紅豆前處理及乳酸菌發酵條件對γ-胺基丁酸生成之影響

Abstract

本研究旨在利用高產GABA的乳酸菌株來提高紅豆基質中的GABA含量，期望能得到高GABA含量的機能性發酵紅豆乳。主要是以國產紅豆高雄八號作為發酵基質，探討不同的發酵前處理條件對於備製發酵基質的影響。本研究，建立了高效液相層析色譜分析樣品中GABA定性和定量的方法，利用冷酒精萃取法將樣品中的GABA萃取後，使用異硫氰酸苯脂 (PITC) 衍生試劑進行衍生化，用逆相C18分離管柱 (Alltima C18 5U, 250 × 4.6 nm) 進行樣品分離分析，在紫外光波長254 nm下進行測量分析。結果顯示樣品中的GABA能夠被管柱分離，並且在0~0.075 mg/ml的GABA標準品濃度範圍內具有良好的線性關係，其線性方程式為Y = 600000000X + 103248 (R2 = 0.9996) 。樣品測定之相對標準差為1.77%，加樣平均回收率達96.8%。由上述可知，所使用的測定方法穩定且再現性良好，可用於樣品中GABA的定量分析。浸泡、發芽或冷衝擊等三種不同的發酵前處理方法均可有效提升紅豆內GABA的含量，其中又以冷衝擊前處理方法：將紅豆於35℃下浸泡24小時後，再於-20℃下冷藏24小時，接著於30℃下融解24小時處理，提升GABA含量為最高，為201.23 mg/100 g，是未經處理紅豆的150倍。因此選用此條件進行後續的發酵實驗。結果顯示，以2%的混合菌株Lactococcus lactis subsp. lactis 和Lactobacillus rhamnosus GG.進行混合發酵，提升GABA含量的能力為較佳。試驗顯示最適發酵基質添加物組成為：4% (w/v) 蔗糖、4% (w/v) 酵母菌萃取物，於起始pH 6.8、30℃下發酵24小時，可得最高的GABA含量為65.77 mg/100 ml，為未經發酵且未經優化之紅豆基質的19倍。在儲存期間GABA的含量持續的上升，尤其是前12天上升較快，之後趨於緩慢，在28天儲存期結束時，GABA產量達68.21 mg/100 ml，為未經發酵且未經優化之紅豆基質 (3.43 mg/100 ml) 的20倍。

The Adzuki bean Kaohsiung 8 was applied to produce fermented functional beverage. The pretreatments/fermentation processes of Adzuki bean Kaohsiung 8 in enrichment of GABA were investigated. The methods for qualitative and quantitative analyses of GABA by HPLC were established. The GABA was extracted from fermented broth or red bean with 70% cold EtOH, derivatized to PICT-GABA with PICT, and then was subsequently separated with C18 separation column (Alltima C18 5U, 250 × 4.6 nm). The content of GABA was determined with UV absorbance detector at 254 nm. Under the condition described above, the HPLC separation gave a well-resolved and symmetrical PITC-GABA peak. The retention time was 11 min and reproducible. Peak area values showed good reproducibility with relative standard deviation (RSD) values at 1.77%. The calibration curve for PITC-GABA was calculated by linear least-squares squares regression. The regression equation was Y = 600000000X + 103248 (R2 = 0.9996) when the initial GABA concentration in the range 0~0.0075 mg/ml. The result indicated that the method described was specific and useful for the determination of GABA in the red bean sample and fermented broth. The GABA content in Adzuki bean Kaohsiung 8 could be enriched effectively by following three pre-fermentation treatments：immersing, germinating and cold shock. The highest GABA content, 201.23 mg/100 g was obtained by immersing red bean in water at 35℃ for 24 h, freezing at -20℃ for 24 h, and then thawing at 30℃ for 24 h. The GABA content of red bean increased 150 fold by this cold shock treament compared to non-treated red bean. The optimum fermentation bacterial seed culture was 2% mixture of Lactococcus lactis subsp. lactis and Lactobacillus rhamnosus GG. The optimum medium constitution was as follows: sucrose 4% (w/v), yeast extract 4% (w/v); and the fermentation condition were as follows: initial pH 6.8, temperature 30℃, fermentation time 24 h. The yield of GABA after optimization reached 65.77 mg/100 ml that increased by 19-fold compared to non-optimization. The GABA contents in improved fermented red bean had little changed between the day 0 and day 28 stored at 4℃: It increased gently in first 12 days. The GABA content of fermented red bean (68.21 mg/100 ml) increased 20 fold by this improved red bean substrate and condition compared to non fermented red bean.