臺灣博碩士論文加值系統

鮪魚蒸煮為鮪魚罐頭加工之必要程序，所產生的煮汁(約含4.0% 蛋白質；1噸鮪魚可產生160公斤蒸煮汁)通常予以廢棄，不僅蛋白質資源損失且造成廢水處理上相當的負擔。因此，本研究擬應用鮪魚蒸煮液中蛋白質為原料開發具有經濟價值之抗微生物活性物質。實驗應用三種商業蛋白質水解酵素Orientase、Alcalase 2.4L、Flavourzyme進行對鮪魚蒸煮液進行水解，並進行水解物之抗菌試驗。實驗所使用菌株包括Staphylococcus aureus、Bacillus subtilis、Escherichia coli、Pseudomonas aeruginosa、Candida albicans、Salmonella enterica subsp. Enterica、Listeria innocua、 Shiegella sonnei 等八株病原菌。結果顯示Alcalase 2.4L蛋白酶水解物具較佳抗微生物活性，(製備條件為：E/S = 1/100 (w/v)，50℃，pH 7.0，3 h)。將蛋白質水解物經由膠過濾層析分離可獲得四個抗微生物活性較高的區分物：(Tuna cooking juice Alcalaes 1，TA1)，(Tuna cooking juice Alcalaes 2，TA2)， (Tuna cooking juice Alcalaes 3，TA3)，(Tuna cooking juice Alcalaes 4，TA4)。結果顯示具有最高活性的區分物為TA2 (M.W.1060~6500)，其對上述八株病原菌之最小抑制濃度 (minimal inhibitory concentration，MIC)分別為800、750、800、750、850、800、850、800 (μg/mL)。爲了解水解物經胃腸道中抗菌活性之變化，使用腸胃消化酵素進行消化試驗。結果顯示，區分物TA2 經胃蛋白酶(pepsin)水解(pH 3.0，37℃，6 h)後，抗微生物活性下降17.6% ~ 26.7%；再經胰蛋白酶(trypsin)、胰凝乳蛋白酶(chymotrysin)水解(pH 8.0，37℃，12 h)後，對八株病原菌抗微生物活性無明顯差異，因此推測區分物TA2在胃消化道中，消化酵素反應造成抗微生物活性的下降。區分物TA2經由HPLC純化可獲得抗微生物活性更高的抑制物，分離後所得五個主要區分物為：TA2H-1、TA2H-2、TA2H-3、TA2H-4、TA2H-5，其中TA2H-1對Candida albicans及Shiegella sonnei具有良好之抑制效果；TA2H-4對其餘六種菌株具有良好之抑制效果，經胺基酸序列分析結果為：
1. Lys-Asp-Ser-Pro-Gly-Gly-Gln-Asp-Arg-Arg ；
2. Pro-Ser-Cys-Trp-Thr-Phe-Gly-Phe-Ser-Glu-Asn-His；
3. Pro-Ser-Ser-Pro-Arg-Glu-Glu-Val-Ser-Leu-Asp-Leu-Asp。
綜合以上結果顯示，鮪魚蒸煮液之蛋白質可做為開發製備抗微生物胜肽之來源。

Tuna cooking is a processing of necessity procedures for canned tuna, The side product (about a 4.0% protein; 1 ton tuna can produce 160 kg of cooking juice) is normally discarded, this waste not only brings the loss of resources and the resulting protein waste water treatment is also considerable burden. Therefore, this study applied the protein from tuna cooking juice for the development of economic value anti-microbial substances. Three commercial protease Orientase, Alcalase 2.4L and Flavourzyme are used in this study. Eight bacteria, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Salmonella enterica subsp. Enterica, Listeria innocua and Shiegella sonnei are choose for the experiment studies. The results showed that protease Alcalase 2.4L hydrolysates reveal better anti-microbial activity, (preparation conditions: E / S = 1 / 100 (w / v), 50 ℃, pH 7.0, 3 h). Higher antimicrobial activity inhibitors of protein hydrolysates are obtained by gel filtration chromatography, the four major fractions after the separation are：(Tuna cooking juice Alcalaes 1, TA1), (Tuna cooking juice Alcalaes 2, TA2), (Tuna cooking juice Alcalaes 3, TA3), (Tuna cooking juice Alcalaes 4, TA4). The results showed that TA2 fraction reveal the highest activity (MW1060 ~ 6500), its minimal inhibitory concentration (minimal inhibitory concentration, MIC) on eight pathogens were 800, 750, 800, 750, 850, 800, 850, 800 (μg/mL). To understand the changes of hydrolysate in the gastrointestinal tract, digestive enzymes are applied to digest hydrolysis. The results showed that the anti-microbial activity of fractions TA2 is decreased in 17.6% ~ 26.7% sequence by pepsin (pepsin) hydrolysis (pH 3.0, 37℃, 6 h); While treating with trypsin (trypsin) and chymotrypsin (chymotrysin) hydrolysis (pH 8.0, 37℃, 12 h), the anti-microbial activity remained unchanged. The revelation expermental gave that the anti-microbial Activity of fractions TA2 decreased in the stomach and the digestive tract by digestive enzyme. The TA2 fraction is further purified by HPLC to obtained a higher antimicrobial activity inhibitor, the five major fractions are labeled after the separation as: TA2H-1, TA2H-2, TA2H-3, TA2H-4, TA2H-5, where TA2H -1 shows inhibitory effect on Shiegella sonnei and Candida albicans , and TA2H-4 have inhibitory effect on the remaining six strains, the amino acid sequence analysis of the results:
1.Lys-Asp-Ser-Pro-Gly-Gly-Gln-Asp-Arg-Arg, 2.Pro-Ser-Cys-Trp-Thr-Phe-Gly-Phe-Ser-Glu-Asn-His,
3. Pro-Ser-Ser-Pro-Arg-Glu-Glu-Val -Ser-Leu-Asp-Leu-Asp.
These results provided that tuna cooking juice can be applied to the development of the source of antimicrobial peptides.

謝誌………………………………………………………………………I
中文摘要 ………………………………………………………………II
英文摘要 ………………………………………………………………IV
圖表索引………………………………………………………………IX
壹、 前言 ………………………………………………………………1
貳、 文獻回顧 …………………………………………………………2
一、 鮪魚簡介 ……………………………………………………2
二、 抗微生物胜肽(Antimicrobial peptide, AMPs)…………4
三、 蛋白質酵素水解……………………………………………8
四、 蛋白質的水解方法…………………………………………9
五、 蛋白質水解物的應用………………………………………11
六、 蛋白質水解物的生物活性 (biological activity) …12
參、目的………………………………………………………………16
肆、 材料與方法………………………………………………………17
一、 研究材料 …………………………………………………17
(1) 鮪魚蒸煮液……………………………………………17
(2) 酵素……………………………………………………17
(3) 試藥……………………………………………………18
(4) 微生物…………………………………………………19
(5) 培養基…………………………………………………19
二、 實驗方法 …………………………………………………20
(一) 一般成分分析……………………………………………20
(1) 水分…………………………………………………20
(2) 灰分…………………………………………………20
(3) 粗蛋白質……………………………………………21
(4) pH值…………………………………………………21
(5) 粗脂肪………………………………………………21
(二) 水解率(degree of hydrolysis DH) 之測定………22
(1) 胺基態氮測定 ……………………………………22
(2) 水解前之總氮含量………………………………22
(三) 最小抑制濃度( MIC )………………………………23
(四) 抗微生物操作法……………………………………23
(1) 試驗菌株懸浮液製備…………………………23
(2) 培養皿製備……………………………………24
(3) 培養條件及測定………………………………24
(五) 鮪魚蒸煮液蛋白質酵素水解物之分子量分佈…24
(六) 鮪魚蒸煮液抗菌測定法…………………………24
(七) 模擬消化試驗……………………………………25
(八) 鮪魚蒸煮液之區分物具抑菌活性胜肽之分離與純化25
(九) 胺基酸序列分析………………………………………26
三、 實驗架構 ………………………………………………27
伍、 結果與討論 …………………………………………………29
一、由鮪魚蒸煮液製備抗微生物活性物質…………………29
(一) 鮪魚蒸煮液一般成分分析…………………………29
(二) 選擇水解鮪魚蒸煮液之蛋白酶……………………31
(三) 水解率(degree of hydrolysis DH) 之測定……43
(四) alcalase水解物之區分物抗微生物活性………45
(五) alcalase水解物之區分物最小抑制濃度( MIC )50
二、鮪魚蒸煮液蛋白質水解物之模擬消化試驗…………54
(一) pepsin 對區分物抗微生物活性之影響 ………54
(二) trypsin 及chymotrypsin 對區分物抗微生物活性之影響…54
三、鮪魚蒸煮液蛋白質區分物之抗微生物胜肽之純化與序列分 析…61

陸、 結論………………………………………………………………74
柒、 參考文獻…………………………………………………………76

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