Abstract
Gamma-aminobutyric acid (GABA) is a non-protein amino acid with numerous health benefits, such as reducing hypertension, treating diabetes, and providing neuroprotection. GABA can be produced by lactic acid bacteria (LAB), but not all LAB produce GABA. Therefore, it is necessary to screen LAB isolates with the potential to be GABA producers. GABA was analyzed qualitatively using thin-layer chromatography (TLC) and quantitatively by Ultraviolet-visible (UV-Vis) spectrophotometer. Ten LAB isolates were evaluated for their ability to produce GABA. Among these, Pediococcus acidilactici YKP4 produced the highest concentration of GABA. P. acidilactici YKP4 as the highest GABA producer and Lacticaseibacillus rhamnosus BD2 as a probiotic candidate were further studied to increase GABA production in de Man Rogosa Sharpe (MRS) broth by providing monosodium glutamate (MSG) as the precursor and applying different initial pH and incubation time. The highest production of GABA by P. acidilactici YKP4 (3.30±0.03 g/L) was obtained by the addition of 1% MSG while that of L. rhamnosus BD2 (2.67±0.07 g/L) by 2% MSG with initial pH of 5.0 and incubation time of 72 h. Application of both LAB as a starter culture for milk fermentation showed that GABA production in milk was lower than in MRS broth. Enrichment of milk with MSG increased GABA from 1.80±0.03 g/L to 1.94±0.02 g/L in P. acidilactici YKP4 with 1% MSG, and from 1.15±0.03 g/L to 1.33±0.04 g/L in L. rhamnosus BD2 with 2% MSG. Besides, P. acidilactici YKP4 and L. rhamnosus BD2 were considered as potential LAB for GABA production and can be applied to ferment milk as a functional food.
Keywords:
Fermentation; Functional food; GABA-producing LAB;
L. rhamnosus
;
P. acidilactici
; Starter culture
HIGHLIGHTS
• P. acidilactici YKP4 and L. rhamnosus BD2 were LAB as potential GABA producers
• MSG concentration, initial pH, and incubation time affected GABA production in both LAB
• Both LAB can be applied to enrich GABA in fermented milk.
1 Introduction
Gamma-aminobutyric acid (GABA) is a bioactive, non-protein amino acid that acts as an inhibitory neurotransmitter in the central nervous system of mammals and has additional health-related functional properties. GABA has numerous bio-activities, such as providing neuroprotection (Li et al., 2016Li, W., Wei, M., Wu, J., Rui, X., & Dong, M. (2016). Novel fermented chickpea milk with enhanced level of γ-aminobutyric acid and neuroprotective effect on PC12 cells. PeerJ, 4, e2292. PMid:27602272. http://doi.org/10.7717/peerj.2292
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), reducing hypertension (Kawakami et al., 2018Kawakami, K., Yamada, K., Yamada, T., Nabika, T., & Nomura, M. (2018). Antihypertensive effect of γ-aminobutyric acid-enriched brown rice on spontaneously hypertensive rats. Journal of Nutritional Science and Vitaminology, 64(1), 56-62. PMid:29491273. http://doi.org/10.3177/jnsv.64.56
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; Suwanmanon & Hsieh, 2014Suwanmanon, K., & Hsieh, P. C. (2014). Effect of γ-aminobutyric acid and nattokinase-enriched fermented beans on the blood pressure of spontaneously hypertensive and normotensive Wistar-Kyoto rats. Yao Wu Shi Pin Fen Xi, 22(4), 485-491. PMid:28911464. http://doi.org/10.1016/j.jfda.2014.03.005
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), treating diabetes (Shang et al., 2018Shang, W., Si, X., Zhou, Z., Strappe, P., & Blanchard, C. (2018). Wheat bran with enriched gamma-aminobutyric acid attenuates glucose intolerance and hyperinsulinemia induced by a high-fat diet. Food & Function, 9(5), 2820-2828. PMid:29693103. http://doi.org/10.1039/C8FO00331A
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; Untereiner et al., 2019Untereiner, A., Abdo, S., Bhattacharjee, A., Gohil, H., Pourasgari, F., Ibeh, N., Lai, M., Batchuluun, B., Wong, A., Khuu, N., Liu, Y., Rijjal, D. A., Winegarden, N., Virtanen, C., Orser, B. A., Cabrera, O., Varga, G., Rocheleau, J., Dai, F. F., & Wheeler, M. B. (2019). GABA promotes β-cell proliferation, but does not overcome impaired glucose homeostasis associated with diet-induced obesity. The FASEB Journal, 33(3), 3968-3984. PMid:30509117. http://doi.org/10.1096/fj.201801397R
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), and possessing anticancer properties (Song et al., 2016Song, L., Du, A., Xiong, Y., Jiang, J., Zhang, Y., Tian, Z., & Yan, H. (2016). γ-Aminobutyric acid inhibits the proliferation and increases oxaliplatin sensitivity in human colon cancer cells. Tumour Biology, 37(11), 14885-14894. PMid:27644246. http://doi.org/10.1007/s13277-016-5367-5
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). It also acts as an antioxidant (Tang et al., 2018Tang, X., Yu, R., Zhou, Q., Jiang, S., & Le, G. (2018). Protective effects of γ-aminobutyric acid against H2O2-induced oxidative stress in RIN-m5F pancreatic cells. Nutrition & Metabolism, 15(1), 60. PMid:30202421. http://doi.org/10.1186/s12986-018-0299-2
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; Zhu et al., 2019Zhu, Z., Shi, Z., Xie, C., Gong, W., Hu, Z., & Peng, Y. (2019). A novel mechanism of Gamma-aminobutyric acid (GABA) protecting human umbilical vein endothelial cells (HUVECs) against H2O2-induced oxidative injury. Comparative Biochemistry and Physiology. Toxicology & Pharmacology, 217(348), 68-75. PMid:30500452. http://doi.org/10.1016/j.cbpc.2018.11.018
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), anti-inflammatory (Sokovic Bajic et al., 2019Sokovic Bajic, S., Djokic, J., Dinic, M., Veljovic, K., Golic, N., Mihajlovic, S., & Tolinacki, M. (2019). GABA-producing natural dairy isolate from artisanal zlatar cheese attenuates gut inflammation and strengthens gut epithelial barrier in vitro. Frontiers in Microbiology, 10, 527. PMid:30936860. http://doi.org/10.3389/fmicb.2019.00527
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), and protects the intestinal wall (Chen et al., 2015Chen, Z., Xie, J., Hu, M. Y., Tang, J., Shao, Z. F., & Li, M. H. (2015). Protective effects of γ-aminobutyric acid (GABA) on the small intestinal mucosa in heat-stressed Wenchang chicken. Journal of Animal and Plant Sciences, 25(1), 78-87.). Additionally, it prevents sleep difficulties and depression (Ngo & Vo, 2019Ngo, D. H., & Vo, T. S. (2019). An updated review on pharmaceutical properties of gamma-aminobutyric acid. Molecules, 24(15), 2678. PMid:31344785. http://doi.org/10.3390/molecules24152678
http://doi.org/10.3390/molecules24152678...
). GABA is present in both animals and plants, but its concentration is relatively low, around µg/g in animals and mg/g in plants (Ramos-Ruiz et al., 2018Ramos-Ruiz, R., Poirot, E., & Flores-Mosquera, M. (2018). GABA, a non-protein amino acid ubiquitous in food matrices. Cogent Food & Agriculture, 4(1), 1-89. http://doi.org/10.1080/23311932.2018.1534323
http://doi.org/10.1080/23311932.2018.153...
). Microorganisms synthesize GABA through glutamate decarboxylation, a process catalyzed by the enzyme glutamic acid decarboxylase (GAD; EC 4.1.1.15) (Wu & Shah, 2017Wu, Q., & Shah, N. P. (2017). High γ-aminobutyric acid production from lactic acid bacteria: Emphasis on Lactobacillus brevis as a functional dairy starter. Critical Reviews in Food Science and Nutrition, 57(17), 3661-3672. PMid:26980301. http://doi.org/10.1080/10408398.2016.1147418
http://doi.org/10.1080/10408398.2016.114...
). Microorganisms are preferable to animals and plants for producing GABA because they grow faster, can produce GABA quicker, and require less space for cultivation (Cui et al., 2020Cui, Y., Miao, K., Niyaphorn, S., & Qu, X. (2020). Production of gamma-aminobutyric acid from lactic acid bacteria: A systematic review. International Journal of Molecular Sciences, 21(3), 1-21. PMid:32028587. http://doi.org/10.3390/ijms21030995
http://doi.org/10.3390/ijms21030995...
). Besides being more efficient, GABA production by microorganisms is a more promising method than chemical synthesis because it is eco-friendly, requires lower costs, and obtains higher yields (Zhao et al., 2014Zhao, W. R., Huang, J., Peng, C. L., Hu, S., Ke, P. Y., Mei, L. H., & Yao, S. J. (2014). Permeabilizing Escherichia coli for whole cell biocatalyst with enhanced biotransformation ability from l-glutamate to GABA. Journal of Molecular Catalysis. B, Enzymatic, 107, 39-46. http://doi.org/10.1016/j.molcatb.2014.05.011
http://doi.org/10.1016/j.molcatb.2014.05...
). Lactic acid bacteria (LAB) are the most widely used for GABA production among microorganisms.
Generally, LAB are widely used as starter cultures for food fermentation due to their specific metabolism activities and they are generally recognized as safe (GRAS) bacteria (Luo et al., 2021Luo, H., Liu, Z., Xie, F., Bilal, M., Liu, L., Yang, R., & Wang, Z. (2021). Microbial production of gamma-aminobutyric acid: Applications, state-of-the-art achievements, and future perspectives. Critical Reviews in Biotechnology, 41(4), 491-512. PMid:33541153. http://doi.org/10.1080/07388551.2020.1869688
http://doi.org/10.1080/07388551.2020.186...
). The production of GABA by LAB has made it possible to create fermented foods with high GABA concentrations, resulting in improved functional properties. Several fermented foods, including yoghurt (Shan et al., 2015Shan, Y., Man, C. X., Han, X., Li, L., Guo, Y., Deng, Y., Li, T., Zhang, L. W., & Jiang, Y. J. (2015). Evaluation of improved γ-aminobutyric acid production in yogurt using Lactobacillus plantarum NDC75017. Journal of Dairy Science, 98(4), 2138-2149. PMid:25622870. http://doi.org/10.3168/jds.2014-8698
http://doi.org/10.3168/jds.2014-8698...
), black soybean milk (Ko et al., 2013Ko, C. Y., Lin, H. T. V., & Tsai, G. J. (2013). Gamma-aminobutyric acid production in black soybean milk by Lactobacillus brevis FPA 3709 and the antidepressant effect of the fermented product on a forced swimming rat model. Process Biochemistry (Barking, London, England), 48(4), 559-568. http://doi.org/10.1016/j.procbio.2013.02.021
http://doi.org/10.1016/j.procbio.2013.02...
), litchi juice (Wang et al., 2021Wang, D., Wang, Y., Lan, H., Wang, K., Zhao, L., & Hu, Z. (2021). Enhanced production of γ-aminobutyric acid in litchi juice fermented by Lactobacillus plantarum HU-C2W. Food Bioscience, 42, 101155. http://doi.org/10.1016/j.fbio.2021.101155
http://doi.org/10.1016/j.fbio.2021.10115...
), fermented mulberry juice (Kanklai et al., 2020Kanklai, J., Somwong, T. C., Rungsirivanich, P., & Thongwai, N. (2020). Screening of GABA-producing lactic acid bacteria from thai fermented foods and probiotic potential of Levilactobacillus brevis f064a for gaba-fermented mulberry juice production. Microorganisms, 9(1), 1-17. PMid:33374175. http://doi.org/10.3390/microorganisms9010033
http://doi.org/10.3390/microorganisms901...
), som-pak (a Thai fermented vegetable) (Tanamool et al., 2020Tanamool, V., Hongsachart, P., & Soemphol, W. (2020). Screening and characterisation of gamma-aminobutyric acid (GABA) producing lactic acid bacteria isolated from Thai fermented fish (Plaa-som) in Nong Khai and its application in Thai fermented vegetables (Som-pak). Food Science and Technology, 40(2), 483-490. http://doi.org/10.1590/fst.05419
http://doi.org/10.1590/fst.05419...
), and kung-som (a Thai fermented shrimp) (Sanchart et al., 2017Sanchart, C., Rattanaporn, O., Haltrich, D., Phukpattaranont, P., & Maneerat, S. (2017). Lactobacillus futsaii CS3, a new GABA-producing strain isolated from Thai fermented shrimp (Kung-Som). Indian Journal of Microbiology, 57(2), 211-217. PMid:28611499. http://doi.org/10.1007/s12088-016-0632-2
http://doi.org/10.1007/s12088-016-0632-2...
), have been enriched with GABA through the use of GABA-producing LAB as starter cultures.
Currently, the isolation and characterization of GABA-producing LAB from different fermented foods have been widely investigated and discussed by Yogeswara et al. (2020)Yogeswara, I. B. A., Maneerat, S., & Haltrich, D. (2020). Glutamate decarboxylase from lactic acid bacteria - a key enzyme in GABA synthesis. Microorganisms, 8(12), 1-24. PMid:33287375. http://doi.org/10.3390/microorganisms8121923
http://doi.org/10.3390/microorganisms812...
and Cui et al. (2020)Cui, Y., Miao, K., Niyaphorn, S., & Qu, X. (2020). Production of gamma-aminobutyric acid from lactic acid bacteria: A systematic review. International Journal of Molecular Sciences, 21(3), 1-21. PMid:32028587. http://doi.org/10.3390/ijms21030995
http://doi.org/10.3390/ijms21030995...
. Nonetheless, screening the most efficient GABA-producing LAB remains imperative due to the varied LAB groups present in fermented foods, which depend on the production methods and raw ingredients used (Seo et al., 2013Seo, M.-J., Lee, J.-Y., Nam, Y.-D., Lee, S.-Y., Park, S.-L., Yi, S.-H., Lee, M.-H., Roh, S. W., Choi, H.-J., & Lim, S.-I. (2013). Production of γ-aminobutyric acid by Lactobacillus brevis 340G isolated from kimchi and its application to skim milk. Food Engineering Progress, 17(4), 418-423. http://doi.org/10.13050/foodengprog.2013.17.4.418
http://doi.org/10.13050/foodengprog.2013...
). Production of GABA by LAB relies on the LAB strain and is affected by the presence of precursor (monosodium glutamate/MSG), pH of the medium, and incubation time (Binh et al., 2014Binh, T. T. T., Ju, W. T., Jung, W. J., & Park, R. D. (2014). Optimization of γ-amino butyric acid production in a newly isolated Lactobacillus brevis. Biotechnology Letters, 36(1), 93-98. PMid:24078124. http://doi.org/10.1007/s10529-013-1326-z
http://doi.org/10.1007/s10529-013-1326-z...
; Lim et al., 2017Lim, H. S., Cha, I. T., Roh, S. W., Shin, H. H., & Seo, M. J. (2017). Enhanced production of gamma-aminobutyric acid by optimizing culture conditions of Lactobacillus brevis HYE1 isolated from kimchi, a Korean fermented food. Journal of Microbiology and Biotechnology, 27(3), 450-459. PMid:27880963. http://doi.org/10.4014/jmb.1610.10008
http://doi.org/10.4014/jmb.1610.10008...
). The concentration of glutamate in MRS broth, which is generally utilized for LAB cultivation, is minimal (Wu & Shah, 2015Wu, Q., & Shah, N. P. (2015). Gas release-based prescreening combined with reversed-phase HPLC quantitation for efficient selection of high-γ-aminobutyric acid (GABA)-producing lactic acid bacteria. Journal of Dairy Science, 98(2), 790-797. PMid:25497828. http://doi.org/10.3168/jds.2014-8808
http://doi.org/10.3168/jds.2014-8808...
). The pH of the medium is crucial factor for GABA production, with the optimal pH range for GABA production being between 4.5 and 5.5 (Yogeswara et al., 2020Yogeswara, I. B. A., Maneerat, S., & Haltrich, D. (2020). Glutamate decarboxylase from lactic acid bacteria - a key enzyme in GABA synthesis. Microorganisms, 8(12), 1-24. PMid:33287375. http://doi.org/10.3390/microorganisms8121923
http://doi.org/10.3390/microorganisms812...
). Previous research has demonstrated the addition of MSG (Tanamool et al., 2020Tanamool, V., Hongsachart, P., & Soemphol, W. (2020). Screening and characterisation of gamma-aminobutyric acid (GABA) producing lactic acid bacteria isolated from Thai fermented fish (Plaa-som) in Nong Khai and its application in Thai fermented vegetables (Som-pak). Food Science and Technology, 40(2), 483-490. http://doi.org/10.1590/fst.05419
http://doi.org/10.1590/fst.05419...
), initial pH (Li et al., 2010Li, H., Qiu, T., Gao, D., & Cao, Y. (2010). Medium optimization for production of gamma-aminobutyric acid by Lactobacillus brevis NCL912. Amino Acids, 38(5), 1439-1445. PMid:19787432. http://doi.org/10.1007/s00726-009-0355-3
http://doi.org/10.1007/s00726-009-0355-3...
; Lim et al., 2017Lim, H. S., Cha, I. T., Roh, S. W., Shin, H. H., & Seo, M. J. (2017). Enhanced production of gamma-aminobutyric acid by optimizing culture conditions of Lactobacillus brevis HYE1 isolated from kimchi, a Korean fermented food. Journal of Microbiology and Biotechnology, 27(3), 450-459. PMid:27880963. http://doi.org/10.4014/jmb.1610.10008
http://doi.org/10.4014/jmb.1610.10008...
), and incubation time (Binh et al., 2014Binh, T. T. T., Ju, W. T., Jung, W. J., & Park, R. D. (2014). Optimization of γ-amino butyric acid production in a newly isolated Lactobacillus brevis. Biotechnology Letters, 36(1), 93-98. PMid:24078124. http://doi.org/10.1007/s10529-013-1326-z
http://doi.org/10.1007/s10529-013-1326-z...
) as potential means to enhance GABA production in LAB.
Indonesia has a variety of fermented foods that involve LAB in their production. The LAB used in this study was obtained from fermented foods such as tempeh, fermented mustard, sticky rice tape, and other sources such as kefir granules and breast milk. Two potential probiotics, Lacticaseibacillus rhamnosus BD2, and Lactobacillus kefiri YK4, were derived from kefir granules (Yusuf et al., 2019Yusuf, D., Nuraida, L., Dewanti-Hariyadi, R., & Hunaefi, D. (2019). In vitro characterization of lactic acid bacteria from Indonesian kefir grains as probiotics with cholesterol-lowering effect. Journal of Microbiology and Biotechnology, 30(5), 726-732. PMid:32482938. http://doi.org/10.4014/jmb.1910.10028
http://doi.org/10.4014/jmb.1910.10028...
) and were found to possess high proteolytic activities (Rubak et al., 2020Rubak, Y. T., Nuraida, L., Iswantini, D., & Prangdimurti, E. (2020). Angiotensin-I-converting enzyme inhibitory peptides in milk fermented by indigenous lactic acid bacteria. Veterinary World, 13(2), 345-353. PMid:32255978. http://doi.org/10.14202/vetworld.2020.345-353
http://doi.org/10.14202/vetworld.2020.34...
). LAB isolated from kefir granules produced antioxidant peptides (Yusuf et al., 2021Yusuf, D., Nuraida, L., Dewanti-Hariyadi, R., & Hunaef, D. (2021). In vitro antioxidant and α-glucosidase inhibitory activities of Lactobacillus spp. isolated from Indonesian kefir grains. Applied Food Biotechnology, 8(1), 39-46. http://doi.org/10.22037/afb.v8i1.30367
http://doi.org/10.22037/afb.v8i1.30367...
) and angiotensin-converting enzyme (ACE) inhibitors (Rubak et al., 2020Rubak, Y. T., Nuraida, L., Iswantini, D., & Prangdimurti, E. (2020). Angiotensin-I-converting enzyme inhibitory peptides in milk fermented by indigenous lactic acid bacteria. Veterinary World, 13(2), 345-353. PMid:32255978. http://doi.org/10.14202/vetworld.2020.345-353
http://doi.org/10.14202/vetworld.2020.34...
). Nevertheless, the ability of these LAB to produce GABA is yet to be determined. Therefore, this study aims to obtain LAB isolates from different foods to determine their potential as GABA producers and identify the factors that contribute to the GABA production in LAB.
2 Material & methods
2.1 LAB isolates and LAB growth medium
The LAB isolates used in this study were obtained from the SEAFAST (Southeast Asian Food and Agricultural Science and Technology) Center, IPB University. The isolates of kefir granules origin were L. kefiri YK4, L. rhamnosus BD2, Pediococcus acidilactici YKP4, Lactococcus lactis subsp. lactis BD17, and Limosilactobacillus fermentum JK13 (Yusuf et al., 2019Yusuf, D., Nuraida, L., Dewanti-Hariyadi, R., & Hunaefi, D. (2019). In vitro characterization of lactic acid bacteria from Indonesian kefir grains as probiotics with cholesterol-lowering effect. Journal of Microbiology and Biotechnology, 30(5), 726-732. PMid:32482938. http://doi.org/10.4014/jmb.1910.10028
http://doi.org/10.4014/jmb.1910.10028...
), while that from tempeh were Lactiplantibacillus plantarum 1W22408 and L. fermentum S206 (Touw, 2014Touw, K. S. (2014). Identification of dominant lactic acid bacteria during tempeh fermentation and evaluation of their potential as probiotic (Undergraduate thesis). IPB University, Bogor.), from fermented mustard were L. plantarum 4C261 (Kurnia, 2018Kurnia, H. (2018). Advantage of lactic acid bacteria from fermented mustard (Brassica juncea L.) for reducing cholesterol in vitro (Undergraduate thesis). IPB University, Bogor.), and LAB isolate from sticky rice tape, namely L. fermentum BK27 (Hasanah et al., 2019Hasanah, U., Ratihwulan, H., & Nuraida, L. (2019). Sensory profiles and lactic acid bacteria density of tape ketan and tape singkong in Bogor. Agritech, 38(3), 265-272. http://doi.org/10.22146/agritech.30935
http://doi.org/10.22146/agritech.30935...
; Nurchandra, 2018Nurchandra, K. P. (2018). Identification of lactic acid bacteria from tape with polymerase chain reaction and its amylolytic characterization (Undergraduate thesis). IPB University, Bogor.). In addition to that, an isolate from breast milk, L. rhamnosus R23 (Nuraida et al., 2012Nuraida, L., Hana, Hartanti, A. W., & Prangdimurti, E. (2012). Potential of Lactobacillus isolated from breast milk to prevent diarrheae. Journal of Food Technology and Industry, 23(2), 158-164. http://doi.org/10.6066/jtip.2012.23.2.158
http://doi.org/10.6066/jtip.2012.23.2.15...
) was used in this study. These LAB were cultivated in de Man Rogosa Sharpe (MRS) broth (Oxoid, UK) and quantified using MRS agar (Oxoid, UK).
2.2 Inoculum preparation
Inoculum preparation refers to Yusuf et al. (2019)Yusuf, D., Nuraida, L., Dewanti-Hariyadi, R., & Hunaefi, D. (2019). In vitro characterization of lactic acid bacteria from Indonesian kefir grains as probiotics with cholesterol-lowering effect. Journal of Microbiology and Biotechnology, 30(5), 726-732. PMid:32482938. http://doi.org/10.4014/jmb.1910.10028
http://doi.org/10.4014/jmb.1910.10028...
with slight modifications. LAB isolates were refreshed in MRS broth and incubated in an incubator (MMM Medcenter Einrichtungen GmbH Incucell, Germany) at 37 °C for 24 hours with an inoculum concentration of 107 CFU/mL.
2.3 Screening of GABA-producing LAB
As much as 1% (v/v) of a fresh culture of LAB was inoculated into 100 mL MRS broth (in 100 mL Schott Duran bottles) containing 1% (w/v) MSG (HiMedia, India). Before sterilization, the initial pH was adjusted to 5.0 (Yang et al., 2018Yang, E., Fan, L., Yan, J., Jiang, Y., Doucette, C., Fillmore, S., & Walker, B. (2018). Influence of culture media, pH and temperature on growth and bacteriocin production of bacteriocinogenic lactic acid bacteria. AMB Express, 8(1), 10. PMid:29368243. http://doi.org/10.1186/s13568-018-0536-0
http://doi.org/10.1186/s13568-018-0536-0...
) by addition of HCl 1 N (Merck, Germany) gradually. The pH was measured using a pH meter (Horiba Laqua-PH1100, Japan). Incubation was carried out for 48 hours at 37 °C (Rayavarapu et al., 2021Rayavarapu, B., Tallapragada, P., & Ms, U. (2021). Optimization and comparison of γ-aminobutyric acid (GABA) production by LAB in soymilk using RSM and ANN models. Beni-Suef University Journal of Basic and Applied Sciences, 10(1), 14. http://doi.org/10.1186/s43088-021-00100-3
http://doi.org/10.1186/s43088-021-00100-...
). Uninoculated MRS broth was used as a control. GABA was analyzed qualitatively and quantitatively.
Qualitative analysis of GABA was performed by thin layer chromatography (TLC) referring to Qiu et al. (2010)Qiu, T., Li, H., & Cao, Y. (2010). Pre-staining thin layer chromatography method for amino acid detection. African Journal of Biotechnology, 9(50), 8679-8681. http://dx.doi.org/10.5897/AJB10.817
https://doi.org/10.5897/AJB10.817...
with slight modification. A 1 mL culture was taken and centrifuged (Labtron Refrigerated Centrifuge LRF-B20, UK) at 8000 rpm for 10 min at 4 °C. A total of 0.5 µL of supernatant, GABA standard (1%) (Sigma-Aldrich, USA), and MSG solution (1%) were plated on silica gel 60 F254 plates (Merck, Germany). The mobile phase consisted of a mixture of butanol (Merck, Germany), acetic acid (Merck, Germany), and distilled water in a ratio of 5:3:2, respectively. The plate was then sprayed with 1.2% ninhydrin (Merck, Germany) and dried in an oven (Memmert UN55 53L, Germany) at 90 °C for 7 minutes. GABA produced by LAB is indicated by the same Rf value as the GABA standard (Yogeswara et al., 2021Yogeswara, I. B. A., Kittibunchakul, S., Rahayu, E. S., Domig, K. J., Haltrich, D., & Nguyen, T. H. (2021). Microbial production and enzymatic biosynthesis of γ-aminobutyric acid (GABA) using Lactobacillus plantarum FNCC 260 isolated from indonesian fermented foods. Processes, 9(1), 1-17. http://doi.org/10.3390/pr9010022
http://doi.org/10.3390/pr9010022...
). To determine the LAB capable of producing the highest GABA, a quantitative analysis of GABA produced by LAB was then carried out.
Quantitative analysis of GABA was performed based on the method of Li et al. (2009)Li, H., Qiu, T., Cao, Y., Yang, J., & Huang, Z. (2009). Pre-staining paper chromatography method for quantification of γ-aminobutyric acid. Journal of Chromatography. A, 1216(25), 5057-5060. PMid:19446298. http://doi.org/10.1016/j.chroma.2009.04.044
http://doi.org/10.1016/j.chroma.2009.04....
with slight modifications. The spot separated on the TLC plate representing GABA was scraped off and dissolved in a solution containing 1 mL of 75% (v/v) ethanol (Merck, Germany) and 0.6% (w/v) cupric sulfate (Merck, Germany) in a 38:2 (v/v) ratio at 40 °C. Samples were measured for absorbance with a UV-Vis spectrophotometer (Hitachi U-2800, Japan) at a wavelength of 512 nm using a 1 mL cuvette. GABA concentration was calculated based on the GABA standard curve which had a concentration range of 0.5-4.5 g/L and a coefficient of determination (R2) of 0.99. LAB with the highest GABA production and probiotic candidate LAB were used in the next stage.
2.4 Effect of precursor (MSG) on GABA production
LAB inoculum of 1% (v/v) was inoculated into 100 mL MRS broth with an initial pH of 5.0 and MSG concentrations of 0, 1, 2, and 3% (w/v) in 100 mL Schott Duran bottles (Tanamool et al., 2020Tanamool, V., Hongsachart, P., & Soemphol, W. (2020). Screening and characterisation of gamma-aminobutyric acid (GABA) producing lactic acid bacteria isolated from Thai fermented fish (Plaa-som) in Nong Khai and its application in Thai fermented vegetables (Som-pak). Food Science and Technology, 40(2), 483-490. http://doi.org/10.1590/fst.05419
http://doi.org/10.1590/fst.05419...
) and incubated at 37 °C for 72 hours (Binh et al., 2014Binh, T. T. T., Ju, W. T., Jung, W. J., & Park, R. D. (2014). Optimization of γ-amino butyric acid production in a newly isolated Lactobacillus brevis. Biotechnology Letters, 36(1), 93-98. PMid:24078124. http://doi.org/10.1007/s10529-013-1326-z
http://doi.org/10.1007/s10529-013-1326-z...
; Sanchart et al., 2017Sanchart, C., Rattanaporn, O., Haltrich, D., Phukpattaranont, P., & Maneerat, S. (2017). Lactobacillus futsaii CS3, a new GABA-producing strain isolated from Thai fermented shrimp (Kung-Som). Indian Journal of Microbiology, 57(2), 211-217. PMid:28611499. http://doi.org/10.1007/s12088-016-0632-2
http://doi.org/10.1007/s12088-016-0632-2...
). GABA concentration was analyzed quantitatively.
2.5 Effect of initial pH of the medium and incubation time on GABA production
A fresh LAB culture of 1% (v/v) was inoculated into 100 mL of sterile MRS broth (in 100 mL Schott Duran bottles) with the best MSG concentration obtained in the previous stage. The initial pH value of the medium was set to 4.5; 5.0; and 5.5. Incubation was carried out for 0, 24, 48, and 72 hours at 37 °C. Analyses were carried out for LAB count, pH, and GABA concentration. The number of LAB was enumerated using the pour plate method in MRS agar, and incubated at 37 °C for 48 hours. Only plates containing colonies from 25-250 were used for calculation of LAB count (Maturin & Peeler, 2001Maturin, L., & Peeler, J. T. (2001). BAM chapter 3: Aerobic plate count. In Food and Drug Administration – FDA (Ed.), Bacteriological Analytical Manual (BAM) main page. Silver Spring: FDA. Retrieved in 2024, February 7, from https://www.fda.gov/food/laboratory-methods-food/bam-chapter-3-aerobic-plate-count
https://www.fda.gov/food/laboratory-meth...
).
2.6 Application in fermented milk
Inoculum preparation refers to Nursini et al. (2022)Nursini, N. W., Antara, N. S., Sugitha, I. M., & Sujaya, I. N. (2022). Screening of Lactobacillus rhamnosus-producing gamma aminobutyric acid (GABA) isolated from Sumbawa mare milk and its potential application to increase GABA content in fermented milk. Food Research, 6(3), 187-195. http://doi.org/10.26656/fr.2017.6(3).380
http://doi.org/10.26656/fr.2017.6(3).380...
with slight modifications. The starter culture was prepared by inoculating fresh LAB cultures at 2% (v/v) into reconstituted skim cow's milk (Sunlac, Malaysia) (14% total solids) and incubated for 24 hours at 37 °C. The starter culture was then inoculated at 5% (v/v) into the production medium (50 mL reconstituted skim cow's milk in 100 mL Schott Duran bottles) with and without addition of MSG of 1% for P. acidilactici YKP4 and 2% for L. rhamnosus BD2 based on the concentration resulted in the highest GABA concentration. Incubation was carried out at 37 °C for 72 hours. Analysis was conducted on GABA concentration, LAB count, and culture pH at the end of incubation. For GABA analysis, 10 mL of fermented milk was centrifuged (6000 rpm for 30 minutes at 4 °C), then the supernatant was filtered using a 0.22 µm pore size membrane filter (Han et al., 2020Han, M., Liao, W., Wu, S. M., Gong, X., & Bai, C. (2020). Use of Streptococcus thermophilus for the in situ production of γ-aminobutyric acid-enriched fermented milk. Journal of Dairy Science, 103(1), 98-105. PMid:31668446. http://doi.org/10.3168/jds.2019-16856
http://doi.org/10.3168/jds.2019-16856...
). The concentration of GABA in the supernatant was analyzed using the method previously described.
2.7 Statistical analysis
The experiment was conducted with two repetitions. Statistical analysis was performed using SPSS 16 software with a one-way Analysis of Variance (ANOVA) test for the first, second, and third stages. If a significant difference was found, the Duncan Multiple Range Test (DMRT) was conducted at a 5% significance level. For the fourth stage, an independent sample t-test was performed at a 5% significance level.
3 Results & discussion
3.1 Screening of GABA-producing LAB
All LAB isolates, derived from various foods, are capable of producing GABA, as demonstrated by the GABA spot with an identical Rf value to the standard GABA Rf value (Table 1). The quantitative GABA analysis was conducted to identify high GABA-producing LAB. The concentration of GABA produced in this study ranged from 1.05 to 2.32 g/L, which is notably higher than that of Park et al. (2014)Park, S. Y., Lee, J. W., & Lim, S. D. (2014). The probiotic characteristics and GABA production of Lactobacillus plantarum K154 isolated from kimchi. Food Science and Biotechnology, 23(6), 1951-1957. http://doi.org/10.1007/s10068-014-0266-2
http://doi.org/10.1007/s10068-014-0266-2...
who used L. plantarum K154, producing 0.20 g/L of GABA. In the previous study, 3% MSG concentration was employed, and the LAB was incubated at 37 °C for 18 hours (Park et al., 2014Park, S. Y., Lee, J. W., & Lim, S. D. (2014). The probiotic characteristics and GABA production of Lactobacillus plantarum K154 isolated from kimchi. Food Science and Biotechnology, 23(6), 1951-1957. http://doi.org/10.1007/s10068-014-0266-2
http://doi.org/10.1007/s10068-014-0266-2...
). This variation may arise as GABA production is affected by LAB strains and the conditions for fermentation (Cui et al., 2020Cui, Y., Miao, K., Niyaphorn, S., & Qu, X. (2020). Production of gamma-aminobutyric acid from lactic acid bacteria: A systematic review. International Journal of Molecular Sciences, 21(3), 1-21. PMid:32028587. http://doi.org/10.3390/ijms21030995
http://doi.org/10.3390/ijms21030995...
).
GABA-producing LAB from various foods after 48 hours of incubation at 37 °C using MRS broth medium and 1% MSG concentration.
The results showed that P. acidilactici YKP4 produced the highest concentration of GABA among the LAB, reaching a concentration of 2.30 g/L after 48 hours of incubation. Additionally, previous studies have indicated that P. acidilactici DS15 and P. pentosaceus HN8 produced GABA concentrations of 0.31 g/L and 9.06 g/L, respectively (Anggraini et al., 2019Anggraini, L., Marlida, Y., Wizna, W., Jamsari, J., & Mirzah, M. (2019). Optimization of nutrient medium for Pediococcus acidilactici DS15 to produce GABA. Journal of World’s Poultry Research, 9(3), 139-146. http://doi.org/10.36380/jwpr.2019.17
http://doi.org/10.36380/jwpr.2019.17...
; Ratanaburee et al., 2013Ratanaburee, A., Kantachote, D., Charernjiratrakul, W., & Sukhoom, A. (2013). Selection of γ-aminobutyric acid-producing lactic acid bacteria and their potential as probiotics for use as starter cultures in Thai fermented sausages (Nham). International Journal of Food Science & Technology, 48(7), 1371-1382. http://doi.org/10.1111/ijfs.12098
http://doi.org/10.1111/ijfs.12098...
). On the other hand, L. rhamnosus BD2, L. kefiri YK4, and L. plantarum 4C261 as probiotic candidates LAB (Kurnia, 2018Kurnia, H. (2018). Advantage of lactic acid bacteria from fermented mustard (Brassica juncea L.) for reducing cholesterol in vitro (Undergraduate thesis). IPB University, Bogor.; Yusuf et al., 2019Yusuf, D., Nuraida, L., Dewanti-Hariyadi, R., & Hunaefi, D. (2019). In vitro characterization of lactic acid bacteria from Indonesian kefir grains as probiotics with cholesterol-lowering effect. Journal of Microbiology and Biotechnology, 30(5), 726-732. PMid:32482938. http://doi.org/10.4014/jmb.1910.10028
http://doi.org/10.4014/jmb.1910.10028...
) produced GABA amounted to 1.33, 1.32, and 1.31 g/L, respectively, that were not significantly different (p > 0.05). Previous research has shown that L. rhamnosus BD2 has high α-glucosidase inhibitory activity, as well as the ability to produce antioxidant peptides (Yusuf et al., 2021Yusuf, D., Nuraida, L., Dewanti-Hariyadi, R., & Hunaef, D. (2021). In vitro antioxidant and α-glucosidase inhibitory activities of Lactobacillus spp. isolated from Indonesian kefir grains. Applied Food Biotechnology, 8(1), 39-46. http://doi.org/10.22037/afb.v8i1.30367
http://doi.org/10.22037/afb.v8i1.30367...
) and reduce cholesterol levels in vitro (Yusuf et al., 2019Yusuf, D., Nuraida, L., Dewanti-Hariyadi, R., & Hunaefi, D. (2019). In vitro characterization of lactic acid bacteria from Indonesian kefir grains as probiotics with cholesterol-lowering effect. Journal of Microbiology and Biotechnology, 30(5), 726-732. PMid:32482938. http://doi.org/10.4014/jmb.1910.10028
http://doi.org/10.4014/jmb.1910.10028...
). The production of GABA by L. rhamnosus BD2 enhances the physiological function of LAB besides inhibiting pathogenic bacteria causing enteric infections. For the subsequent stage, two LAB isolates were selected: P. acidilactici YKP4 as high GABA producer and L. rhamnosus BD2, considered a potential probiotic candidate.
3.2 Effect of precursor addition (MSG) on GABA production
Figure 1 demonstrated that supplementing MSG enhanced GABA production in both chosen LAB. The GAD enzyme in LAB decarboxylates glutamate into GABA, hence adding MSG stimulates GABA production. LAB utilizes glutamate as a protective mechanism against acidic compounds that accumulate in the MRS broth medium. Undissociated acidic compounds can efficiently enter the cell membrane. The pH value inside the cell is higher than that outside the cell, causing the acid to dissociate into protons (H+) and acid ions. This results in a decrease in pH inside the cell (Cotter & Hill, 2003Cotter, P. D., & Hill, C. (2003). Surviving the acid test: Responses of gram-positive bacteria to low pH. Microbiology and Molecular Biology Reviews, 67(3), 429-453. PMid:12966143. http://doi.org/10.1128/MMBR.67.3.429-453.2003
http://doi.org/10.1128/MMBR.67.3.429-453...
). To counteract this, LAB utilizes glutamate to form GABA and balance the pH inside the cell. Glutamate present in the MRS broth medium enters the cell through GadC, and then is decarboxylated by GadA/GadB, consuming protons in the process to form GABA. The produced GABA is subsequently discharged by the cell via GadC (Yogeswara et al., 2020Yogeswara, I. B. A., Maneerat, S., & Haltrich, D. (2020). Glutamate decarboxylase from lactic acid bacteria - a key enzyme in GABA synthesis. Microorganisms, 8(12), 1-24. PMid:33287375. http://doi.org/10.3390/microorganisms8121923
http://doi.org/10.3390/microorganisms812...
). This maintains the pH balance of the cell, resulting in efficient LAB metabolism, allowing LAB to survive in an acidic environment.
GABA production by P. acidilactici YKP4 and L. rhamnosus BD2 in MRS broth with various concentrations of MSG after 72 hours of incubation time at 37 °C. Different letters indicate significant differences at the 5% test level using the Duncan test with two replicates.
The highest GABA yield for P. acidilactici YKP4 was 3.25 g/L reached by the addition of 1% MSG concentration. The results of 1% addition of MSG obtained in this step was higher than presented in Table 1 was considered due to application of longer fermentation time in this step. When the concentration of MSG added to the medium exceeded 1%, it decreased the production of GABA. In the case of L. rhamnosus BD2, the highest GABA production (2.52 g/L) was reached with addition of a 2% MSG, but decreased with concentrations greater than 2% (Figure 1). This suggests that MSG concentrations above 1% suppress GABA production in P. acidilactici YKP4, whereas MSG concentrations above 2% inhibit GABA production in L. rhamnosus BD2. As shown in the study by Seo et al. (2013)Seo, M.-J., Lee, J.-Y., Nam, Y.-D., Lee, S.-Y., Park, S.-L., Yi, S.-H., Lee, M.-H., Roh, S. W., Choi, H.-J., & Lim, S.-I. (2013). Production of γ-aminobutyric acid by Lactobacillus brevis 340G isolated from kimchi and its application to skim milk. Food Engineering Progress, 17(4), 418-423. http://doi.org/10.13050/foodengprog.2013.17.4.418
http://doi.org/10.13050/foodengprog.2013...
, L. brevis 340G produced 7.09 g/L of GABA at a concentration of 3% MSG, which decreases when MSG concentrations are above 3%. High MSG concentrations elevate the osmotic pressure of LAB cells leading to water loss and eventually cellular dehydration. When cells lack water, LAB growth is inhibited and their metabolism is disrupted, including a decrease in GABA production (Stecker et al., 2022Stecker, D., Hoffmann, T., Link, H., Commichau, F. M., & Bremer, E. (2022). L-Proline synthesis mutants of Bacillus subtilis overcome osmotic sensitivity by genetically adapting l-arginine metabolism. Frontiers in Microbiology, 13(June), 908304. PMid:35783388. http://doi.org/10.3389/fmicb.2022.908304
http://doi.org/10.3389/fmicb.2022.908304...
; Yang et al., 2008Yang, S. Y., Lü, F. X., Lu, Z. X., Bie, X. M., Jiao, Y., Sun, L. J., & Yu, B. (2008). Production of γ-aminobutyric acid by Streptococcus salivarius subsp. thermophilus Y2 under submerged fermentation. Amino Acids, 34(3), 473-478. PMid:17514494. http://doi.org/10.1007/s00726-007-0544-x
http://doi.org/10.1007/s00726-007-0544-x...
). However, other LAB were able to adapt to high concentrations of MSG. L. brevis CRL 1942 was capable of generating a significant GABA yield of ≈26 g/L by utilizing an MSG concentration of 4.6% (Villegas et al., 2016Villegas, J. M., Brown, L., Savoy de Giori, G., & Hebert, E. M. (2016). Optimization of batch culture conditions for GABA production by Lactobacillus brevis CRL 1942, isolated from quinoa sourdough. Lebensmittel-Wissenschaft + Technologie, 67, 22-26. http://doi.org/10.1016/j.lwt.2015.11.027
http://doi.org/10.1016/j.lwt.2015.11.027...
). These findings suggest that specific concentrations of MSG may promote GABA production. In addition to the concentration of MSG, the concentration of GAD enzyme and its catalytic properties may influence the efficiency of GAD enzyme in producing GABA (Mazzoli et al., 2010Mazzoli, R., Pessione, E., Dufour, M., Laroute, V., Giuffrida, M. G., Giunta, C., Cocaign-Bousquet, M., & Loubière, P. (2010). Glutamate-induced metabolic changes in Lactococcus lactis NCDO 2118 during GABA production: Combined transcriptomic and proteomic analysis. Amino Acids, 39(3), 727-737. PMid:20174841. http://doi.org/10.1007/s00726-010-0507-5
http://doi.org/10.1007/s00726-010-0507-5...
). The concentration of MSG that increased GABA production by both LAB isolates in this study was lower compared to the findings of other studies. Based on the results, the MSG concentration used in the next stage was 1% for P. acidilactici YKP4 and 2% for L. rhamnosus BD2.
3.3 Effect of initial pH of the medium and incubation time on GABA production
The study revealed an increase in GABA concentration throughout the 72-hour incubation time. It could be noted that P. acidilactici YKP4 exhibited greater GABA production than L. rhamnosus BD2 during the incubation time (Figures 2 and 3). The highest GABA concentration with a concentration of 3.30 g/L was achieved by P. acidilactici YKP4 at 72 hours incubation time, initial pH of 5.0, and 1% MSG concentration. In L. rhamnosus BD2, the maximum GABA concentration was 2.67 g/L achieved after 72 hours of incubation, with an initial pH of 5.0 and 2% MSG concentration.
Effect of incubation time and initial pH (◆) 4.5, (◼) 5.0, and (▲) 5.5 of MRS broth containing 1% MSG on GABA production (A), total LAB (B), and pH change (C) by P. acidilactici YKP4 at 37 °C for 72 hours. Different superscripts indicate significant differences between incubation times at the 5% test level using the Duncan test with two replicates.
Effect of incubation time and initial pH (◇) 4.5, (◻) 5.0, and (△) 5.5 of MRS broth containing 1% MSG on GABA production (A), total LAB (B), and pH change (C) by L. rhamnosus BD2 at 37 °C for 72 hours. Different superscripts indicate significant differences between incubation times at the 5% test level using the Duncan test with two replicates.
Based on the results of this study, the growth of P. acidilactici YKP4 and L. rhamnosus BD2 exhibited a similar pattern at varying pH levels (Figures 2 and 3). Both LAB experienced a steep increase in growth for the initial 24 hours and slightly decreased until the end of the 72-hour incubation. In contrast to the growth GABA production continuously increased after the growth ceased. Lim et al. (2018)Lim, H. S., Seo, D. H., Cha, I. T., Lee, H., Nam, Y.-D., & Seo, M. J. (2018). Expression and characterization of glutamate decarboxylase from Lactobacillus brevis HYE1 isolated from kimchi. World Journal of Microbiology & Biotechnology, 34(3), 44. PMid:29500614. http://doi.org/10.1007/s11274-018-2427-6
http://doi.org/10.1007/s11274-018-2427-6...
found that as LAB transition to the stationary phase, they express GadA, GadB, and GadC genes, which allow them to produce GABA. The initial pH value of the medium did not affect the growth of the LAB (Figures 2B and 3B), however, it was observed that GABA production (Table 2) was affected, with the highest amount achieved when the initial pH was 5.0 after 72 h incubation that significantly different with other pHs. Although GABA production serves as a mechanism of protection for LAB against acidic conditions in order to maintain their viability, however when the initial pH was too low, the GABA production was lower, especially for P. acidilactici YKP4.
Effect of pH on the GABA production and viable cells of P. acidilactici YKP4 and L. rhamnosus BD2 after 72 h incubation in MRS broth with addition of MSG.
Figures 2 and 3 show that the pH of cultures decreased after 24 hours of incubation. This was due to a sharp increase in LAB numbers, resulting in acid production and subsequent pH decrease. At 48 and 72 hours of incubation, the pH increased slightly, and GABA production continued to increase, while LAB growth ceased. This may occur because the acidic MSG undergoes decarboxylation by the GAD enzyme present in the cytoplasm to produce alkaline GABA, which is then released into the MRS medium via the GABA antiporter. This aligns with prior studies indicating that alkaline GABA is transported into the growth medium via the antiporter, leading to a slight increase in pH in the medium. The increase in pH cannot be attributed to ammonia production, as LAB lacks glutamate dehydrogenase which produces NH3 (Cotter & Hill, 2003Cotter, P. D., & Hill, C. (2003). Surviving the acid test: Responses of gram-positive bacteria to low pH. Microbiology and Molecular Biology Reviews, 67(3), 429-453. PMid:12966143. http://doi.org/10.1128/MMBR.67.3.429-453.2003
http://doi.org/10.1128/MMBR.67.3.429-453...
; Seo et al., 2013Seo, M.-J., Lee, J.-Y., Nam, Y.-D., Lee, S.-Y., Park, S.-L., Yi, S.-H., Lee, M.-H., Roh, S. W., Choi, H.-J., & Lim, S.-I. (2013). Production of γ-aminobutyric acid by Lactobacillus brevis 340G isolated from kimchi and its application to skim milk. Food Engineering Progress, 17(4), 418-423. http://doi.org/10.13050/foodengprog.2013.17.4.418
http://doi.org/10.13050/foodengprog.2013...
). Furthermore, pH is one of the most important factors in GABA production, as GAD activity in LAB is very active at pH 4.5-5.5 (Yogeswara et al., 2020Yogeswara, I. B. A., Maneerat, S., & Haltrich, D. (2020). Glutamate decarboxylase from lactic acid bacteria - a key enzyme in GABA synthesis. Microorganisms, 8(12), 1-24. PMid:33287375. http://doi.org/10.3390/microorganisms8121923
http://doi.org/10.3390/microorganisms812...
). Li et al. (2010)Li, H., Qiu, T., Gao, D., & Cao, Y. (2010). Medium optimization for production of gamma-aminobutyric acid by Lactobacillus brevis NCL912. Amino Acids, 38(5), 1439-1445. PMid:19787432. http://doi.org/10.1007/s00726-009-0355-3
http://doi.org/10.1007/s00726-009-0355-3...
conducted research revealing that the initial pH of fermentation has an effect on GABA production. Nonetheless, the optimal initial pH for GABA production differs for each species or strain. L. brevis NCL912 has the ability to produce GABA with maximum yield at an initial pH of 5.0, as reported by Li et al. (2010)Li, H., Qiu, T., Gao, D., & Cao, Y. (2010). Medium optimization for production of gamma-aminobutyric acid by Lactobacillus brevis NCL912. Amino Acids, 38(5), 1439-1445. PMid:19787432. http://doi.org/10.1007/s00726-009-0355-3
http://doi.org/10.1007/s00726-009-0355-3...
. In addition, L. brevis HYE1 can produce GABA optimally at an initial pH of 4.74, according to Lim et al. (2017)Lim, H. S., Cha, I. T., Roh, S. W., Shin, H. H., & Seo, M. J. (2017). Enhanced production of gamma-aminobutyric acid by optimizing culture conditions of Lactobacillus brevis HYE1 isolated from kimchi, a Korean fermented food. Journal of Microbiology and Biotechnology, 27(3), 450-459. PMid:27880963. http://doi.org/10.4014/jmb.1610.10008
http://doi.org/10.4014/jmb.1610.10008...
, and L. paracasei 15C has been found to produce GABA with maximum production at an initial pH of 5.5, as reported by Franciosi et al. (2015)Franciosi, E., Carafa, I., Nardin, T., Schiavon, S., Poznanski, E., Cavazza, A., Larcher, R., & Tuohy, K. M. (2015). Biodiversity and γ-aminobutyric acid production by lactic acid bacteria isolated from traditional alpine raw cow’s milk cheeses. BioMed Research International, 2015, 625740. PMid:25802859. http://doi.org/10.1155/2015/625740
http://doi.org/10.1155/2015/625740...
.
During this study, the production of GABA was also influenced by the incubation time. Within the 24–72-hour incubation period, GABA production consistently increased despite the inhibition and cessation of LAB growth (reaching the stationary phase). This suggests that LAB growth during the stationary phase does not affect GABA production. When the growth of LAB is deceased, they actively employ glutamate to generate GABA to protect themselves from acidic conditions. The pH value at the end of the incubation time shows the acidification of the MRS medium, achieving 4.6 in P. acidilactici YKP4 and 4.5 in L. rhamnosus BD2 from the initial pH of 5.0 (Figures 2C and 3C). This pH condition brought the growth of both LAB isolates to a halt. The growth conditions are in compliance with the study carried out by Mataragas et al. (2003)Mataragas, M., Metaxopoulos, J., Galiotou, M., & Drosinos, E. H. (2003). Influence of pH and temperature on growth and bacteriocin production by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442. Meat Science, 64(3), 265-271. PMid:22063012. http://doi.org/10.1016/S0309-1740(02)00188-2
http://doi.org/10.1016/S0309-1740(02)001...
which indicated the inhibition of the growth of Leuconostoc mesentroides L124 and Lactobacillus curvatus 442 when the pH level is below 5.0.
The research found that the greatest time for GABA production was at 72 hours of incubation. The present results correspond with previous studies (Binh et al., 2014Binh, T. T. T., Ju, W. T., Jung, W. J., & Park, R. D. (2014). Optimization of γ-amino butyric acid production in a newly isolated Lactobacillus brevis. Biotechnology Letters, 36(1), 93-98. PMid:24078124. http://doi.org/10.1007/s10529-013-1326-z
http://doi.org/10.1007/s10529-013-1326-z...
; Sanchart et al., 2017Sanchart, C., Rattanaporn, O., Haltrich, D., Phukpattaranont, P., & Maneerat, S. (2017). Lactobacillus futsaii CS3, a new GABA-producing strain isolated from Thai fermented shrimp (Kung-Som). Indian Journal of Microbiology, 57(2), 211-217. PMid:28611499. http://doi.org/10.1007/s12088-016-0632-2
http://doi.org/10.1007/s12088-016-0632-2...
) which also demonstrated that 72 hours was the most effective incubation period for GABA production. Sanchart et al. (2017)Sanchart, C., Rattanaporn, O., Haltrich, D., Phukpattaranont, P., & Maneerat, S. (2017). Lactobacillus futsaii CS3, a new GABA-producing strain isolated from Thai fermented shrimp (Kung-Som). Indian Journal of Microbiology, 57(2), 211-217. PMid:28611499. http://doi.org/10.1007/s12088-016-0632-2
http://doi.org/10.1007/s12088-016-0632-2...
discovered that L. futsaii CS3 produced the highest concentration of GABA, approximately 25 g/L, in MRS medium after 72 hours of incubation. Another study demonstrated that incubation for more than 72 hours did not result in significant differences in GABA production (Binh et al., 2014Binh, T. T. T., Ju, W. T., Jung, W. J., & Park, R. D. (2014). Optimization of γ-amino butyric acid production in a newly isolated Lactobacillus brevis. Biotechnology Letters, 36(1), 93-98. PMid:24078124. http://doi.org/10.1007/s10529-013-1326-z
http://doi.org/10.1007/s10529-013-1326-z...
). The results of this and previous studies suggest that environmental conditions during incubation greatly influence GABA production and that each LAB have unique capabilities in this respect.
3.4 Application in fermented milk
The addition of MSG to skim milk resulted in increased GABA production by both cultures (Figure 4). Specifically, the addition of 1% MSG into skim milk increased GABA production significantly (p < 0.05) by P. acidilactici YKP4 from 1.80±0.03 g/L to 1.94±0.02 g/L, meanwhile, the addition of 2% MSG also increased significantly (p < 0.05) GABA production by L. rhamnosus BD2 from 1.15±0.03 g/L to 1.33±0.04 g/L. Using skim milk supplemented with MSG as a substrate for fermentation, previous research showed an increase in GABA production by different species of LAB. Seo et al. (2013)Seo, M.-J., Lee, J.-Y., Nam, Y.-D., Lee, S.-Y., Park, S.-L., Yi, S.-H., Lee, M.-H., Roh, S. W., Choi, H.-J., & Lim, S.-I. (2013). Production of γ-aminobutyric acid by Lactobacillus brevis 340G isolated from kimchi and its application to skim milk. Food Engineering Progress, 17(4), 418-423. http://doi.org/10.13050/foodengprog.2013.17.4.418
http://doi.org/10.13050/foodengprog.2013...
found that GABA production by L. brevis 340G increased from 0.08 g/L to 0.48 g/L after 48 hours of incubation in skim milk with the addition of 1% MSG. Research carried out by Somkuti et al. (2012)Somkuti, G. A., Renye Junior, J. A., & Steinberg, D. H. (2012). Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110. Journal of Industrial Microbiology & Biotechnology, 39(7), 957-963. PMid:22427071. http://doi.org/10.1007/s10295-012-1114-0
http://doi.org/10.1007/s10295-012-1114-0...
also demonstrated an increase in GABA production in skim milk supplemented with 1% MSG by Streptococcus thermophilus ST110 from 0.003 g/L to 0.07 g/L. The content of free glutamate present in skim milk is inadequate to produce significant levels of GABA (Seo et al., 2013Seo, M.-J., Lee, J.-Y., Nam, Y.-D., Lee, S.-Y., Park, S.-L., Yi, S.-H., Lee, M.-H., Roh, S. W., Choi, H.-J., & Lim, S.-I. (2013). Production of γ-aminobutyric acid by Lactobacillus brevis 340G isolated from kimchi and its application to skim milk. Food Engineering Progress, 17(4), 418-423. http://doi.org/10.13050/foodengprog.2013.17.4.418
http://doi.org/10.13050/foodengprog.2013...
), thus, the addition of glutamate increases the precursor for GABA production that results in significant increases in GABA production in skim milk. Although in the present research GABA production (1.94 g/L) in skim milk with the addition of MSG was higher than that of reported in the previous studies (Seo et al., 2013Seo, M.-J., Lee, J.-Y., Nam, Y.-D., Lee, S.-Y., Park, S.-L., Yi, S.-H., Lee, M.-H., Roh, S. W., Choi, H.-J., & Lim, S.-I. (2013). Production of γ-aminobutyric acid by Lactobacillus brevis 340G isolated from kimchi and its application to skim milk. Food Engineering Progress, 17(4), 418-423. http://doi.org/10.13050/foodengprog.2013.17.4.418
http://doi.org/10.13050/foodengprog.2013...
; Somkuti et al., 2012Somkuti, G. A., Renye Junior, J. A., & Steinberg, D. H. (2012). Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110. Journal of Industrial Microbiology & Biotechnology, 39(7), 957-963. PMid:22427071. http://doi.org/10.1007/s10295-012-1114-0
http://doi.org/10.1007/s10295-012-1114-0...
), however, the percentage increased in GABA production was lower than those two previous research (8% vs 500% and 2233%, respectively). This may be due to a different composition of skim milk and incubation temperature, besides different cultures between the present research and two previous studies. Increase of MSG concentration from 1% to 1.5% in combination with addition of casein hydrolysate (1 g/L) in skim milk has been demonstrated to increase 93% (from 2.80 g/L to 5.40 g/L) GABA production by S. thermophilus (Han et al., 2020Han, M., Liao, W., Wu, S. M., Gong, X., & Bai, C. (2020). Use of Streptococcus thermophilus for the in situ production of γ-aminobutyric acid-enriched fermented milk. Journal of Dairy Science, 103(1), 98-105. PMid:31668446. http://doi.org/10.3168/jds.2019-16856
http://doi.org/10.3168/jds.2019-16856...
). This research confirms that beside LAB strains and fermentation conditions that affect GABA production, enrichment of nutrient may also needed to increase GABA production.
GABA production by P. acidilactici YKP4 (A) and L. rhamnosus BD2 (B) in skim milk and skim milk + MSG medium after 72 hours incubation time at 37 °C. The different letters indicate significant differences based on a t-test with a 5% significance level with two replicates.
Furthermore, there was no notable difference in the total number of LAB between the skim milk medium containing MSG and the one without MSG (p > 0.05) after the incubation time ended for both P. acidilactici YKP4 and L. rhamnosus BD2 (Figure 5). These findings suggest that MSG did not impede LAB growth at the end of the incubation period (Figure 5), but did affect GABA production (Figure 4). GABA production by both LAB isolates in skim milk supplemented with MSG was comparatively lower than in MRS medium supplemented with MSG (refer to Table 3). This is believed to be attributed to dissimilarities in the initial pH value employed. Specifically, while the initial pH of the MRS + MSG medium was brought down to pH 5.0 by a gradual addition of HCl, the initial pH of the skim milk + MSG medium could not be regulated due to clotting when acid was included, resulting in an initial measurement of pH 6.5 (Figure 6). The initial pH value significantly affects GABA production, with pH 4.5-5.5 being the most conducive for GABA production (Yogeswara et al., 2020Yogeswara, I. B. A., Maneerat, S., & Haltrich, D. (2020). Glutamate decarboxylase from lactic acid bacteria - a key enzyme in GABA synthesis. Microorganisms, 8(12), 1-24. PMid:33287375. http://doi.org/10.3390/microorganisms8121923
http://doi.org/10.3390/microorganisms812...
). These findings align with those of Seo et al. (2013)Seo, M.-J., Lee, J.-Y., Nam, Y.-D., Lee, S.-Y., Park, S.-L., Yi, S.-H., Lee, M.-H., Roh, S. W., Choi, H.-J., & Lim, S.-I. (2013). Production of γ-aminobutyric acid by Lactobacillus brevis 340G isolated from kimchi and its application to skim milk. Food Engineering Progress, 17(4), 418-423. http://doi.org/10.13050/foodengprog.2013.17.4.418
http://doi.org/10.13050/foodengprog.2013...
, who noted lower GABA production by L. brevis 340G in skim milk + MSG medium (0.48 g/L) compared to the optimized MRS medium (7.09 g/L). LAB isolates generate acid during fermentation, resulting in reduced pH levels of milk. The final pH value of skim milk added with MSG, both fermented by P. acidilactici YKP4 and L. rhamnosus BD2, tended to be higher than without MSG (Figure 6) and this correlated with the production of GABA as a mechanism for reducing acid in the medium.
Total LAB of P. acidilactici YKP4 (A) and L. rhamnosus BD2 (B) in skim milk and skim milk + MSG medium after 72 hours incubation time at 37 °C. The different letters indicate significant differences based on a t-test with a 5% significance level with two replicates.
Comparison of GABA levels in MRS + MSG and skim milk + MSG medium after 72 hours of incubation at 37 °C.
Decrease in pH of skim milk and skim milk + MSG media fermented by P. acidilactici YKP4 (A) and L. rhamnosus BD2 (B) after 72 hours incubation time at 37 °C. The different letters indicate significant differences based on a t-test with a 5% significance level with two replicates.
4 Conclusions
Lactic acid bacteria are promising GABA producers, however, its capability relies on the strain and fermentation conditions. Indeed, P. acidilactici YKP4 was found to be the highest GABA producer among the isolates of kefir grain origin. However, L. rhamnosus BD2, a potential probiotic candidate, also showed as a promising GABA producer. The ability to produce GABA will enhance its health beneficial effect. MSG as a precursor plays an important role in increasing GABA production. As can be seen, different LAB responses differently to the precursor. MSG concentration of 1% was sufficient to increase GABA production by P. acidilactici YKP4 while L. rhamnosus BD2 needed 2%. Initial pH and incubation time were responded similarly by the two LAB with the maximum production at an initial pH of 5.0 and 72 h fermentation. Considering the growing demand for fermented food as functional food with specific health beneficial effect, enriched fermented milk with GABA is becoming one of the promising product. Thus, P. acidilactici YKP4 and L. rhamnosus BD2 are the potential to be used as starter culture for GABA-enriched fermented milk. However, further exploration is needed to optimize the MSG concentration and sensory aspects of the fermented milk.
Acknowledgements
The authors are grateful to the staff of the SEAFAST Center Laboratory and the Biopharmaca Laboratory, IPB University, for their valuable assistance and support in executing this research.
-
Cite as:
Wicaksono, S., Nuraida, L., & Faridah, D. N. (2024). Tapping the potential of lactic acid bacteria: optimizing gamma-aminobutyric acid production for enhanced health benefits in fermented milk. Brazilian Journal of Food Technology, 27, e2024015. https://doi.org/10.1590/1981-6723.01524 -
Funding:
Directorate General of Higher Education, Research and Technology; Ministry of Education, Culture, Research and Technology under the Master Thesis Research scheme with number 082/E5/PG.02.00.PT/2022 dated May 10th, 2022.
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Associate Editor:
Publication Dates
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Publication in this collection
13 Sept 2024 -
Date of issue
2024
History
-
Received
07 Feb 2024 -
Accepted
28 May 2024