臺灣博碩士論文加值系統

摘要

γ-氨基丁酸（GABA）是一種四碳非蛋白氨基酸，廣泛分佈於自然界中;它是一種重要的生物活性調節劑，被認為是脊椎動物中樞神經系統的主要抑制性神經遞質，是微生物將谷氨酸去羧基後的代謝產物。許多傳統食物經過微生物發酵後都含有GABA，GABA是安全且環保的，並且還可以提供富含GABA的新健康食品。 GABA的生物合成主要是透過谷氨酸脫羧酶（GAD）對谷氨酸進行α-脫羧，因此最佳發酵條件主要是取決於酶的生化特性。根據文獻顯示，微生物發酵生產GABA主要是由乳酸菌（LAB）生產的，已被證明是有效的食品添加劑，乳酸菌具有在胃腸道中生長和益生菌作用的能力。影響微生物發酵生產GABA的主要因素包括：溫度、pH、發酵時間、攪拌速率和碳源。本研究主要在探討培養條件對Lactobacillus brevis 和Lactobacillus plantarum生產GABA之影響。實驗結果顯示，Lb. brevis 在30oC、pH5.0和150 rpm下培養48小時，GABA產量為420 mg / L。而Lb. plantarum在37 oC、 pH 5.0和0rpm下培養48小時，GABA產量則為295 mg / L。此外，為了提升GABA產量，本研究利用添加葡萄糖與谷氨酸鈉（MSG）來增加GABA產率。結果顯示：Lb. brevis在添加2%MSG與1%葡萄糖可生產GABA為 478 mg / L.；Lb. plantarum在添加4%MSG與1%葡萄糖可生產GABA為415 mg / L。最後我們探討發酵時間和接種量對GABA之影響，其中，Lb. brevis的較佳發酵條件為2% MSG、4%葡萄糖、 150 rpm、pH 5.0和接種量為4% 下培養30 h，可增加GABA產量至 481 mg/L ；而Lb. plantarum的較佳發酵條件為4% MSG、4%葡萄糖、0 rpm，pH 5.0和4%接種量下培養24 h，可增加GABA產量至 486 mg/L。

Gamma-aminobutyric acid (GABA) is a four carbon non-protein amino acid that is widely distributed in nature; it is an important bioactive regulator, and it is considered a major inhibitory neurotransmitter of the vertebrate central nervous system. As a metabolic product of microorganisms produced by the decarboxylation of glutamic acid. A wide range of traditional foods produced by microbial fermentation contain GABA, in which GABA is safe and eco-friendly, and also has the option of providing new health-benefited foods enriched with GABA. GABA’s biosynthesis is primarily through the α-decarboxylation of glutamate by glutamate decarboxylase (GAD), therefore the optimal fermentation condition is mainly based on the biochemical properties of the enzyme. Previous studies have shown that the major GABA production by microbial fermentation is produced by lactic acid bacteria (LAB) that has proven effective as a food additive; this microorganism has the ability to grow and act like probiotics in the gastrointestinal tract. The main factors that can affect GABA production by microbial fermentation include temperature, pH, fermentation time, agitation rate, and carbon sources. This research aimed to investigate the effects of culture conditions mentioned above on GABA production by Lactobacillus brevis and Lactobacillus plantarum. The results showed that GABA production yield was 420 mg/L (48 h at 30oC, pH 5.0 and 150 rpm) by Lb. brevis, and 295 mg/L (48 h at 37oC, pH 5.0 and 0 rpm) by Lb. plantarum. To increase the production yield using a precursor like Monosodium Glutamate (MSG) as a catalyst and the addition of glucose the results showed gamma-aminobutyric acid production was 478 mg/L by Lb. brevis (2% MSG/ 1% Glucose) and 415 mg/L by Lb. plantarum (4% MSG/4%Glucose) after that we tested fermentation time (range 6 hours) and inoculum percentage the final results showed an increase in GABA production (486 mg/L and 481 mg/L) by Lb. plantarum (4%MSG, 4% glucose, 0rpm. 5.0 pH, 24h, 4% inoculum) and Lb. brevis (2%MSG, 4% glucose, 0rpm. 5.0 pH, 30h, 4% inoculum) respectively.
Keywords: Gamma-aminobutyric acid (GABA), culture conditions, carbon sources, lactic acid bacteria, microbial fermentation, Monosodium glutamate (MSG), Lactobacillus brevis, Lactobacillus plantarum.

Table of Contents
Title Page i
摘要 ii
ABSTRACT iv
Table of Contents vi
List of Figures x
List of Tables xii
CHAPTER 1 INTRODUCTION 1
1.1 Background 1
1.2 MOTIVATION 2
CHAPTER 2 LITERATURE REVIEW 4
2.1 History of GABA 5
2.2 Gamma-Aminobutyric acid (GABA) 6
2.3 GABA receptors. 6
2.3.1 GABAA receptors 7
2.3.2 GABAB receptors. 7
2.4 GABA’s synthesis 9
2.5 GABA’s synthesis by Lactic Acid Bacteria 9
2.6 Properties of GABA 11
2.7 Glutamate 14
2.8 L-glutamate decarboxylase (GAD) 14
2.9 GABA Producers 15
2.9 Lactic Acid Bacteria 16
2.10 Echerichia coli 18
2.11 Factors affecting GABA synthesis 18
2.11.1 Effect of pH on GABA yield 19
2.11.2 Effect of temperature 20
2.11.3 Effect of media additives 21
2.11.4 Effect of Monosodium Glutamate on GABA yield 21
2.11.5 Effect of coenzyme - pyridoxal phosphate (PLP) on the activity of GAD 22
2.12 Effect of Carbon Sources on the yield of GABA 23
2.13 Effect of fermentation time on GABA yield. 24
2.14 Effect of the inoculum concentration on the GABA yield. 24
2.15 Ways to produce GABA 25
2.15.1 Different methods of bacteria cultivation 27
2.15.2 Pure culture 27
2.16 Analysis of GABA 27
2.16.1 High Performance Liquid Chromatography (HPLC) 27
2.16.2 Derivatization reagent for the analysis of GABA 28
CHAPTER 3 MATERIALS AND METHODS 29
3.1 Reagents 29
3.2 Equipment 30
3.3 Composition media (Man Rogose and Shape). 32
3.4 Strain 33
3.4.1 Strain with plastid origin 33
3.4.2 Strains of preservation (Keep Bacteria) 33
3.5 Strain activation 33
3.6 Gamma-aminobutyric acid (GABA) culture 34
3.7 High performance liquid chromatography (HPLC) analysis method 34
3.7.1 HPLC analysis conditions and column 34
3.8 GABA calibration curve 35
3.9 Production of gamma-aminobutyric acid (GABA) 35
3.10 Application of culture conditions on γ-aminobutyric acid (GABA) 37
3.10.1 Different Culture Temperature on Yield of GABA 37
3.10.2 pH control on the yield of GABA 38
3.10.3 Carbon Sources 38
3.10.4 Inoculum 39
CHAPTER 4 RESULTS AND DISCUSSION 40
4.1 Results 40
4.1.1 HPLC Analysis 40
4.1.2 Effect of Different Culture Temperature on GABA Production 42
4.1.3 Effect of Different Agitation rate on GABA Production 45
4.1.4 Effect of Different Culture pH on GABA Production 48
4.1.5 Effect of Monosodium Glutamate (MSG) on GABA Production. 51
4.1.6 Effect of Carbon Sources on GABA Production. 54
4.1.7 Effect of Glucose on GABA Production. 55
4.1.8 Effect of Incubation time on GABA Production. 59
4.1.9 Effect of Inoculum percentage on GABA Production. 62
4.2 Discussion 65
4.2.1 HPLC Analysis 65
4.2.2 Different Culture Temperature on GABA Production 67
4.2.3 Different Agitation rate on GABA Production 68
4.2.4 Different Culture pH on GABA Production 71
4.2.5 Monosodium Glutamate (MSG) on GABA Production. 73
4.2.6 Carbon Sources on GABA Production. 75
4.2.7 Effect of Glucose on GABA Production. 76
4.2.8 Effect of Fermentation time on GABA Production. 76
4.2.9 Inoculum percentage on GABA Production. 77
CHAPTER 5 CONCLUSIONS 79
5.1 Conclusions 79
CHAPTER 6 FUTURE WORK 81
CHAPTER 7 REFERENCES 83



List of Figures
Figure 1. 1 Chemical GABA structure.. 1
Figure 1. 2:Fish bone chart optimal conditions on GABA production. 3
Figure 2. 1: Function of GABA in the neuron. The GABA nervous system in C. elegans . 8
Figure 2. 2: Structure of repressors. Neuropharmacology: Anxiety Disorders.. 8
Figure 2. 3: Synthesis of Gama aminobutyric acid..9
Figure 2. 4: Acid lactic bacteria pathway . 10
Figure 2. 5: GABA pathway shunt metabolism in prokaryotes and GAD activity.. 11
Figure 2. 6: The stimulation of GABA synthesis through increases in cytosolic H+ or Ca 2+ levels. Ca 2+ entry into the cytosol is mediated by Ca 2+ channels located in the plasma membrane or tonoplast. Opening of these channels may occurs in response to cold shock, mechanical stimulation or the binding of ligands. 20
Figure 2. 7: a) Pyridoxine b) Pyridoxal c) Pyridoxamine Structure.. 23
Figure 2. 8: Synthesis of Pyridoxal phosphate (PLP) to produce GABA. 23
Figure 2. 9: GABA is derivatized with reagent FDNB . 28
Figure 3. 1: Calibration curve of GABA standard 35
Figure 4. 1: HPLC analysis (A) is a GABA standard (B) is GABA samples produced by Lb. brevis (C) is GABA samples produced by Lb. plantarum. 41
Figure 4. 2: Effect of temperature on GABA production by Lb. plantarum 43
Figure 4. 3: Effect of temperature on GABA production by Lb. brevis 44
Figure 4. 4: Effect of agitation rate on GABA production by Lb. plantarum 46
Figure 4. 5: Effect of agitation rate on GABA production by Lb. brevis 47
Figure 4. 6: Effect of initial pH on GABA production by Lb. plantarum 49
Figure 4. 7: Effect of initial pH on GABA production by Lb. brevis 50
Figure 4. 8: Effect of addition of MSG on GABA production by Lb. plantarum 52
Figure 4. 9: Effect of addition of MSG on GABA production by Lb. brevis 53
Figure 4. 10: Effect of Carbon Sources on GABA Production (2% of Glucose, Lactose, Maltose, Arabinose, Fructose, Sucrose and without carbon source (MRS)). 55
Figure 4. 11: Effect of Carbon Sources (Glucose) on GABA Production by Lb. plantarum 57
Figure 4. 12 : Effect of Carbon Sources (Glucose) on GABA Production by Lb. brevis 58
Figure 4. 13: : Effect of the percentage of fermentation time on GABA Production by Lb. plantarum 60
Figure 4. 14: Effect of the percentage of fermentation time on GABA Production by Lb. brevis 61
Figure 4. 15: Effect of the percentage of inoculum on GABA Production by Lb. plantarum 63
Figure 4. 16. Effect of the percentage of inoculum on GABA Production by Lb. brevis 64


List of Tables

Table 2. 1: Physiological functions of GABA tested on animals and humans . 13
Table 2. 2: The variability in the architecture of the bacterial glutamate decarboxylase (GAD) system. 15
Table 2. 3: Production of GABA by different microorganisms. Production of GABA (γ-aminobutyric acid) by microorganisms. 25
Table 3. 1: Composition media of Man Rogose and Sharpe (broth and agar). 32
Table 3. 2: HPLC Condition in this research 34
Table 3. 3: Formulation of 0.5% 1-fluoro-2,4-dinitrobenzene (FDNB) 36
Table 3. 4: Formulation of 0.5 (mol/l) Sodium Bicarbonate Solution (NaHCO3) 36
Table 3. 5: Formulation 0.02 (mol / L) phosphate buffer solution 36
Table 3. 6: 0.02 mol/L Phosphate buffer 37
Table 4.2. 1 Comparison of culture conditions and GABA productivity 74

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