extracted with 4 ml 75 % (v/v) ethanol/0.6 % (w/v) cupric sulfate (38:2, v/v) at 40 °C. The absorbance of the supernatant was read at 512 nm. Purity of the product was also determined by HPLC (Li et al. 2009).

Results and discussion Optimization of culture condition for GABA production MRS media containing 6 % (w/v) L-glutamic acid (GA) at various initial pHs from 3.5 to 7.0 were used for cultivation of L. brevis K203 at 37 °C to test pH effect on cell growth and GABA production (Cho et al. 2007). The effect of temperature was measured from 20 to 55 °C at pH 5.25. For the effect of glutamate concentration, MRS media containing 4–12 % (w/v) GA and monosodium glutamate (MSG) were used (Li et al. 2010). To compare GABA productivity for various carbon sources, MRS media containing 6 % (w/v) GA with glucose, maltose, sucrose, and soluble starch at 1 and 2 % (w/v) were prepared. For the effect of nitrogen, 1 % (w/v) protease peptone, 1 % (w/v)

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Isolation and identification of GABA producing bacteria Fourteen strains were isolated from kimchi as tentative GABA producers (Supplementary Fig. 1a). Among them, three strains K203, K304 and K305 produced high concentration of GABA. Strain K203 completely converted glutamate to GABA within 72 h cultivation (Supplementary Fig. 1b) and was identified as L. brevis. Currently, a few GABA-producing LAB species have been isolated from kimchi, including L. brevis OPY-1 (Park and Oh 2005), L. brevis GABA 100 (Kim et al. 2009), Lactococcus lactis B (Lu et al. 2009), and Lactobacillus buchneri MS (Cho et al. 2007).


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Fig. 1 The effect of the initial pH on cell growth (a) and GABA production (b). Bacteria were grown in 100 ml flasks containing MRS medium with 6 % GA at 37 °C for 120 h. The 100 % represents 33.8 mg/ml GABA

Optimization of culture conditions for GABA production Growth of L. brevis K203 reached the stationary phase after 48 h at all pH values except those below pH 4.5 (Fig. 1a). The highest populations were at pH 5.0 and 5.25. The optimal initial pH for GABA production was 5.25 (Fig. 1b). GABA production rapidly decreased at pH values below 4.5 or above 6.5, suggesting pH dependence of cell growth and GABA production. At the initial pH of 5.0 and 5.25, the entire GA was converted to GABA within 96 h (data not shown). This reaction would be carried out by a glutamic acid decarboxylase that has been recognized in Lactobacillus spp. (Li and Cao 2010). The optimum temperature for growth of L. brevis K203 was 35 °C. The strain grew well from 20 to 40 °C but not above than 45 °C (data not shown). The highest production of GABA was obtained at 37 °C (34.8 mg/ml) and 40 °C (33.1 mg/ml). The GABA production by K203 was affected by the concentration of GA or MSG (Fig. 2a). When GA

Fig. 2 Effect of glutamate (a) and carbon source (b) on GABA production by L. brevis K203. Bacteria were grown in 100 ml flasks containing MRS medium with GA or various carbon source at initial pH 5.25 and 37 °C for 120 h. The 100 % represents 33.6 mg/ml (a) and 32.3 mg/ml GABA (b)

concentration increased from 4 to 6 % (w/v), GABA production increased significantly. However, above 8 %, GABA production decreased. The extra high concentration of GA was inhibitory for the bacterium growth and for GAD activity. Maltose (4 %) was the best carbon source for GABA production with a 16 % increase of GABA (from 27.8 to 32.3 mg/ml), compared to standard MRS medium (2 % D-glucose) (Fig. 2b). The optimal carbon sources for GABA production varied according to the LAB strains, for examples, 1 % glucose for L. buchneri (Cho et al. 2007), 5.5 % glucose for L. brevis NCL912 (Li et al. 2009) and 4 % sucrose for L. sakei B2-16 (Kook et al. 2010).

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Fig. 3 Effects of nitrogen sources (1 %) (a) and nitrogen concentration (0.5–4 %) (b) on GABA production by L. brevis K203. Bacteria were grown in 100 ml flasks containing MRS medium with 6 % GA and nitrogen source at initial pH 5.25 and 37 °C for 120 h. The 100 % represents 34.0 mg/ml (a) and 41.7 mg/ml GABA (b)

Addition (1 %) of casein peptone, meat extract and yeast extract gave high GABA production (31.3–34 mg/ ml) (Fig. 3a). Exceptionally low production of GABA was observed from skim milk, malt extract, soybean powder and corn meal. Simple, soluble and digest-ready nitrogen sources were better for the growth of L. brevis K203 and GABA production. GABA production was also affected by the concentration of nitrogen source (Fig. 3b). GABA production increased according to the concentration; 32.6, 34 and 41.7 mg/ml at 0.5, 1 and 2 % of yeast extract, respectively. However, at 4 % yeast extract, it decreased to 38.4 mg/ml. The same patterns of GABA production were observed for casein peptone and meat extract (Fig. 3b). In a previous study, 3 % (w/v) soy peptone was the optimal nitrogen source for GABA production with L. brevis NCL912 (Li et al. 2009). Lactobacillus brevis K203 grew well up to 0–7 % (w/v) NaCl which was expected as it was isolated from kimchi, a salty fermented vegetable food. NaCl at 1 %

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Fig. 4 Time course of cell growth (a), pH change (b), and GABA production (c) during fermentation in a jar fermenter (3 l) under the optimized condition. The pH was adjusted to 6.0 during cultivation (b)

(w/v) was the best for GABA production (42 mg/ml). Addition of 1 % (w/v) CaCl2 also gave a 9 % enhancement in GABA production. In case of L. brevis NCL912, addition of CaCl2, MgSO4, vitamin C and nicotinic acid had no significant effect on GABA production (Li et al. 2009). Addition of Tween 80 (2 ml/l), a growth-stimulating factor for LABs, enhanced GABA productivity (36.3 mg/ml GABA). Addition of 0.01 and 0.02 mM PLP, a coenzyme of glutamic acid decarboxylase, gave 10 and 20 % enhancement of GABA production, respectively, compared with no PLP addition. The optimum conditions for GABA production by L. brevis K203 are therefore as follows: 6 % (w/v) glutamic acid, 4 % (w/v) maltose, 1 % (w/v) casein peptone, 1 % (w/v) meat extract, 0.5 % (w/v) yeast extract, 1 % (w/v) NaCl, 1 % (w/v) CaCl2, 2 ml Tween 80/l and 0.02 mM pyridoxal 50 -phosphate at initial pH of 5.25 and 37 °C.


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Table 1 Purification of GABA from the culture broth Step

Amount

Recovery (%)

Purity (%)

Culture broth

190 ml

100

44.2

Decolorization

165 ml

88.8

52.6

Ethanol precipitation IEC

50 ml 5.89 g

71 69.8

70 96.6

GABA production in a jar fermenter When grown in a jar fermenter (3 l) under optimized conditions, the bacterium reached the stationary phase within 24 h (Fig. 4a). Accompanied with GABA formation, pH of the liquid medium increased rapidly and thus pH adjustment was needed (Fig. 4b). Within 72 h cultivation (24 h earlier than flask cultures), GABA reached its highest concentration of 44.4 mg/ml (Fig. 4c). The net conversion rate was calculated as 99.7 %, based on the theoretical GABA yield from 6 % (w/v) GA. Until now, the highest conversion rate of GA to GABA was 97.3 % by L. brevis NCL912 (Li et al. 2010). GABA (5.89 g) was obtained as white powder from 200 ml culture broth, as summarized in Table 1. The purification of GABA from the fermented broth was completed with 69.9 % recovery and 96.7 % purity, suggesting that protocol is simple but effective.

Conclusions Lactobacillus brevis K203, a GABA-producing LAB, was isolated and characterized from kimchi. Culture conditions were optimized for GABA production with L. brevis K203. Under the optimized condition, the yield of GABA reached 44.4 g/l after 72 h fermentation with the bioconversion rate 99.7 %, the highest record up to now. GABA was purified by ion exchange chromatography with 69.9 % recovery and 96.7 % purity from the culture broth. Acknowledgments This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ007983), Rural Development Administration, Republic of Korea.

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