芋頭農業資材最佳化糖解產物於嗜酸乳酸菌發酵與其生理活性之研究

Optimization of Glucose Release from Taro Agricultural Resource for Lactobacillus acidophilus Cultivation and Evaluation of Its Biological Activity Properties

潘筱蕙(Xiao-Hui Pua)

指導教授 ： 鄭光成

共同指導教授 ： 謝淑貞(Shu-Chen Hsieh)

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.00898

摘要

由於農業與工廠製造過程所產生的農業廢棄物日益增加，如何永續利用農業廢棄物逐漸獲得世人關注。這些廢棄物在經過適當的前處理之後成為具有潛力的農業資材，並可用於再生能源之生產。另一方面，近期的研究指出，非活性乳酸菌體對於抗腫瘤及免疫調節有一定的功效，然而有關培養基對非活性乳酸菌的影響之研究卻非常有限。此研究利用酵素水解芋頭農業資材中的澱粉生成葡萄糖並以此作為乳酸菌培養基之碳源培養嗜酸乳酸菌BCRC 14079再探討不同培養基對於其熱致死菌、菌胞內液及胞外多醣之抗腫瘤和免疫調節功效的影響。本實驗以Box-Behnken設計之反應曲面法最適化澱粉液化生產還原糖得知澱粉液化最適化條件為在溫度79.2oC下加入9 mL/L之 α-澱粉酶反應5小時，可產30.57 g/L 還原糖。經過液化後，以在溫度60 oC下加入0.3 mL/L葡萄苷澱粉水解酵素反應3小時可達最高產量，產出60.14 g/L葡萄糖。本研究探討了6種農業飼料作為替代培養基之氮來源的效果，調配出具有較低成本且環保的乳酸菌培養基。這6種飼料分別為動物性之肉骨粉、雞肉粉及魚骨粉與植物性之玉米酒糟粉、玉米蛋白粉及大豆粉。結果顯示，以37 g/L葡萄糖（來自芋頭農業資材水解物）為碳來源混合25 g/L之玉米蛋白粉並額外添加1 g/L酵母萃取物之CGMY1培養基可產出log 9.20 CFU/mL嗜酸菌。MTT assay細胞存活率分析結果顯示CGM培養基來源之熱致死菌及胞外多醣對抑制人類大肠癌細胞HT-29及Caco-2的活性最高，且熱致死菌的功效高於胞外多醣。NF-ΚB與COX-2螢光系統檢測顯示最能刺激螢光素報導體的表現為CGM培養基所得之熱致死細胞，而CGMY1培養基所得之胞外多醣次之，最後為MRS培養基所得之胞內液。本研究證實芋頭農業資材可被開發成一具有經濟價質且環保的乳酸菌培養基，並可以此培養基生產具有更高抗腫瘤及免疫調節功效之非活性乳酸菌體。

關鍵字

芋頭農業資材 ； 嗜酸乳酸菌 ； 熱致死菌 ； 胞外多醣 ； 免疫調節 ； 抗腫瘤

並列摘要

Food manufacturing sectors, using starch crop, fruits and vegetables as input, has generated a huge amount of field waste and processing wastes that can be utilized as agricultural resources. Yet, the current applications of taro agricultural resources (TAR) was limited as fertilizers. On the other hand, recent studies have revealed that non-viable probiotics have many therapeutic activities including anti-tumor and immunoregulatory properties. However, studies on the influence of culturing medium towards the biological activities of non-viable probiotics were scarce. In this study, TAR was utilized to produce glucose as carbon source for Lactobacillus acidophilus BCRC 14079 cultivation and the anti-tumor and immunomudulatory property of non-viable probiotics namely heat-killed cell (HKC), cytoplasmic fraction (CF) and exopolysaccharide (EPS) were evaluated. Optimum parameter for liquefaction determined by Box-Behnken Design Response Surface Methodology (BBD-RSM) was 79.2oC of temperature, 9 mL/L of α-amylase enzyme solution and 5 h of reaction time, producing 30.57 g/L of reducing sugar. For saccharification, dose of amyloglucosidase, temperature, and saccharification time were determined as 0.3mL/L of amyloglucosidase enzyme solution, 60oC and 3 h, resulting in approximately 60.14 g/L of glucose. Various alternative nitrogen sources, namely bone meal (BM), fish meal (FM), chicken meal (CM), dried distillers grains with solubles (DDGS), corn gluten meal (CGM), and soy meal (SM) were evaluated to substitute yeast extract (YE) as nitrogen source in order to produce a more economic and environmental friendly medium for lactic acid bacteria (LAB) cultivation. The most potential novel TAR medium for enhanced LAB growth, CGMY1 medium, constitutes of approximately 37 g/L of glucose (TAR hydrolyzate) and CGM supplemented with 1 g/L of YE, producing log 9.20 CFU/mL of LAB cells. MTT assay result showed HKC and EPS from CGM medium exhibited the highest anti-proliferative effect towards colon adenocarcinoma cells HT-29 and Caco-2. In addition, the anti-tumor effect of HKC was better than EPS. The results of luciferase-based NF-ΚB and COX-2 system indicated that HKC from CGM medium stimulates the most expression of luciferin reporter in both systems, followed by EPS from CGMY1 medium and CF from MRS medium. In conclusion, this study demonstrated the potential of utilizing TAR for L. acidophilus cultivation and the production of non-viable probiotics with enhanced anti-tumor and/or immunoregulatory property by using novel TAR mediums.

並列關鍵字

Taro agricultural resource ； Lactobacillus acidophilus ； Heat-killed cell ； Exopolysaccharide ； Immune-regulation ； Anti-tumor

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