發酵與脫水技術對納豆的酵素活性與機能性成分的影響 翁添滿、陳明造
E-mail: [email protected]
摘 要
本研究分成六個實驗以探討發酵與脫水技術對納豆酵素活性與機能性成分的影響。首先，研究不同發酵時間對納豆中大豆 蛋白質的影響，結果顯示，蛋白質水解率和TCA可溶性氮隨著發酵時間增加而增加，蛋白質溶解率則先下降然後增加；在 未發酵樣品中大豆蛋白之電泳圖可見清晰的片段，而發酵24小時後樣品之大豆蛋白明顯被分解，分子量20 kDa以上大豆蛋 白消失不見；必需胺基酸含量經過發酵36小時後下降，而48小時發酵後除histidine含量下降外全部都上升。 其次探討?燥方 法對納豆酵素活性的影響，納豆分別經烘乾、凍乾及真空乾燥後，測定其凝乳?、蛋白?、脂解?、納豆激?等酵素活性和超 氧陰離子清除力。結果顯示，乾燥納豆除超氧陰離子清除力下降外，其他酵素活性都和未乾燥納豆相近甚至更高，因此我 們認為本研究的乾燥方法適合乾燥納豆粉末的製作。並以相同方法乾燥，分析納豆功能性成分，結果顯示上述乾燥方法不 會影響納豆γ-聚谷氨酸（γ-PGA）和異黃酮的含量，但是對納豆中血管收縮素轉換?抑制能力（ACE-I）有影響。 另外探 討蒸煮大豆接種B. subtilis 後，分別在pH值5、7、9，溫度37、40、43℃，經發酵24、36、48小時後測定納豆的凝乳?、蛋 白?、血管素收縮轉換?抑制能力和胺基酸成分。結果發現蒸煮大豆在pH 9、40℃發酵36小時後，胺基酸含量較未發酵蒸煮 大豆低，因此建議可依不同的目的和所需要的產物來選定不同的發酵條件。 研究蒸煮大豆先接種R. oligosporus再接種B.
subtilis兩階段發酵後酵素活性和大豆蛋白的變化，結果得知無論單菌發酵或兩階段發酵，樣品中可溶性氮和蛋白水解率隨 著發酵時間而增加；兩階段發酵過程樣品的胺基酸變化無規律性，產品凝乳作用比單菌發酵樣品高；由電泳圖顯示經過24 小時兩階段發酵樣品中分子量高過20 kDa 的大豆蛋白消失不見。 比較商業凝乳?與 B. subtilis、 R. oligosporus 發酵所產生的 酵素，在純化及凝乳效力的差異，結果顯示商業凝乳?所產生的凝乳塊具有最高的黏度及凝乳張力，凝乳時間也最短，
由R. oligosporus產生的酵素居次。B. subtilis產生的酵素具有最強的蛋白水解活性，而商業凝乳?的凝乳作用最好。
關鍵詞 : 血管收縮素轉換?抑制能力、枯草芽孢桿菌、生物特性、乾燥方法、酵素活性、發酵條件、異黃酮、商業凝乳?、
凝乳、納豆、純化、根黴菌、γ-聚谷氨酸、大豆蛋白、天貝 目錄
CONTENTS COVER INSIDE PAGE SIGNATURE PAGE 中文摘要 iii ABSTRACT v ACKNOWLEDGEMENTS viii TABLE OF CONTENTS ix LIST OF FIGURES xii LIST OF TABLES xiv 1. GENERAL INTRODUCTION 1 2.
LITERATURE REVIEW 5 2.1 Introduction of fermented product 5 2.2 Bacillus subtilis fermentation in soybean 6 2.3 Rhizopus oligosporus fermentation in soybean 8 2.4 Enzymatic and functional compounds of Natto and Tempeh 10 2.4.1 Nattokinase 10 2.4.2 Protease 12 2.4.3 Lipase 14 2.4.4 Milk-clotting enzyme 15 2.4.5 γ-PGA 16 2.4.6 ACE inhibitory 17 2.4.7 Isoflavone 18 2.4.8 Superoxide dismutase 20 3. CHANGE OF PROTEIN IN NATTO （A FERMENTED SOYBEAN FOOD）AFFECTED BY DURATION OF FERMENTATION 22 3.1 Introduction 22 3.2 Materials and Methods 24 3.3 Results and Discussion 29 3.4 Conclusions 36 4. EFFECTS OF DRYING METHODS ON ENZYMATIC ACTIVITY OF SOY-FERMENTED FOOD (NATTO) BY Bacillus subtilis 37 4.1 Introduction 37 4.2 Materials and Methods 38 4.3 Results and Discussion 41 4.4 Conclusions 51 5. EFFECT OF DRYING METHODS ON γ-PGA, ISOFLAVONE CONTENTS AND ACE INHIBITORY ACTIVITY OF NATTO (A FERMENTED SOYBEAN) 53 5.1 Introduction 53 5.2 Materials and Methods 55 5.3 Results and Discussion 58 5.4 Conclusions 64 6. EFFECT OF FERMENTATION CONDITIONS ON BIOACTIVE PROPERTIES OF STEAMED SOYBEAN INOCULATED WITH Bacillus subtilis ( NATTO ) 67 6.1 Introduction 67 6.2 Materials and Methods 68 6.3 Results and Discussion 72 6.4 Conclusions 77 7. EFFECT OF TWO-STEP FERMENTATION BY Rhizopus oligosporus AND Bacillus subtilis ON BIOLOGICAL ACTIVITIES OF FERMENTED STEAMED SOYBEAN 80 7.1 Introduction 81 7.2 Materials and Methods 83 7.3 Results and Discussion 90 7.4 Conclusions 103 8. COMPARISON ON MILKING-CLOTTING ACTIVITY OF PROTEINASE PRODUCED BY Bacillus subtilis var. natto AND Rhizopus oligosporus WITH COMMERCIAL RENNET 106 8.1 Introduction 107 8.2 Materials and Methods 107 8.3 Results and Discussion 111 8.4 Conclusions 131 9. GENERAL
CONCLUSION 133 9.1 Conclusions 133 9.2 Recommendations 134 REFERENCES 135 LIST OF FIGURE Fig. 3.1 Relationship between degree of hydrolysis of fermented soybean and the fermenting time 32 Fig. 3.2 Change in the SDS-PAGE electrophoretogram of soy protein in natto at different fermentation time 35 Fig. 7.1 Relationship between the degree of hydrolysis and fermenting time of fermented soybean 95 Fig. 7.2 Change of SDS-PAGE electrophoretogram of soya protein of Rhizopus oligosporus at different fermentation 102 Fig. 7.3 Change of SDS-PAGE electrophoretogram of soya protein in two-step
fermentation of Rhizopus oligosporus and Bacillus subtilis var. natto at different fermentation 104 Fig. 8.1 Effects of pH and temperature on clotting viscosity by the crude enzyme from soybean fermented by Bacillus subtilis var. natto 115 Fig. 8.2 Effects of pH and temperature on clotting viscosity by the crude enzyme from soybean fermented by Rhizopus oligosporus 116 Fig. 8.3 Effects of pH and temperature on curd tension by the crude enzyme from soybean fermented by Bacillus subtilis var.natto 117 Fig. 8.4 Effects of pH and temperature on curd tension by the crude enzyme from soybean fermented by Rhizopus oligosporus 118 Fig. 8.5 Chromatographic pattern of milk-clotting enzyme from soybean fermented by Bacillus subtilis var. natto on DEAE-Sepharose CL-6B gel 121 Fig. 8.6 Chromatographic pattern of milk-clotting enzyme from soybean fermented by Bacillus subtilis var. natto on Sephacryl S-100HR gel 122 Fig. 8.7 Chromatographic pattern of milk-clotting enzyme from soybean fermented by Rhizopus oligosporus on DEAE-Sepharose CL-6B gel 125 Fig. 8.8 Chromatographic pattern of milk-clotting enzyme from soybean fermented by Rhizopus oligosporus on Sephacryl S-100HR gel 126 Fig. 8.9 SDS-PAGE electrophoretograms of milk and curd formed by enzymes 130 Fig 8.10 The microstructures of (A) rennet (B) Rhizopus oligosporus (c) Bacillus subtilis var. natto 132 LIST OF TABLE Table 3.1 Effects of fermentation time on TCA-N and protein solubility of soy protein in natto 30 Table 3.2 Effects of fermentation time on amino acid composition of soy protein in natto 33 Table 4.1 Effects of drying methods on the numbers of Bacillus subtilis 42 Table 4.2 Effects of drying methods on milk-clotting activity of natto 44 Table 4.3 Effects of drying methods on total activity of Nattokinase of natto 45 Table 4.4 Effects of drying methods on protease activity of Natto 47 Table 4.5 Effects of drying methods on lipase activity of natto 49 Table 4.6 Effects of drying methods on superoxide anion scavenging activity of natto 50 Table 5.1 Effects of drying methods on γ-PGA content of natto 60 Table 5.2 Effect of drying methods on total isoflavone content of Natto 61 Table 5.3 Effects of drying methods on the numbers of Bacillus subtilis 63 Table 5.4 Effects of drying methods on ACE inhibitory activity of natto 65 Table 6.1 Effects of fermentation condition on milk-clotting activity of natto 73 Table 6.2 Effects of fermentation condition on proteolytic activity of natto 75 Table 6.3 Amino acid composition of natto fermented under different condition 76 Table 6.4 Degree of hydrolysis of soy protein and ACE inhibitory activity of natto under different fermentate condition 79 Table 7.1 Effect of different fermentation times on TCA-N and solubilization ratio of soybean protein of the two-step fermented soybean and natto 91 Table 7.2 Effect of fermentation time on amino acid composition of soy protein in natto and the two-step fermented soybeans 93 Table 7.3 Comparison on the milk-clotting activity of natto, tempeh and the two-step culture fermented soybean 97 Table 7.4 Comparison on the lipase activity of the dried products obtained from different fermentation times by different drying methods with the dried natto fermented for 48 h 98 Table 7.5 Comparison on the protease activity of the dried products obtained from different fermentation times by different drying methods with the dried natto fermented for 48 h 100 Table 8.1 Effects of pH and temperature on clotting time by the crude enzyme from soybean fermented by Bacillus subtilis var. natto 112 Table 8.2 Effects of pH and temperature on clotting time by the crude enzyme from soybean fermented by Rhizopus oligosporus 113 Table 8.3 Comparison on time, viscosity and curd tension of clotting milk by the milk-clotting enzymes produced by Bacillus subtilis var.
natto, Rhizopus oligosporus and rennet 119 Table 8.4 Purification of milk-clotting enzyme from soybean fermented by Bacillus subtilis var. natto 123 Table 8.5 Purification of milk-clotting enzyme from soybean fermented by Rhizopus oligosporus 127 Table 8.6 Comparison of milk-clotting activity to proteolytic activity ratio 128
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