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標題: 放線菌5012之角蛋白酶及不同前處理(20%酒精及0.05M NaOH)對雞腳皮膜分解效果之研究
Study of Keratinase from Actinomycetes 5012 and Pre-treatment (20% alcohol and 0.05M sodium hydroxide) on Digestive Efficiency of Chicken Scutate Scales
作者: 陳碩文 
Chen, Shou-Wen 
關鍵字: 腳皮膜;scutate scale;角蛋白;放線菌;keratin;Actinomycetes
出版社: 動物科學系所
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腳皮膜(Scutate scales)為貼附於鳥類腳部表皮的一層附屬物,功能為保護與隔絕外在環境。文獻指出腳皮膜係由表皮細胞凋亡後,由角蛋白所組成的鱗狀多層次結構,其中具有含大量脂質之液胞。隨著家禽業的發達,角蛋白之廢棄物因結構穩定而難以分解,而造成嚴重的汙染問題。過去之報告多探討如何處理及應用羽毛、豬毛…等角蛋白廢棄物於動物飼料來源,並無報告可指出雞腳皮膜之處理方式及應用。因此,本研究將分為兩部分進行,第一部分先探討雞腳皮膜之一般化學組成,再以雞腳皮膜作為培養基質,與本研究室所分離之放線菌5012進行培養,藉以誘導提升其角蛋白酶之活性並尋求其最佳作用的受質酵素比;第二部分則先以酒精(20 %)與氫氧化鈉(0.05 M)做不同前處理,再與放線菌5012所萃取之粗酵素液進行培養14天,再藉由雞腳皮膜經酵素處理後其溶液中可溶性蛋白質、受質之可消化蛋白與掃描式電子顯微鏡顯微結構觀察等結果來評估其對雞腳皮膜的分解效果。第一部分結果顯示:一般化學組成方面,雞腳皮膜以20 %酒精處理組有最高之粗蛋白含量,氫氧化鈉處理組在灰分上有顯著較其他組別為高,而所有處理組皆有約20%之粗脂肪含量,顯示雞腳皮膜豐富的脂質含量。另外,以2%雞腳皮膜作為培養基質與放線菌5012進行培養,於第七天則有最高之角蛋白酶活性(29.0 U),顯示適量雞腳皮膜的添加具有提升放線菌5012分泌角蛋白酶的效果。在受質酵素比為100:1時,培養液中可測得最高的可溶性蛋白,此結果亦顯示此為最佳的受質酵素作用比例。乾燥及酒精之前處理組於放線菌5012粗酵素處理14天後能顯著提升雞腳皮膜之可消化蛋白質,其中又以乾燥後樣品經14天處理組有最高可消化蛋白質(53.72%)。掃描式電子顯微鏡顯微結構觀察中,顯示鹼前處理之雞腳皮膜表面有蛋白質碎片沉積的結果,顯示鹼處理對雞腳皮膜結構會產生破壞;而各處理組的雞腳皮膜在經過放線菌5012粗酵素處理14天後,外觀皆有明顯的分解情形,顯示放線菌5012所產的角蛋白酶對雞腳皮膜具有高度分解效果。綜觀上述,乾燥雞腳皮膜經放線菌5012粗酵素處理14天之固形物有最高的可消化蛋白質(53.72%)。若先以鹼處理36小時後再經放線菌5012粗酵素處理七天則培養液中有最高之蛋白態可溶性蛋白質;另外亦顯示,先以鹼處理36小時後經放線菌5012粗酵素處理十四天則培養液中有最高之總蛋白質含量。因此,雞腳皮膜可藉由不同前處理及配合放線菌5012粗酵素處理進而提升其可消化蛋白質及可溶性蛋白質等效果以增加其未來在動物飼料的利用價值。

Scutate scale is a layer on epidermis of avian that protected and isolated from outer environment. Scutate scales came from apoptosis of epidermis cells and became a scaly structure with mounts of lipid. With the development of poultry industry, wastes composed of keratin are rapidly accumulated and resulted in a serious pollution problem on environment. In the past, using keratin wastes as feedstuff had been reported and generally focused on poultry feather and pig hair. No report or information about chicken scutate scales was evidenced to apply in animal feedstuff. However, chicken scutate scales were not only composed of high protein also contained aboundant amount of lipid. Therefore, the objective of this study was divided into two parts, the first part was to evaluate the protease and keratinase activity of Actinomycetes 5012 was incubated with chicken scutate scales. The second part was use of 20% alcohol, 0.05M sodium hydroxide for 24 and 36 hours as pretreatment then treated by different ratio of enzyme and substrate (raw keratinase from Actinomycetes 5012 / chicken scutate scales) and tried to obtain the optimum condition depended on soluble protein in solution and digestive protein in chicken scutate scales. In the chemical compositions, chicken scutate scales with 20% alcohol pretreatment had the highest protein content and both of chicken scutate scales with sodium hydroxide pretreatments also had higher ash value than the others. However, all samples showed high mount of ether extract (about 20%) and this result indicated the lipid in chicken scutate scales was belonged to wax which was high molecular weight in fat. The results of the first part were showed that Actinomycetes 5012 and 2% scutate scale in substrate had the highest keratinase activity (29.0 U) at day 7 during incubation. The highest soluble protein of the ratio of enzyme and substrate was found in 100:1 and the value was . The digestive protein of dry chicken scutate scales and 20% alcohol pretreatment increased when treated with enzymes during incubation. Dry chicken scutate scales treated with enzyme for 14 days had the highest digestive protein (53.72%) in all treatments. Nevertheless, chicken scutate scales treated with sodium hydroxide had higher soluble protein in solution but lower digestive protein existed in substrate after enzyme treatment. In the scanning electron microscope observation, some protein fractions were found on the suface of chicken scutate scales with alkine and enzyme treatment for 14 days. Also, some significantly destroied traces were exhibited on the surface of chicken scutate scales in all treatment after incubating with raw enzymes from Actinomycetes 5012 in this research. In conclusion, use of raw enzymes from Actinomycetes 5012 and alkine pretrement was an effiecient performing method to increase the digestive protein and soluble protein of chicken scutate scales and also to elevate added value of chicken scutate scales in animal feed in the future.
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