利用Lactobacillus plantarum NCD2 生產gamma-胺基丁酸之較適生產條件探討及其製作發酵乳可行性之研究

紀玉婷

doi:10.6845/NCHU.2015.00644

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利用Lactobacillus plantarum NCD2 生產gamma-胺基丁酸之較適生產條件探討及其製作發酵乳可行性之研究

Study on production of -aminobutyric Acid (GABA) by Lactobacillus plantarum NCD2 and its application in fermented milk

紀玉婷 , Masters　　Advisor：方繼　　

繁體中文 DOI： 10.6845/NCHU.2015.00644

gamma-胺基丁酸（GABA ；乳酸菌； L. plantarum ；發酵乳；菌株特性； gamma-aminobutyric acid (GABA) ； Lactic acid bacteria ； L. plantarum ； cultured milk ； probiotic characteristic

Abstract │ Reference (136) │ Times Cited (1) │ Altmetrics

Abstract 〈TOP〉

本研究目的乃從本土醃漬蔬菜分離乳酸菌中，篩選出具有生產-胺基丁酸（-aminobutyric acid, GABA）能力者，並針對此菌株建立生產GABA之較適化條件。另針對其大量生產效率及該乳酸菌之菌株特性方面進行探討。最後將其用來製備成發酵乳，探討其成為新興GABA產品之可能性。

首先利用薄層層析法（thin-layer chromatography, TLC）及高效能液相層析法（high performance liquid chromatography, HPLC）篩選出高GABA產量乳酸菌，其經API 50 CHL測定後，確定菌種為Lactobacillus plantarum，因此後續實驗將其以L. plantarum NCD2命名表示。經較適生產條件探討後，得知其生產GABA之較適化培養基配方為（1 l）：60 g MSG、20 g fructose、20 g yeast extract、1.0 g Tween 80、2.0 g ammonium citrate、2.0 g disodium hydrogenphosphate、5.0 g sodium acetate、0.1 g magnesium sulfate、0.05 g manganese sulfate。培養條件為：37oC、3天、起始pH 為6.0。其GABA產量可達8.38  0.80 mg/ml。

本研究經由結合L. plantarum NCD2及米基質如米糠及米胚芽進行發酵，結果顯示米基質之添加並未能提升其生產GABA之能力。2 L發酵槽培養中，兩次實驗所產生之GABA產量分別為6.77 mg/ml及6.18 mg/ml，顯示L. plantarum NCD2具良好之放大培養潛力。

L. plantarum NCD2發酵乳之較適製備方法為以8% (w/v)脫脂奶粉為奶粉添加量，單獨以L. plantarum NCD2發酵而成，其GABA含量可達2.86  0.08 mg/ml。此方式製備之發酵乳經14天4 oC冷藏後，除L. plantarum NCD2菌數呈現些微下降外，其GABA含量及pH值皆與第0天相近。喜好性感官品評方面，L. plantarum NCD2 發酵乳無論在色澤、香氣、風味、酸度及整理接受度上，普遍較不受消費者喜愛。

L. plantarum NCD2菌株特性探討方面，其於pH 2.0 耐酸實驗中顯示對酸之耐受性不佳，在經過3小時作用後，菌數下降至剩餘102 CFU/ml。耐膽鹽結果顯示，L. plantarum NCD2對0.3 % 膽鹽具有良好之抵抗能力。L. plantarum NCD2經由抗病原菌實驗證明，其對Esherichia coli、Salmonella enterica、Staphylococcus aureus及Listeria monocytogenes之抗菌能力分別為28.14 %、10.98 %、44.88 %及15.76 %。除此，L. plantarum NCD2 被證實除ampicillin 外，對vancomycin、ciprofloxacin、polymyxin B、kanamycin及streptomycin皆有不同程度之抵抗能力，尤以對vancomycin 之抗性最佳，其濃度需高於2 mg/ml方可對L. plantarum NCD2產生抑制作用。

Parallel Abstract 〈TOP〉

The objective of this study was to screen for lactic acid bacteria which have -aminobutyric acid (GABA) producing capacity from mustard pickle isolates. The optimal culture conditions for GABA production were investigated. Besides, the GABA producing capacity in benchtop fermentor scale and probiotics characteristic were studied as well. Finally, this particular strain was used to make cultured milk with enhanced GABA content.

A strain of lactic acid bacteria with high GABA producing capacity was  isolated through thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) method. It had been identified as Lactobacillus plantarum by API 50 CHL kit and was named as L. plantarum NCD2 in this study. The best medium compositions for GABA production by L. plantarum NCD2 were with medium containing (per l): 60 g MSG, 20 g fructose, 20 g yeast extract, 1.0 g Tween 80, 2.0 g ammonium citrate, 2.0 g disodium hydrogenphosphate, 5.0 g sodium acetate, 0.1 g magnesium sulfate, 0.05 g manganese sulfate. With above medium (pH 6.0) and cultivated at 37oC for 3 days, 8.38  0.80 mg/ml of GABA was produced by L. plantarum NCD2.

Rice germ or rice husk were supplemented to the cultivation medium to investigate the production of γ-aminobutyric acid (GABA) by L. plantarum NCD2. However, the results showed that no improvement of GABA production was observed. For the 2-L fermentor scale experiment, GABA reached 6.46 mg/ml and, indicating the potential of GABA production by L. plantarum NCD2 in scale-up fermentation.

For making fermented milk with enhanced GABA content, 8% (w/v) skim milk powder was supplemented to cultivation medium with L. plantarum NCD2 alone. GABA content reached 2.86  0.08 mg/ml through these cultivation. During 14 days 4oC cold storage, besides lactic acid bacteria number slightly decreased, no significant difference was found in pH value and GABA concentration of L. plantarum NCD2 cultured milk when compare to the group of 0 day. The sensory evaluation results showed that consumers disliked L. plantarum NCD2 cultured milk regardless of color, aroma, flavor, acidity and overall acceptance.

Viability of L. plantarum NCD2 was only remained 102 CFU/ml after exposed to pH 2.0 acid buffer for 3 hours. Results indicated that L. plantarum NCD2 exhibited low acid resistance ability. However, it exhibited high bile salts tolerance in bile salt resistance experiment. L. plantarum NCD2 displayed inhibition toword E. coli, S. enterica, S. aureus, and L. monocytogenes at rates of 28.14 %, 10.98 %, 44.88 % and 15.76 %. L. plantarum NCD2 possessed resistant to several antibiotics such as Vancomycin, Ciprofloxacin, Polymyxin B, Kanamycin and Streptomycin except Ampicillin with different level. L. plantarum NCD2 showed the greatest resistant to Vancomycin among these tested antibiotics with minimal inhibitory concentration of higher than 2 mg/ml.

Reference ( 136 ) 〈TOP〉

王仁辰。2013。利用米胚芽中麩胺酸脫羧酶與味精反應以提升 -胺基丁酸（GABA）生成量之探討。國立中興大學食品暨應用生物科技學系碩士論文。
連結：
周韋廷。2011。菇柄多醣對乳酸菌之助生性及應用在益生菌發酵乳之研究。國立中興大學食品暨應用生物科技學系碩士論文。
連結：
黃靖堯。2007。台灣酸菜中高胞外多醣益生乳酸菌之篩選與其醃漬液製作酸菜醃蛋可行性之研究。國立中興大學食品暨應用生物科技學系碩士論文。
連結：
Adeghate E., & Ponery A. S. (2002). GABA in the endocrine pancreas: cellular localization and function in normal and diabetic rats. Tissue & Cell. 34:1-6.
連結：
Amrouche T., Boutin Y., Prioult G., Fliss I. (2006). Effects of bifidobacterial cytoplasm, cell wall and exopolysaccharide on mouse lymphocyte proliferation and cytokine production. International Dairy Journal. 16:70–80.
連結：

Times Cited (1) 〈TOP〉

伍筱琪(2016)。以紅豆和蕎麥製作機能性類優格產品。中興大學食品暨應用生物科技學系所學位論文。2016。1-112。

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