Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/25264
標題: 以鴨蛋白開發具抗氧化性產品之研究
Studies on development of duck egg white products with antioxidative activities
作者: 陳怡兆
Chen, Yi-Chao
關鍵字: Duck egg white
鴨蛋白
Yeast
Hydrolysates
Antioxidant
酵素
酵母菌
水解液
抗氧化性
出版社: 動物科學系所
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摘要: 本研究嘗試以鴨蛋白為原料開發具抗氧化性之產品,以提昇鴨蛋產品價值。試驗則以商業酵素(papain、trypsin、chymotrypsin、alcalase及flavourzyme)進行鴨蛋白之水解、並進一步自行篩選具蛋白水解能力之酵母菌株及其酵素之萃取,並應用於鴨蛋白之水解試驗及其抗氧化測定等。 以papain、trypsin、chymotrypsin、alcalase及flavourzyme等5種酵素進行鴨蛋白水解試驗,結果僅papain對鴨蛋白具有水解能力,其水解液(DEWHP)亦具有抗氧化性。此外,高E/S比有助於鴨蛋白之水解,而此結果亦可從電泳分析中得知。DEWHP的亞麻油酸自氧化抑制率及超氧陰離子清除率均可達70%以上,Fe2+螯合力可達90%以上,而其DPPH (α,α-diphenyl-β-picrylhydrazyl)自由基清除率及還原力則無顯著效果,高胜肽含量則是DEWHP抗氧化性提昇的主因。 自克弗爾乳中分離之4種不同形態之酵母菌株(YK-1~YK-4)以RapID Yeast Plus System鑑定同屬於Candida kefyr,其菌體均具有很強的valine arylamidase及cystine arylamidase等蛋白酶活性,亦具有磷酸酶活性及半乳醣苷酶活性,且在酪蛋白發酵試驗中,亦能利用酪蛋白溶液中較小的蛋白質片斷,而使酪胺酸的含量增加。 Candida kefyr YK-1、YK-2、YK-3及YK-4等篩選菌株在鴨蛋白液中之生長曲線相似,且於培養48小時可達菌體生長之對數期,此時其發酵液的TCA-可溶性蛋白質含量增加,其中以YK-3的鴨蛋白發酵液(DEWHF)的TCA-可溶性蛋白質含量最高,且具有最佳的亞麻油酸自氧化抑制率(32.48%),並於電泳分析中亦可發現YK-3之DEWHF在25-37 kDa處的蛋白質增加,此顯示YK-3對鴨蛋白的利用能力優於其它試驗菌株,同時其發酵液亦具有較佳的抗氧化性。 進一步將YK-3菌株利用凍融法(frozen-thawed)、球磨法及超音波震碎法進行破菌處理以萃取酵素。球磨法處理420秒無益於破碎率之提昇,利用凍融法則YK-3破碎率為22.62%,若併用超音波震碎600秒處理,破碎率可大幅提昇至55.06%。而萃得之YK-3細胞壁蛋白酶活性約為胞內酵素的33.2倍,顯著優於胞內酵素(P<0.05),其最適作用條件為60℃及pH 6.0,屬酸性蛋白酶。再者,YK-3細胞壁蛋白酵素具有aminopeptidase的活性最高(252 nM/min),tripeptidase (58.1 nM/min)及endopeptidase (51.6 nM/min)次之,dipeptidase的活性則最低(21.3 nM/min)。 以 YK-3之細胞壁粗酵素(CWE)應用於鴨蛋白之水解,其最佳添加量為3%,而其水解液(DEWHcwe)之亞麻油酸自氧化抑制率為52.35%高於YK-3之DEWHF (32.48%)。在CWE的穩定性測試方面,CWE重覆使用1次之DEWHcwe 的TCA-可溶性蛋白減少約30%,重覆使用2次則幾乎不具有鴨蛋白的水解能力,而此亦反應在其抗氧化性的測定上,即CWE 重覆使用1次之DEWHcwe仍具有37.1%的亞麻油酸自氧化抑制率,略高於YK-3之DEWHF (32.48%)相當,CWE重覆使用2次之DEWcwe則不具抗氧化性。
In this study, we try to promote the value of duck egg by developing the duck egg white (DEW) product with antioxidative activity. The experiment tasks include that DEW was hydrolyzed using commercial enzymes (papain, trypsin, chymotrypsin, alcalase, and flavourzyme), yeasts with proteolysis activity which isolated from kefir and stinky tofu, the extraction of enzymes from screened yeasts and the protease activities assessed, then the proteases from screened yeasts were applied to hydrolyze DEW and evaluate their antioxidative activities. Duck egg white (DEW) hydrolysates were prepared by five enzymes (papain, trypsin, chymotrypsin, alcalase, and flavourzyme) and investigated their antioxidative activities. DEW was hydrolyzed with papain (DEWHP) had the highest peptides contents among the five enzymatic treatments. Besides, the higher enzyme to substrate ratio (E/S ratios) contributed to elevate the hydrolysis degree of DEW effectively. Similar results were also discovered in Tricine-SDS-PAGE. In antioxidative properties, the DEWHP showed more than 70% of inhibitory activity on the linoleic acid peroxidation and superoxide anion scavenging. Moreover, the Fe2+ chelating effects of DEWHP could be up to 90%, while no significant difference were observed in DPPH (α,α-diphenyl-β-picrylhydrazyl) radicals scavenging and reducing ability. The results suggested that higher peptides contents benefited to possess the strong antioxidative activity in DEWHP. In the yeasts isolated from kefir, four colonies (YK-1~YK-4) with various morphologies were screened and all of them were identified as Candida kefyr by RapID Yeast Plus System. Furthermore, the yeasts (YK-1~YK-4) exhibited strong activities of valine arylamidase and cystine arylamidase, meanwhile their phosphatase and galactosidase were noticed in the enzymatic assays using API ZYM system. Compared with control treatment (the casein solution without fermentation), the casein solutions fermented with the yeasts (YK-1~YK-4) showed lower levels of TCA-soluble protein and higher levels of free amino acid. The results of this work suggested that the small peptide fragments in casein solution could be metabolized and hydrolyzed into free amino acids by screened yeasts. The hydrolysates of duck egg white fermented by Candida kefyr strains (YK-1, YK-2, YK-3, YK-4) screened from kefir milk were prepared and their antioxidative activities were investigated. Results showed that all test strains were similar in growth curves and cells grew to stationary phase at 48 hours of incubation in DEW. TCA-soluble protein contents of duck egg white hydrolysates fermented (DEWHF) by yeasts were higher than unfermented treatment. The TCA-soluble protein content of DEWHF by YK-3 was the highest among all strains. In SDS-PAGE protein profiles, there were no obvious different in all strains treatments except DEWHF by YK-3 found the intensity increasing slightly of bands between 25-37 kDa. In antioxidative activities, the peroxidation inhibition of linoleic acid emulsion system in all strains treatments were observed and DEWHF by YK-3 had the best (inhibitory activity was 32.48%). Taken together, YK-3 was most efficient in DEW utilization among all test strains and its DEWHF possessed the strongest antioxidative activity. Further study, the extraction of enzyme from YK-3 strain was carried out in cell disruption using frozen-thawed, bead mill and sonication methods. Results demonstrated that 420 seconds bead milling for cell disruption was in vain. The disruption rate was 22.62% using frozen-thawed method, and the distruption rate was promoted sharply to 55.06% by combination of frozen-thawed and sonication methods. In protease activities, the protease activity from cell wall of YK-3 was 33.2-folds than that of cytoplasm. It was revealed that cell wall enzyme exhibited noticeably higher than cytoplasm. Optimal activity of cell wall protease exhibited at 60℃ and pH 8.0. It belongs to an acid protease and possesses aminopeptidase (252 nM/min), tripeptidase (58.1 nM/min), and endopeptidase (51.2 nM/min) in sequence. Optimal dose of cell wall enzyme (CWE) from YK-3 was 3% for DEW hydrolysis. The peroxidation inhibition of linoleic acid emulsion system in DEW hydrolysates prepared by CWE hydrolysis (DEWHcwe) was 52.3%, which was higher than that of DEWHF by YK-3 (inhibitory was 32.48%). In CWE stability test, TCA-soluble protein of DEWHcwe by re-used once CWE was 30% off comparing with DEWHcwe by fresh CWE. Re-used twice CWE was few ability for DEW hydrolysis, which was same trend in antioxidative activity. DEWHcwe by re-used once CWE still exhibited 37.1% inhibitory on the linoleic acid peroxidation, that was slight more than DEWHF by YK-3 (32.48%), and DEWHcwe by re-used twice CWE did not possess antioxidative activity.
URI: http://hdl.handle.net/11455/25264
其他識別: U0005-2807200906284600
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