臺灣博碩士論文加值系統

豬肝是豬屠宰的副產品，富含蛋白質及各種營養素，然而台灣較少利用。酶水解用於回收及改善來自魚類和動物副產物的蛋白質之營養及功能特性，以產生機能性成分。超音波已被用於改善蛋白質水解及修飾酶活性。因此，本研究之目的是評估不同酶，超音波處理和水解條件對豬肝酶水解產生水解產物的影響，並進一步評估其抗氧化活性。
試驗一，評估使用Alcalase®，木瓜蛋白酶，胃蛋白酶或紅麴黴菌微生物懸浮液 (分別為APLH，PaPLH，PePLH和MPLH) 在各種水解時間 (3, 6和12h) 後製備的豬肝蛋白水解物之抗氧化活性。結果顯示APLH之產率及胜肽含量最高，PaPLH的水解率高於其他組別。MPLH具有最高的2, 2-diphenyl-1-picrylhydrazyl (DPPH)自由基清除活性及還原能力，APLH及PaPLH具有較MPLH更高的亞鐵離子螯合能力，所有水解產物的分子量小於10 kDa。PaPLH的總胺基酸及疏水性胺基酸含量最高。15個從MPLH獲得的抗氧化胜肽片段含有一個或多個胺基酸 (酪胺酸，色胺酸，丙胺酸，脯胺酸，蛋胺酸，賴胺酸，天冬胺酸，半胱胺酸，纈胺酸，亮胺酸或組胺酸等)。儘管由紅麴黴菌微生物懸浮液水解的水解產物顯示出最高的DPPH自由基清除活性及還原力，但是其產量和水解率最低。因此，選擇具有能產生最高產量、胜肽含量及和亞鐵離子螯合能力之Alcalase®進行後續試驗。
試驗二中，進而藉由超音波處理來提高水解產物的抗氧化活性。在試驗中，評估超音波預處理 (0, 15, 30, 45及60 s) 對使用Alcalase®的豬肝蛋白水解物 (PLPH) 的抗氧化活性之影響。隨著超音波處理時間的延長，PLPH的水解率及胜肽含量增加。60秒超音波預處理的水解產物顯示出最高的水解率及胜肽含量。45秒超音波預處理的水解產物顯示出最高的亞鐵離子螯合能力及還原力。30秒超音波預處理的水解產物顯示出最高的DPPH自由基清除活性及在亞麻油酸自氧化系統中較高的抑制活性；所有水解物胜肽的分子量小於6,200 Da。
試驗三進一步藉由運用反應曲面法(Response surface methodology; RSM) 來評估不同水解參數 (E/S比，pH值和溫度) 對超音波輔助酶水解豬肝蛋白質水解物(PLH)的影響。結果顯示E/S比、pH值及溫度對豬肝水解物的抗氧化活性有顯著影響(P <0.01)。當E/S比1.4% (v/w)、溫度55.5 °C及初始pH值10.15時，能產生具有最高DPPH自由基清除活性的PLH，其水解率、DPPH自由基清除活性、亞鐵離子螯合能力、還原力、分子量及疏水性胺基酸含量分別為24.12%、79%、98.18%、0.601、< 5,400 Da、45.7%。
由本研究結果得知，藉由使用紅麴黴菌微生物懸浮液、應用超音波處理及選擇最佳水解條件進行酶水解時，可自豬肝中獲得性能優良的抗氧化水解物，具有預防食品中脂質氧化的潛在應用。

Porcine liver, a by-product of pig slaughtering, is rich in proteins and various nutrients. However, it is rarely used in Taiwan. Enzymatic hydrolysis is used to recover and improve the nutritional and functional properties of proteins from fish and animal byproducts to yield functional components. Ultrasonic has been used to improve protein hydrolysis and modify enzyme activities. Therefore, the objective of this study was to evaluate the effects of different enzymes, ultrasonic treatments, and hydrolysis conditions on the enzymatic hydrolysis of porcine liver for the production of hydrolysates and further characterization of their antioxidant activities.
In the experiment 1, the antioxidant activity of porcine liver protein hydrolysates (PLPH) prepared by using Alcalase®, papain, pepsin, or Monascus purpureus microbial suspension (APLH, PaPLH, PePLH, and MPLH, respectively) after various hydrolysis time (3, 6, and 12 h) was evaluated. The results exhibited that the highest yield and peptide content were obtained from APLH, whereas the degree of hydrolysis (DH) of PaPLH was higher than the others. MPLH had the highest 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and reducing power, whereas APLH and PaPLH had higher ferrous ion-chelating ability than did MPLH. The molecular weights of all the hydrolysates were <10 kDa. The PaPLH exhibited the highest contents of total amino acids and hydrophobic amino acids. Fifteen antioxidant fractions obtained from MPLH contained one or more of the following amino acids in their sequences: Tyr, Trp, Ala, Pro, Met, Lys, Asp, Cys, Val, Leu, and His. Even though the hydrolysate hydrolyzed by Monascus purpureus microbial suspension exhibited the highest DPPH free radical scavenging activity and reducing power, it had the lowest yield and DH. Therefore, Alcalase® which exhibited the highest yield, peptide content and ferrous ion-chelating ability has been selected to hydrolyze porcine liver proteins in the next experiment.
In the experiment 2, ultrasonic was applied on the enzymatic hydrolysis in order to improve the antioxidant activity of the porcine liver hydrolysate. The effect of the ultrasonic pretreatment (0, 15, 30, 45, and 60 s) on the antioxidant activity of PLPH employing Alcalase® was evaluated. The results exhibited the DH and peptide contents of the PLPHs increased as the time of ultrasonication increased. The hydrolysate pretreated with ultrasonication for 60 s exhibited the highest DH and peptide contents. The hydrolysate pretreated with ultrasonication for 45 s exhibited the highest ferrous ion chelating ability and reducing power. The hydrolysate pretreated with ultrasonication for 30 s exhibited the highest DPPH radical scavenging activity and the higher inhibitory activity in the linoleic acid autoxidation system. The molecular weight of the peptides in the hydrolysates was less than 6.2 kDa.
In the experiment 3, by using response surface methodology (RSM), the effect of various hydrolysis parameters (E/S ratio, pH, and temperature) on the ultrasonic-assisted enzymatic hydrolysis of PLPH was evaluated. The results exhibited that E/S ratio, pH, and temperature significantly affected the antioxidant activity of the hydrolysate (P < 0.01). The optimal conditions for producing PLPH with the highest scavenging activity by using RSM were as follows: E/S ratio, 1.4% (v/w); temperature, 55.5°C; and initial pH, 10.15. Under these conditions, the degree of hydrolysis, DPPH free radical scavenging activity, ferrous ion chelating ability, and reducing power of PLPHs were 24.12%, 79%, 98.18%, and 0.601, respectively. The molecular weight of PLPH produced under these optimal conditions was less than 5,400 Da and contained 45.7% hydrophobic amino acids.
In conclusion, favorable antioxidant hydrolysates from porcine liver enzymatic hydrolysis, which have a potential application for retarding lipid oxidation in food products, can be obtained by using Monascus purpureus microbial suspension, applying of ultrasonic treatment as well as selecting of the optimal hydrolysis conditions.

誌 謝 辭 i
摘 要 ii
ABSTRACT iv
TABLE OF CONTENTS vii
LIST OF TABLES xi
LIST OF FIGURES xii
CHAPTER 1 1
1.1 Animal by-products/Porcine liver 2
1.2 Monascus spp. 3
1.3 Enzymes 3
1.3.1 Aspartic proteinase 4
1.3.2 Alcalase® 4
1.3.3 Pepsin 5
1.3.4 Papain 5
1.4 Protein hydrolysates 6
1.4.1 Enzymatic hydrolysis 7
1.4.2 Factors affecting enzymatic hydrolysis of proteins 8
1.5 Ultrasonication 11
1.5.1 Introduction 11
1.5.2 The effects of ultrasonication on protein structures, functionality, and performance 12
1.6 Response surface methodology (RSM) 14
Aim of this study 15
CHAPTER 2 17
2.1 Abstract 18
2.2 Introduction 18
2.3 Materials and methods 21
2.3.1 Preparation of M. purpureus microbial suspension 21
2.3.2 Preparation of porcine liver hydrolysates using various proteases 21
2.3.3 Measurement of yields and DH of hydrolysates 22
2.3.4 Determination of peptide content 22
2.3.5 Determination of antioxidant activities 23
2.3.6 Determination of amino acid composition and molecular weight distribution of PLPHs 23
2.3.7 Determination of antioxidant peptide sequence 24
2.3.8 Statistical analysis 24
2.4 Results and Discussion 24
2.4.1 Enzymatic hydrolysis 24
2.4.2 Antioxidant properties of hydrolysates 26
2.4.3 The molecular weight distribution of protein hydrolysates 28
2.4.4 Amino acid composition of hydrolysates 29
2.4.5 Characterization of antioxidant peptides 30
CHAPTER 3 40
3.1 Abstract 41
3.2 Introduction 41
3.3 Materials and methods 43
3.3.1 Materials 43
3.3.2 Ultrasonic pretreatment of porcine liver 43
3.3.3 Determination of soluble protein content 44
3.3.4 Preparation of porcine liver protein hydrolysates 44
3.3.5 Determination of the DH of hydrolysates 45
3.3.6 Determination of peptide contents 46
3.3.7 Determination of antioxidant activities 46
3.3.8 Inhibition of linoleic acid autoxidation 46
3.3.9 Determination of the molecular weight distribution of hydrolysates 47
3.3.10 Statistical analysis 48
3.4 Results and discussion 48
3.4.1 DH, soluble protein content, and peptide content 48
3.4.2 Antioxidant activities of hydrolysates 50
3.4.3 Molecular weight distribution of protein hydrolysates 52
CHAPTER 4 59
4.1 Abstract 60
4.2 Introduction 61
4.3 Materials and methods 62
4.3.1 Preparation of PLPHs 62
4.3.2 RSM 62
4.3.3 DH measurement 63
4.3.4 Antioxidant activity measurement 63
4.3.5 Determination of PLPH molecular weight distribution and amino acid composition 64
4.3.6 Statistical analysis 64
4.4 Results and discussion 64
4.4.1 Optimization of the hydrolysis process 64
4.4.2 Visual analysis of the RSM experiment 66
4.4.3 Optimization of technical conditions 67
4.4.4 DH and antioxidant activity 68
4.4.5 Molecular weight and amino acid composition of the PLPH produced under optimal conditions 69
CHAPTER 5 77
5.1 Conclusion 78
5.2 Future research 79
REFERENCES 80

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