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研究生:羅曼莉
研究生(外文):Rommanee Thammasena
論文名稱:除鹽鹹鴨蛋白粉水解液之分離胜肽片段其抗氧化性及抗菌活性之評估與在肉品上的應用
論文名稱(外文):Evaluation of antioxidative and antibacterial activity of peptide fractions derived from hydrolysate of desalted duck egg white powder and its application in meat system
指導教授:劉登城
指導教授(外文):Deng-Cheng Liu
口試委員:陳文賢吳勇初程仁華譚發瑞
口試委員(外文):Wen-Shyan ChenYun-Chu WuJen-Hua ChengFa-Jui Tan
口試日期:2019-01-15
學位類別:博士
校院名稱:國立中興大學
系所名稱:動物科學系所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:173
中文關鍵詞:抗氧化抗菌胜肽片段除鹽鹹鴨蛋白生鮮豬排
外文關鍵詞:desalted duck egg whitepeptide fractionantioxidativeantibacterialfresh pork chop system
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本篇之研究目的為 : 一、評估除鹽鹹鴨卵白(DDEW)及除鹽鹹鴨卵白粉(DDEWP)之功能性,二、評估由DDEWP分解之胜肽片段之抗氧化及抗菌活性,三、挑選具有成為天然保存劑潛力之胜肽片段,四、將此片段應用於生鮮豬排中並於10℃環境下保存3天對豬排品質之影響。
試驗一為鹹鴨卵白及鹹鴨卵白粉之除鹽效率及其功能性之評估。試驗結果顯示,超過濾除鹽處理(ultrafiltration treatment) 能夠去除鹹鴨卵白中92.93%之鹽分,使除鹽完畢之鹹鴨卵白之鹽度降至0.65%。鹹鴨卵白之營養成分(nutrient content)及胺基酸(amino acid) 皆顯著的低於新鮮鴨卵白。鹹鴨卵白及除鹽鹹鴨卵白於試驗中顯示出良好之起泡力 (foaming ability),然而其乳化力 (emulsifying capacity) 顯著的低於新鮮鴨卵白;且鹹鴨卵白及除鹽鹹鴨卵白之膠體強度 (gel strength)、硬度(hardness) 及彈性 (elasticity) 也較新鮮鴨卵白為低。
試驗二將除鹽鹹鴨卵白粉利用胃蛋白酶 (pepsin, PEP)、枯草桿菌酶 (Bacillus spp., BA) 納豆激酶(natokinase, NAT) 等三種蛋白酶以三種不同濃度 (0.1、0.3及0.5%) 分別進行水解。試驗結果顯示,於所有處理組中,除鹽鹹鴨卵白粉之水解程度(degree of hydrolysis, DH) 隨水解時間及蛋白酶濃度之增加而上升;且水解產物之抗氧化活性及抗菌活性受到蛋白酶濃度及水解時間之影響。於三種蛋白酶水解產物中,利用PEP進行水解之處理組具有最高的水解物產率,而利用BA及NAT進行水解之處理組之水解程度則低於5%;利用PEP進行水解之水解產物於清除DPPH自由基能力(DPPH radical scavenging activity)顯著的高於BA及NAT處理組,而其亞鐵離子清除能力(<37%)則顯著的低於BA(>37-92%)及NAT處理組(30-79%);此外,使用PEP進行水解之水解產物之還原力(reducing power) 顯著的高於BA及NAT處理組。於抗菌活性之結果顯示,透過PEP進行水解之水解產物無法有效的抑制大腸桿菌(Escherichia coli)、沙門氏菌(Salmonella typhimurium)及綠膿桿菌(Pseudomonas aeruginosa)。其中,僅濃度0.1%之PEP進行降解之處理組對於金黃色葡萄球菌具有良好之抗菌活性。試驗一之結果顯示由蛋白酶對除鹽鹹鴨卵白進行水解之水解產物具有較高的水解程度、水解物產率、清除DPPH自由基能力及還原力,且對於金黃色葡萄球菌具有良好的抑制作用。
試驗三將DDEWP以0.3% 之PEP水解9小時,並將水解產物以超過濾方式根據不同分子量(MWCO)分為四個片段。本篇試驗旨在探討F-I (Mw >100 kDa), F-II (Mw 30-100 kDa), F-III (Mw 10-30 kDa) 及 F-IV (Mw <10 kDa) 之抗氧化及抗菌活性及其作為天然保存劑並應用於肉品之潛力。於四個片段中,F-IV顯示出最高的清除DPPH自由基能力、超氧陰離子自由基清除能力(superoxide radical scavenging activities)及亞鐵離子清除能力(Fe2+- chelating activity)。四個處理組之還原能力依序為F-IV > F-III > F-II > F-I。其中,F-IV對於四種選定之微生物(金黃色葡萄球菌、沙門氏菌、大腸桿菌及綠膿桿菌)具有較大的抑制圈;四個牲肽對金黃色葡萄球菌、沙門氏菌、大腸桿菌之最小抑菌濃度為150 mg/mL,但對綠膿桿菌的抑菌作用最為敏感,其最小抑菌濃度為75 mg/mL。
由於F-IV於試驗四中顯示出最佳之抗氧化及抗菌能力,因此,試驗四旨於探討F-IV片段應用於新鮮豬排中於10℃中保存3天對其品質之影響。新鮮豬排隨機分配為6組,分別為control (無添加)、water (添加RO水)、DDEWP (添加150 ml/mL之DDEWP)、PEP150 (添加150 ml/mL之DDEWP水解產物)、F-IV100 (添加100 mg/mL之F-IV 胜肽片段)及F-IV150 (添加100 mg/mL之F-IV 胜肽片段)。本試驗將含有胜肽片段之水溶液噴灑於豬排表面,維持豬排表面之水溶液最終濃度於150 mg/cm2,而F-IV100組之表面最終濃度則維持於100 mg/cm2。本試驗之測定項目包括pH值, TBARS (2-thiobarbituric acid reactive substances), VBN (volatile basic nitrogen), 總生菌數(total plate counts, TPC), 乳酸菌數(lactic acid bacteria counts, LAB), 色澤 (L*, a*, b*) 及感官品評(sensory evaluation)。結果顯示,TBARS及VBN具有濃度依賴性,F-IV100及F-IV150組較control組於保存過程中具有較低之總生菌數及乳酸菌數。於色澤方面,各處理組間於保存過程中均無顯著差異(P > 0.05)。感官品評的結果顯示,F-IV100及F-IV150組於保存試驗末期具有較強之酸性風味;此外,F-IV150處理組於2日保存期間內相較於control組具有較高的風味(flavor)、質地(texture)及總接受度(overall acceptability) 。試驗三之結果顯示F-IV胜肽片段之水解產物於冷藏條件下能夠顯著的維持豬排之品質。
試驗五旨在探討DDEWP透過PEP水解產生之F-IV胜肽片段對於四種選定食源性致病微生物(金黃色葡萄球菌、沙門氏菌、大腸桿菌及綠膿桿菌)於冷藏保存豬排之抗菌活性。新鮮豬排分別與四種選定之微生物進行培養,並分別噴灑滅菌水(sterilize water)、150 mg/mL之DDEWP (PEP150)、100 mg/mL之F-IV胜肽片段(F-IV100)及150 mg/mL之F-IV胜肽片段(F-IV150),維持豬排表面之水溶液最終濃度於150 mg/cm2、F-IV100組之表面最終濃度維持於100 mg/cm2,並將試驗結果與未噴灑之negative control組進行比較。結果顯示,F-IV100及F-IV150組能夠顯著的抑制金黃色葡萄球菌、沙門氏菌、大腸桿菌及綠膿桿菌之生長,且菌數隨噴灑之濃度提升而下降。於貯藏過程中,F-IV100 及F-IV150組之菌數低於control組、water組及DDEWP組。其中,沙門氏菌及金黃色葡萄球菌對於其抑制能力較為敏感,然而對於大腸桿菌則顯示出較低的抑制活性。試驗結果亦顯示PEP150組能夠於貯藏初期抑制食源性致病菌,然而其抑菌活性於貯藏末期則低於F-IV組。
綜合以上所述,超過濾法能夠有效率的降低鹹鴨卵白中之鹽分,且除鹽鹹鴨卵白粉水解產物中,分子量較低之多肽片段(F-IV, Mw <10 kDa)相較於control組,能夠有效的維持豬排於冷藏保存期間之品質。
The objectives of this research were 1) to evaluate the functional properties of desalted duck egg white (DDEW) and desalted duck egg white powder (DDEWP) 2) to determine the antioxidative and antibacterial activity of peptide fractions derived from hydrolysate of DDEWP and 3) to select the best peptide fractions as natural preservatives and 4) to apply in fresh pork chops stored at 10℃ for 3 days.
In experiment I, the desalinated efficiency and functional properties of desalted duck egg white (DDEW) and desalted duck egg white powder (DDEWP) were determined. The result showed that ultrafiltration treatment can be used as desalting to remove 92.93% salt from salted duck egg white and final salt% of desalted duck egg white was 0.65%. The analysis of nutrient content and amino acid of salted duck egg white and desalted sample was significantly lower than those of fresh samples. Although emulsifying capacity of salted and desalted samples exhibited significantly lower than that of fresh sample, an excellent foaming ability was found in salted and desalted samples. Moreover, the texture profiles (gel strength, hardness and elasticity) of salted and desalted samples presented lower values than fresh samples.
In experiment II, DDEWP was hydrolyzed by three proteases, including pepsin (PEP), Bacillus spp. (BA) and natokinase (NAT) with three different concentrations (0.1, 0.3 and 0.5%), individually. The results showed that the degree of hydrolysis (DH) of all treatments increased with increasing hydrolysis time and protease concentrations. The antioxidant and antimicrobial activities of the hydrolysates were affected by type and concentration of protease as well as hydrolysis time. Hydrolysis of PEP significantly (P < 0.05) obtained the highest yield of hydrolysates, however, both of BA and NAT were substantially showed lower DH values and did not exceed 5% still the end of hydrolysis. Among the different hydrolysates, PEP exhibited significantly higher DPPH radical scavenging activity than BA and NAT. All hydrolysates from PEP had lower ferrous ion chelating activity (<37%) and significantly lower than that of NAT (>37-92%) and BA (30-79%). Besides, hydrolysates of PEP presented significantly higher reducing power than BA and NAT. In antimicrobial activities, Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa were not effectively inhibited by all hydrolysates of PEP except of Staphylococcus aureus. Especially, an excellent antibacterial activity against S. aureus was only displayed in hydrolysate of PEP 0.1%. Hydrolysates from pepsin demonstrated significantly better DH, yield, DPPH radical scavenging activity and reducing power, furthermore, had excellent inhibitory on S. aureus due to large clear zone and moderated inhibitory in bactericidal inhibition.
In experiment III, DDEWP was hydrolyzed by pepsin at 0.3% for 9 h then the hydrolysate was further fractionated into four fractions by UF membrane with MWCO. The antioxidant and antibacterial activities of F-I (Mw >100 kDa), F-II (Mw 30-100 kDa), F-III (Mw 10-30 kDa) and F-IV (Mw <10 kDa) were determined to look for a good natural preservative and apply in meat. Among the four fractions, F-IV exhibited the highest DPPH, superoxide radical scavenging activities, Fe2+- chelating activity and reducing power among all fractions and was in order F-IV > F-III > F-II > F-I. Besides, the antimicrobial activity of F-IV also had larger inhibition zone against the growth of four selected bacteria (Staphylococcus aureus, Salmonella typhimurium, Escherichia coli and Pseudomonas aeruginosa), furthermore, the minimal inhibitory concentration (MIC) value was 150 mg/mL and the same to other fractions in this study. Whereas, P. aeruginosa was the most sensitive with MIC value at 75 mg/mL.
In experiment IV, since F-IV fraction of DDEWP hydrolysate exhibited the highest antioxidative and antibacterial activity for the growth of four selected foodborne pathogens in the above results. Therefore, it was selected as natural preservative to apply in fresh pork chops stored at 10℃ for 3 days. In this experiment, fresh pork chops were randomly divided into 6 groups such as control: non treatment, water: sterilized RO water, DDEWP: DDEWP solution at 150 mg/mL, PEP150: DDEWP hydrolysate at 150 mg/mL, F-IV100: F-IV peptide fraction at 100mg/mL and F-IV150: F-IV peptide fraction at 150 mg/mL, individually. Moreover, the spraying solutions were sprayed on the surface of samples in each group with the final concentration at 150 mg/cm2 and 100 mg/cm2 for F100 sample. The pH, TBARS (2-thiobarbituric acid reactive substances), VBN (volatile basic nitrogen), total plate counts (TPC), lactic acid bacteria counts (LAB), instrumental color (L*, a*, b*) and sensory evaluation were determined. The TBARS and VBN values were lower in a dose-dependent manner (P < 0.05). The TPC and LAB counts of F-IV samples had lower than control during storage. The TPC and LAB counts of F-IV150 samples were the lowest number among all treatments. Non-significant difference in the instrumental color among treatments was observed during storage (P > 0.05). Analysis of sensory attributes, pork chops with two F-IV treatments exhibited slight acid flavor and had strong density in F-IV150 treatment at the end of storage. However, samples with F-IV150 also obtained higher flavor, texture and overall acceptance compared with control before 2-day during storage. All results confirmed that the use of F-IV fractions of hydrolysate from DDEWP could effectively maintain the quality of pork chops during refrigerated storage.
In experiment V, the objective was to investigate the antimicrobial activity of peptic hydrolysate fractions (F-IV) from DDEWP against four selected foodborne pathogens including S. aureus, S. typhimurium, E. coli and P.s aeruginosa in fresh pork chop under refrigerated conditions for 3 days. Fresh pork chop was separately inoculated with four selected foodborne pathogens and it was individually sprayed with sterilize water, DDEWP solution at 150 mg/mL (DDEWP), peptic hydrolysate at 150 mg/mL (PEP150), peptic hydrolysate fraction at 100 mg/mL (F-IV100) and peptic hydrolysate fraction at 150 mg/mL (F-IV150) to meet the final concentration at 150 mg/cm2 and 100 mg/cm2 for F-IV100 and the results were compared to fresh pork chop without sprayed any additive (negative control). Results showed that the treatments of F-IV significantly (P < 0.05) inhibited S. aureus, S. typhimurium, E. coli and P. aeruginosa growth. The counts were significantly (P <
TABLE OF CONTENTS
Page
ACKNOWLEDGMENT i
摘要 ii
ABSTRACT v
TABLE OF CONTENTS ix
LIST OF TABLE xii
LIST OF FIGURE xiv
CHAPTER 1:
Introduction 1
1. Relation and Background 2
2. Aim of this study 3
CHAPTER 2:
Literature review 5
1. Salted duck egg (SDE) 6
2. Desalination 6
3. The function of egg white 11
4. Proteases for hydrolysis 14
5. Pepsin 19
6. Proteases of Bacillus strains 19
7. Antioxidant 20
8. Bioactive peptides 22
9. Isolation bioactive peptides 26
CHAPTER 3:
Evaluation of nutrient content, physicochemical and functional properties of desalted duck egg white by ultrafiltration as desalination 30
1. Abstract 31
2. Introduction 31
3. Materials and methods 33
4. Results and discussion 36
CHAPTER 4:
Antioxidant and antimicrobial activities of different enzymatic hydrolysates from desalted duck egg white 51
1. Abstract 52
2. Introduction 53
3. Materials and methods 54
4. Results and discussion 58
CHAPTER 5:
Antioxidant and antimicrobial activities of peptide fractions derived from pepsin hydrolysate of desalted duck egg white powder 72
1. Abstract 73
2. Introduction 73
3. Materials and Methods 74
4. Results and discussion 80
CHAPTER 6:
Effect of hydrolysate fractions derived from desalted duck egg white as a preservative on the quality of pork chops during storage 93
1. Abstract 94
2. Introduction 94
3. Materials and methods 96
4. Results and discussion 101
CHAPTER 7:
Antimicrobial activity of hydrolysate fractions derived from desalted duck egg white against foodborne pathogens in pork chop model 117
1. Abstract 118
2. Introduction 118
3. Materials and methods 120
4. Results and discussion 123
CHAPTER 8:
Conclusion 133
REFERENCES 136
APPENDICES 172
Chapter 1
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Chapter 2
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Chapter 7
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