發芽糙米產製過程中微生物調控及其發酵生理活性產物之探討

Abstract

在發芽糙米的生產過程中所須考慮的幾個重要因子，包含：糙米的品種、種子的活性、發芽的溫度、發芽的時間、生產過程中微生物的控制與指標機能性成分的累積。在此過程中微生物控管與安全性的考量顯得特別的重要，其中又以選擇不影響糙米的發芽能力及符合食品安全性的滅菌方法最為重要；就本實驗而言可選擇的控制方法及符合食品安全的方法並不多，而在以消費者為考量的前提下，傳統食品化學添加劑並不受歡迎。因此選擇以超音波表層清洗，結合0.6 ppm 濃度O3 與循環水的紫外線滅菌流程，將浸漬水(soaking water)總生菌數的濃度控制在103 cfu/ml 以下的標準。當在此條件之下進行黃麴毒素檢測時，所有樣本均未發現黃麴毒素的存在，已符合食品安全標準。此外，更選擇了七個稻米品種，在相同的發芽率篩選標準下比較不同的溫度差，對γ-氨基丁酸(GABA)累積變化之影響。結果顯示，經24小時浸置後以台梗八號品種（TK-8）在37℃的發芽程序最佳，風味也最好，而其所累積的GABA含量亦可達31.12 mg/100g。

利用納豆菌(Bacillus natto) SYH-MT 0379，進行以發芽糙米作為培養前驅物基質之發酵，藉由固態及液態兩種不同發酵方法生產新的機能性活性物質。初期設定以抗菌活性以及抗氧化活性檢測分析做為新機能性活性物質的篩選方法。實驗結果發現，發芽糙米與納豆菌SYH-MT 0379液態發酵所得之產物經過濾後，分別以正丁烷（n-hexane）、乙酸乙酯（ethyl acetate），以及甲醇（methanol）進行生物指標追蹤之萃取沖提可得，乙酸乙酯層中存在具有抑菌（以Candida albicans為標的）及抗氧化活性物質（以DPPH自由基清除為標的），經薄膜色層（TLC）分析分離及高效能液相層析（HPLC）分析對比出一個具有上述活性之新發酵代謝產生物質之吸收波峰，特將其命名為PGBRNFL-I。將乙酸乙酯萃取層部分，以TLC進一步純化該物質後可得一抗菌及抗氧化活性確定之單一化合物。迄今均未曾有報導提及發芽糙米以及納豆菌組合發酵代謝產物之研究，而藉由此方法所得脂溶性機能性活性物質，本研究為首例。PGBRNFL-I物質分子結構相關之有機光譜資料，包括：紫外光光譜（UV）、紅外光光譜 (IR)、含一維氫譜、碳譜以及二維之氫氫譜（1H1H COSY）和碳氫譜（1H13C COSY）之核磁共振光譜（NMR），以及電子衝擊模式之質譜（EI-MS）等，均已收集並進行其構造解析中。

During the production process of the germinated brown rice, several factors must be considered, including the cultivar of the rice, germination activity of seeds, sprouting temperature, sprouting time, the control of microorganisms and accumulation of main functional composition. The control of microorganisms and food safety are very important, especially the disinfection method that does not impair the sprouting capability while meeting food safety. To this study, the available control methods that meet food safety requirements are limited. Given the concerns of the consumers, traditional food additive is not acceptable. Therefore, this study used ultrasonic technique for surface cleaning, and UV disinfection in combination with 0.6 ppm O3 and circulating water to control the total count of microorganisms in soaking water to under103 cfu/ml. The aflatoxin test conducted under such conditions did not detect the presence of aflatoxin, which meets the food safety standard. Among the selected seven rice cultivars tested under different temperatures, TK-8 was rated the best sprouting process and taste after soaked for 24 hours at 37℃, and the accumulated GABA reached as high as 31.12 mg/100g. A combinatory fermentation, combined with germinated brown rice as a functional cultural precursor, was processed using Bacillus natto SYH-MT 0379 as the solely strain in performing solid-state and liquid batch fermentation in search of new active functional substances. In the screening experiment, the antimicrobial and antioxidant activity tests were chosen as the testing models. After filtration the fermentation broth was successively with n-hexane, ethyl acetate and methanol to be present in the ethyl acetate fraction. The active compounds were found extracted TLC together with HPLC analytical program was applied for comparison study and the resulting HPLC profile showed a newly generated absorption peak. After repeated TLC separation, the single substance, named PGBRNFL-I, were found in the ethyl acetate fraction. The characteristics of the compound exhibited the fluorescence signal at UV 254 and 365 nm and the biological functions of antimicrobial (mainly against Candida albicans) and antioxdant (mainly scavenging the DPPH radical) activity. Neither the germinated brown rice itself nor the combinatory fermentation of the germinated brown rice/Bacillus natto system has been reported previously. Up to now, this is the first study dealing with the theme. The spectroscopic data for PGBRNFL-I have been collected including ultraviolet (UV) spectrum, infrared (IR) spectrum, as well as 1-D 1H and 13C, 2-D 1H-1H and 1H-13C COSY nuclear magnetic resonance (NMR) spectra. In addition, an electron impact mass spectrum (EI-MS) was measured to elucidate the molecular fragmentation and its molecular weight. The structure determination by the information of the related spectroscopic data is now undergoing.