TWI608840B - 卵繫帶水解物及其製備方法與用途 - Google Patents
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Description
本發明為一種卵繫帶水解物及其製備方法與用途,尤其係指一種以特定方法製備之卵繫帶水解物,可有效降低肝臟脂肪堆積以及肝臟氧化壓力。
雞蛋是一種便宜又具備高營養價值的食物,能被應用於眾多種類食品的製備,已是生活中不可或缺的食材;液蛋(liquid eggs)為一種去殼蛋品,將雞蛋打破後取出內容物,並依目的分成全蛋、蛋黃及蛋白液,以取代傳統的粒蛋,且液蛋的製造過程中會經過低溫殺菌,在使用上更為安全。卵繫帶(chalaza)是全蛋的組織之一,其功能為固定卵黃,使卵黃位於蛋的中央;卵繫帶富含多種蛋白質,但卵繫帶在液蛋製備的過程中被視為廢棄物而丟棄,十分可惜。
食源性蛋白質水解物係指將含有蛋白質之食品水解後所產生的水解物,這些水解物含有不同的胺基酸或多肽,有些甚至具有保健功能。例如日本專利號JP 5735734(B)號專利係為一種乳清蛋白的水解物,含有分子量小於10kDa以下的多肽或游離胺基酸,具有
改善脂質代謝的功效。中華民國專利TW I425004(B)號專利係一種具有控制體重的豬肝水解物,以酵素水解豬肝後,獲得的產物可以刺激膽囊收縮素分泌而抑制食慾,進而達到降低體重的功效。另外,中華民國專利TW I437999(B)號專利為一種具抗氧化功能之寡糖肽(oligosaccharide peptides),係將菇蕈類植物(例如樟芝)的粗萃物進行水解以獲得具抗氧化功能之寡糖肽。但是原料的種類,以及水解的步驟都會影響到最終產水解物所具有的特性。
今,發明人有鑑於現今富含蛋白質的卵繫帶並無獲得良好的利用,於是乃一本孜孜不倦之精神,並藉由其豐富專業知識及多年之實務經驗所輔佐,而加以改善,並據此研創出本發明。
本發明係有關於一種卵繫帶水解物,其包含90~120毫克/克(mg/g)的游離胺基酸,其中游離胺基酸係包含10~20wt%白胺酸(leucine)、7~14wt%精氨酸(arginine)、8~10wt%苯丙胺酸(phenylalanine)、8~10wt%纈胺酸(valine)以及8~10wt%離胺酸(lysine)。
本發明係有關於一種卵繫帶水解物的製備方法,包含,步驟一:將卵繫帶解凍後利用去離子水洗去雜質,並離心以保留下層之第一產物;步驟二:將第一產物以95℃恆溫加熱10~30分鐘並冷卻,再加入去離子水均質以獲得卵繫帶均質液;步驟三:取100~200克之卵繫帶均質液與水解酵素以酵素受質比1:100~1:500(w/w)混合,水解一段時間之後獲得第一水解液;步驟四:將第一水解液
以95℃恆溫加熱10~30分鐘並冷卻,離心後保留上層之第二水解液;步驟五:過濾並凍乾第二水解液,以獲得卵繫帶水解物。
本發明係有關於一種卵繫帶水解物於製備保護肝臟組成物之用途,係以一有效劑量施予所需個體,以減少脂肪堆積於肝臟或降低肝臟氧化壓力,達到肝臟保護之功效;其中卵繫帶水解物包含90~120毫克/克(mg/g)游離胺基酸,且游離胺基酸包含10~20wt%白胺酸(leucine)、7~14wt%精氨酸(arginine)、8~10wt%苯丙胺酸(phenylalanine)、8~10wt%纈胺酸(valine)以及8~10wt%離胺酸(lysine)。
於本發明之一實施例中,卵繫帶水解物含有15.47%白胺酸,10.89wt%精氨酸,9.74wt%苯丙胺酸,9.64wt%纈胺酸,以及9.09wt%離胺酸。
於本案之一實施例中,卵繫帶水解物進一步包含肌肽(carnosine)以及甲肌肽(anserine)其中之一或兩種以上之組合。
於本發明之一實施例中,肌肽為20~60mg/100g且甲肌肽為95~150mg/100g。
於本案之一實施例中,水解酵素係選自胃蛋白酶(pepsin)、蛋白酶A(protease A)以及保解蛋質素(prozyme 6)至少其中之一者,且酵素受質比為1:200(w/w),作用時間係為30分鐘。
於本案之一實施例中,水解酵素係為蛋白酶A(protease A)。
於本案之一實施例中,脂肪為三酸甘油酯(triglyceride)或總膽固醇(total cholesterol)。
藉此,本案透過一種特定製備方法獲得之卵繫帶水解物,用以降低肝臟脂肪堆積以及降低肝臟氧化壓力,以達到肝臟保護之用途。
第一圖:相同酵素受質比製備之卵繫帶水解物體外抗氧化分析圖。
第二圖:以等量活性的酵素製備之卵繫帶水解物體外抗氧化分析圖。
第三圖:不同酵素受質比之蛋白酶A卵繫帶水解物體外抗氧化分析圖。
第四圖:卵繫帶水解物胜肽含量以及產率分析圖。
第五圖:卵繫帶水解物SDS-PAGE分析圖。
第六圖:小鼠肝臟切片染色分析圖。
第七圖:小鼠肝臟及糞便脂質含量分析圖。
第八圖:小鼠肝臟脂質代謝相關基因表現分析圖。
第九圖:小鼠血清與肝臟抗氧化能力與脂質過氧化情況分析圖。
第十圖:小鼠肝臟抗氧化物質含量分析圖。
第十一圖:小鼠肝臟發炎相關細胞激素含量分析圖。
為令本發明之技術手段其所能達成之效果,能夠有更完整且清楚的揭露,茲詳細說明如下,請一併參閱揭露之圖式:本發明係有關於一種卵繫帶水解物,其包含90~120mg/g的游離胺基酸,其中游離胺基酸係包含10~20wt%白胺酸(leucine)、7~14wt%
精氨酸(arginine)、8~10wt%苯丙胺酸(phenylalanine)、8~10wt%纈胺酸(valine)以及8~10wt%離胺酸(lysine);此外,卵繫帶萃取物進一步包含肌肽(carnosine)以及甲肌肽(anserine)其中之一者,其中肌肽為20~60mg/100g且甲肌肽為95~150mg/100g。
本發明之卵繫帶水解物係以下列方法製得:步驟一:將一卵繫帶解凍後利用去離子水洗去雜質,並離心以保留下層之第一產物;步驟二:將第一產物以95℃恆溫加熱10~30分鐘並冷卻,再加入去離子水均質以獲得一卵繫帶均質液;步驟三:取100~200g之卵繫帶均質液與一水解酵素以酵素受質比1:100~1:500(w/w)混合,水解一作用時間之後獲得一第一水解液;步驟四:將第一水解液以95℃恆溫加熱10~30分鐘並冷卻,離心後保留上層之第二水解液;步驟五:過濾並凍乾第二水解液,以獲得卵繫帶水解物。其中,水解酵素為胃蛋白酶(pepsin)、蛋白酶A(protease A)以及保解蛋質素(prozyme 6)至少其中之一者,且酵素受質比為1:200(w/w),該作用時間為30分鐘。
本案之卵繫帶水解物可用於製備保護肝臟組成物,以一有效劑量施予一所需個體,可降低肝臟脂肪堆積或降低肝臟氧化壓力,以達到肝臟保護的功效;其中卵繫帶水解物係包含90~120mg/g游離胺基酸,其中游離胺基酸係包含10~20wt%白胺酸(leucine)、7~14wt%精氨酸(arginine)、8~10wt%苯丙胺酸(phenylalanine)、8~10wt%纈胺酸(valine)以及8~10wt%離胺酸(lysine)。其中,卵繫帶水解物所降低之肝臟脂肪種類包含三酸甘油酯(triglyceride)或總膽固醇(total cholesterol)。
此外,藉由下述具體實施例,可進一步證明本發明可實際應用之範圍,但不意欲以任何形式限制本發明之範圍。
本實驗所使用之卵繫帶(crude chalaza)由大毅蛋品有限公司(Daiegg Co.Ltd.,Tainan city,Taiwan)提供,每天於液蛋生產線上收集後於-18℃冷凍儲存,再冷凍宅配至國立臺灣大學動物科學技術學系保健畜產品暨肉品研究室,並保存於-20℃冰櫃中。卵繫帶經過解凍、去除雜質與水洗過後,以轉速900G的條件下離心15分鐘,收集下層之卵繫帶,再放入恆溫循環水浴槽以熱水95℃水浴15分鐘,以去除卵繫帶內含的酵素活性,水浴後將樣品置於冰上並冷卻至室溫。於冷卻樣品中加入兩倍重量之去離子水(Distilled deionized water,ddH2O)並以均質機均質1分鐘後以獲得一卵繫帶均質液,將卵繫帶均質液靜置於4℃冰箱以去除均質過程中產生的泡沫。
取150g之卵繫帶均質液(其中包含50g卵繫帶以及100g之去離子水),將卵繫帶均質液的酸鹼值與溫度調整至酵素的理想作用環境,使用的酵素包含胃蛋白酶(pepsin,簡稱PE)、蛋白酶A(protease A,簡稱PA)以及保解蛋質素(prozyme 6,簡稱P6),三種酵素的特性請參見表一。當卵繫帶均質液的溫度與酸鹼值達到各酵素的理想作用條件後,以酵素受質比(ratio of enzyme to substrate)1:100~1:500(w/w)的比例與卵繫帶均質液混合並進行水解反應;水解時間共進行4小時,並在開始水解後0.5、1、1.5、2、2.5、3與4小時收集水解液,並以95℃加熱15分鐘以終止水解反應;冷卻後以900G、4℃之條件離心15分鐘:離心後取上層水解液,以55mm濾紙
過濾後並將濾液酸鹼值調整至pH=7.0,將濾液進行冷凍乾燥以獲得卵繫帶水解物;將卵繫帶水解物保存於-20℃,並進行後續清除自由基能力的分析,分析結果將作為卵繫帶水解物製備方法的選擇依據;為區別上述三種酵素製備之卵繫帶水解物,以PE水解物、PA水解物或P6水解物稱之。
DPPH(2,2-diphenyl-1-picrylhydrazyl)為一種會產生紫羅蘭色的自由基(peroxyl radical),其乙醇溶液在517nm下會有強吸收波峰,若受測物具有抗氧化能力時,可提供氫離子以還原DPPH導致517nm之吸光值(簡稱OD517值)降低,因此若517nm的吸光值愈低即表示受測物的供氫能力越高、抗氧化能力越強。其實驗步驟為:在96孔盤中加入200μL之卵繫帶水解物配置之溶液與100μL之1mM DPPH乙醇溶液,混合均勻並靜置30分鐘後,以酵素免疫分析測讀儀偵測其在517nm波長下之吸光值。此實驗需全程避光,並使用1mg/mL二丁基羥基甲苯(BHT)乙醇溶液與95%乙醇分別作為陽性對照組(positive control)以及陰性對照組(negative control)之樣品,並與卵繫帶水解物之清除DPPH自由基能力做比較;DPPH清除率的
計算公式為:清除率(%)=(1-受測物OD517值/陰性對照組OD517值)X 100%
ABTS[2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)]在過氧化氫(H2O2)及過氧化氫酶(peroxidase)的反應下會生成藍綠色之ABTS+自由基,於波長734nm下有強吸收波峰。當受測物具有抗氧化能力時可抑制顏色的產生,故使吸光值降低;檢測方法為:混合5mL之ABTS(1mM)、5mL過氧化氫酶(44U/mg)、5mL之10%過氧化氫與30mL之去離子水,避光反應60分鐘;取200μL之上述反應液與20μL之受測物,混合並靜置10分鐘,於734nm波長下測定其吸光值;另以不同濃度的維生素E類似物Trolox進行上述反應以繪製出標準曲線,再將受測物之734nm吸光值與標準曲線進行比對,以計算總抗氧化能力(trolox equivalent antioxidant capacity,TEAC),單位為每毫克(mg)水解物含有多少Trolox當量(TE)。
具有抗氧化力之物質可將帶三價鐵離子的鐵氰化鉀(K3[Fe(CN)6])還原為帶二價亞鐵離子的亞鐵氰化鉀(K4[Fe(CN)6].3H2O)後,可進一步與氯化鐵(FeCl3)反應形成普魯士藍(prussian blue),於波長700nm具有最強吸收波峰;藉由測定普魯士藍的含量,可得知受測物的還原力,若受測物吸光值越高即代表還原力越佳。將卵繫帶水解物溶液500μL加入,250μL之0.2M磷酸鹽緩衝液(phosphate buffered saline)與250μL之1%鐵氰化鉀水溶液,混合均勻後置入50℃恆溫水浴槽中反應15分鐘,再取出置於冰上迅速冷卻至室溫;再加入250μL之10%三氯醋酸(trichloroacetic acid,TCA)水溶液,
混合均勻後離心,並抽取500μL上清液,加入250μL去離子水與500μL之0.1%氯化鐵水溶液,混合均於後靜置10分鐘,並測量700nm下之吸收波長,以1mg/mL BHT乙醇溶液作為陽性對照組(positive control)之樣品,與卵繫帶水解物之還原力做比較。
金屬離子會促進氧化反應,進而增加脂質過氧化的機率,在多種金屬離子中,Fe2+經常是最具影響力的助氧化劑,少量的Fe2+便可產生自由基並加速脂質氧化的進行。在抗氧化的研究上常利用Fe2+與菲囉勅(ferrozine)結合後的複合物在562nm之呈色反應,可測得樣品對Fe2+離子的螯合能力。當受試樣品與Fe2+螯合時,便會減少複合物的產生並減少562nm下的吸收波峰。取250μL不同濃度的樣品及控制組,加入3.7mL甲醇,再加入0.1mL之2mM FeCl2,30秒後加入0.2mL之5mM菲囉勅,反應10分鐘後檢測562nm的吸光值,並使用以二胺四乙酸(EDTA)水溶液與去離子水分別作為陽性對照組(positive control)與陰性對照組(negative control);562nm吸光值越低表示樣品螯合亞鐵離子的能力越強。亞鐵離子螯合率之計算公式為:螯合率(%)=[1-(受測物562nm吸光值/陰性對照組562nm吸光值)]X 100%
實驗數據以變異數分析(analysis of variance,ANOVA)進行統計分析,再以最小顯著差異法(least significant difference)方法比較各組之間差異;在分析圖表的呈現上,若兩組之間沒有顯著差異而分類在同一組,則會以相同之小寫英文字母標記,若有些組別被標記為「ab」,表示該組別與「a」組無顯著差異,與「b」組亦無顯著差異,故同時被分類在「a」組與「b」組。
參見第一圖,以相同酵素受質比(1:200)的三種酵素製備之卵繫帶水解物清除DPPH自由基,相對於BHT能力之分析圖;未水解的卵繫帶(水解時間為0小時者),DPPH自由基的清除率為35.12%,但經過三種酵素水解的卵繫帶水解物,隨著水解時間增加,獲得之卵繫帶水解物清除DPPH自由基的能力顯著提升。在相同水解時間的前提下,PE水解物的DPPH清除率最佳,且其能力隨著水解時間而提升,水解兩小時獲得之PE水解物的自由基清除率最高,為90.14%。而水解0.5小時之PA水解物與P6水解物的DPPH自由基清除率最高,PA水解物的清除率為56.3%、P6水解物之清除率45.24%。
第一圖中,未水解之卵繫帶(水解時間為0小時)清除ABTS+之能力只有0.79μmole TE/mg-卵繫帶原料,歷過三種酵素處理後,皆顯著提高卵繫帶水解物清除ABTS+之能力。在反應時間0.5小時後,各組內之的ABTS+清除能力便達高峰,當中又以P6水解物擁有最佳之清除能力,在反應0.5小時後其清除能力便為2.91μmole TE/mg-卵繫帶水解物,其次為PA水解物之2.60μmole TE/mg-卵繫帶水解物,而PE水解物之ABTS+清除能力顯著低於其餘兩組(p<0.05),其清除能力只有1.20μmole TE/mg-卵繫帶水解物。
還原力分析的實驗中,水解前的卵繫帶在700nm的吸光值只有0.05,然而三種酵素處理所得之卵繫帶水解物的還原力均有提升:PA水解物還原能力最強,在水解後0.5小時後吸光值即達0.13,顯著高於其餘兩組(p<0.05);雖然PE水解物與P6水解物還原能力相似,在水解2.0小時達最大值,但只有0.06,表現皆不佳。
因為各種酵素的活性並不相同,本案選用的PE活性為3,000,000unit/g,PA活性為63,000unit/g,以及P6的活性為748,000unit/g,因此上述實驗使用相同酵素受質比為1:200(w/w)的作用條件,實際上加入的活性酵素單位並不相同;為使試驗更加客觀,故使用相同活性之酵素水解卵繫帶,並將所得之卵繫帶水解物再次進行體外抗氧化活性分析;本試驗使用的酵素活性含量為795unit/g-卵繫帶原料,水解時間為0.5小時。
如第二圖所示,在DPPH自由基的清除率上,相對於BHT,PE水解物有16.93%清除率、PA水解物有29.25%,以及P6水解物為3.46%之清除率。在ABTS+自由基清除的實驗結果中,PA水解物的清除力最佳,為1.81μmole TE/mg-水解物,其次為P6水解物之1.15μmole TE/mg-水解物,最末為PE水解物的0.89μmole TE/mg-水解物,代表在相同酵素活性(795unit/g-卵繫帶原料)下,PA水解物清除ABTS+自由基的能力最佳。還原力測試的結果中,PA水解物與P6水解物的還原力皆比PE水解物佳。
如第二圖所示,相對於EDTA,PA水解物與P6水解物的螯合亞鐵離子能力分別為56.60%以及57.96%,而PE水解物則為46.69%,顯著低於PA水解物與P6水解物(p<0.05)。
蛋白酶A(protease A)為一種兼具外肽酶(exopeptidase)與內切蛋白酶(endoproteases)功效的酵素,在分析以上關於三種卵繫帶水解物於體外抗氧化能力測試的結果,PA水解物在清除DPPH自由基、清除ABTS+自由基、螯合亞鐵離子或還原力的測試中皆有良好表現,因此最終選定以蛋白酶A作為後續製備卵繫帶水解物時使用的水解酵素。
為進一步確定本案卵繫帶水解物製備方法中,酵素與受質的最佳比例,便將酵素受質比為1:100~1:500製備之PA水解物進行體外抗氧化能力測試。如第三圖所示,酵素受質比1:100之PA水解物清除DPPH自由基之清除率為38.24%,隨著酵素受質比下降,即1:200、1:300、1:400以及1:500之PA水解物,其DPPH自由基清除率亦會下降,依序為26.47%、23.71%、23.38%、以及8.99%;在ATBS+自由基清除實驗中亦有相同的現象,其ATBS+自由基清除能力,分別為1.90、1.87、1.85、1.80、以及1.63μmole TE/mg-PA水解物;還原力測試結果亦與DPPH或ABTS+自由基清除結果相似,酵素受質比最高者(1:100)具有最佳的還原力,其700nm吸光質為0.094,當酵素受質比下降時,其吸光質依序下降,依照酵素受質比高至低排列分別為0.067、0.036、0.027以及0.026。然而,在亞鐵離子螯合測試中,五種PA水解物的螯合率相差不大,依照酵素受質比高至低排列分別為47.14%、46.78%、47.80%、47.95%、以及47.47%。
卵繫帶水解物的胜肽含量,可用以評估酵素水解能力之高低,與推測受測物中胜肽的含量。利用鄰苯二甲醛(o-phthalaldehyde;OPA)、2-巰基乙醇(β-mercaptoethanol,β-ME)與一級胺(primary amine)結合後產生的藍色的螢光物質在340nm下會有最強吸收波峰,作為評估樣品中胜肽含量之指標。首先取40mg之OPA粉末溶於1mL甲醇,將其加入25mL之100mM之四硼酸鈉(sodium tetraborate decahydrate)、2.5mL之20%十二烷基硫酸鈉(SDS)與100μL之β-ME,混合均勻後加入去離子水並定量至50mL,即為OPA試劑。取10μL不同濃度之甘胺酸-甘胺酸(glycine-glycine)水溶液
(100、50、25、12.5、10、1mg/mL)與200μL之OPA試劑,加入96孔盤後反應兩分鐘,立即測量340nm下之吸光值並做出標準曲線,再將經適當稀釋的受試樣品用上述方法加入OPA試劑進行反應,對照標準曲線後即可推算出胜肽含量。
請參見第四圖,未水解之卵繫帶胜肽含量很低,為6.42mg/g;經過PA水解後,PA水解物中的胜肽含量明顯增加,又以酵素受質比1:100之PA水解物含量最高,為778.65mg/g,當酵素受質比下降時,獲得之PA水解物之胜肽含量亦隨之下降;產率分析的結果中,在水解0.5小時的條件下,五種酵素受質比所製備之卵繫帶水解物,產率依照酵素受質比由高至低排列分別為:5.37%、5.35%、4.83%、3.99%、以及4.04%;其中產率的計算公式為:產率(%)=卵繫帶水解物重量(g)/卵繫帶重量(g)X 100%。
根據實驗二與實驗三之結果,以蛋白酶A(protease A)、酵素受質比1:100進行水解0.5小時,可製備出含有高量胜肽以及具較佳抗氧化能力之卵繫帶水解物。
利用SDS-PAGE,分析卵繫帶水解物的蛋白質水解程度以及蛋白質片段大小的分布。將受測物與分子量標準品(marker)注入以十二烷基硫酸鈉-聚丙烯醯胺製備之凝膠的樣品槽中並進行電泳,電泳完成後將凝膠置入含有0.25%考馬斯亮藍(coomassie brilliant blue)的染劑中進行染色,再以退染劑(含有10%醋酸與50%甲醇之水溶液)退染,並將凝膠照相。
如第五圖所示,未水解之卵繫帶可偵測得多種較大片段的蛋白質(如箭頭所指之處);而卵繫帶以蛋白酶A水解後,其PA水解物中50kDa以上的蛋白質片段幾乎消失,但可額外偵測到14kDa以下的片段,由此結果可確定卵繫帶在經蛋白酶A水解後,大片段的蛋白質皆會被水解。
本實驗委託財團法人食品發展工業研究所進行檢測,主要原理參考Konosu等人在1974年的方法,利用三氯乙酸(trichloroacetic acid,TCA)萃取所需之樣品。將5g的受測物與20mL之7% TCA溶液混合並靜置2分鐘後再以4000G、4℃離心20分鐘,上清液以濾紙過濾,並以7% TCA溶液調整體積至100mL以獲得一TCA混合液;取40mL之TCA混合液加入等量乙醇以洗去TCA,再減壓濃縮將水層去除,最後以去離子水定量至25mL,即成為TCA可溶性抽出物。以0.02N HCl適當稀釋1mL的TCA可溶性抽出物,並以0.2μm膜過濾後,以胺基酸分析儀(Hitachi L8800 amino acid analyzer,Hitachi High-Technologies Co.,Tokyo,Japan)分析之。
參見表二,為未經水解之卵繫帶與PA水解物的游離胺基酸及雙胜肽含量分析表;結果顯示經適當水解後,游離胺基酸由卵繫帶的39.14mg/100g提高至PA水解物的10,827.77mg/100g,且PA水解物的組成以白胺酸(leucine)、精胺酸(arginine)、苯丙胺酸(phenylalanine)、纈胺酸(valine)、以及離胺酸(lysine)為主,並佔總游離胺基酸之15.47wt%、10.89wt%、9.74wt%、9.64wt%、以及9.09wt%;此外,甲肌肽(anserine)以及肌肽(carnosine)的含量也從水解前的未檢出至水解後各別有48.06mg/100g以及106.84mg/100g,證
明本研究所使用之最適條件可大幅提昇水解物中游離胺基酸以及短鏈胜肽的含量。
由臺大醫院實驗動物中心購入的18隻八週齡雄性C57BL/6小鼠,飼養於環境控制在22±2℃、濕度60~80%、明暗週期12小時的動物房中。18隻小鼠隨機分為三組,分別為:(1)對照組(CON):給予正常流質飼糧並任食,每日管灌0.1mL生理食鹽水;(2)酒精組(ALC):給予Lieber-DeCarli酒精流質飼糧並任食,每日管灌0.1mL生理食鹽水;以及(3)卵繫帶水解物組(ALC+PA水解物):給予Lieber-DeCarli酒精流質飼糧並任食,每日管灌0.1mL卵繫帶水解物溶液(100mg PA水解物/kg-體重)。
表三為正常流質飼料與Lieber-DeCarli酒精流質飼料的配方組成:
在小鼠移入動物房一週適應期後開始進行為期8週的試驗,並於第9週將18隻小鼠犧牲,收集血液、肝臟與糞便樣品為後續分析之樣品。在犧牲前一週採集小鼠糞便,以烘箱烘乾保存;實驗結束時前一晚將飼料移除,禁食八小時後以毛細管採血針採血,檢體靜置於冰上一小時後以2500G、4℃之條件離心10分鐘,此步驟重複後取得小鼠之血清;犧牲時記錄小鼠心臟、肝臟、腎臟與腹部脂肪的重量。所有採集的樣品包括血清、糞便與臟器都保存在-80℃冰箱中供後續分析使用。
此研究使用完全隨機設計(Completely Randomized Design),所有數據皆以SAS9.2軟體處理,並以一元變方分析(one way analysis of variance)進行統計分析,當組間差異性顯現時(p<0.05),再以最小顯著差異法(least significant difference)方法比較各組之間差異,實驗之數據結果以平均值±標準差(mean±SD)。在分析圖呈現上,若兩組之間沒有顯著差異而分類在同一組,則會以相同之小寫英文字母標記,若有些組別被標記為「ab」,表示該組別與「a」組無顯著差異,與「b」組亦無顯著差異,故同時被分類在「a」組與「b」組。
於犧牲小鼠時,秤量小鼠的體重、臟器重量、腹腔脂肪墊重量,並將臟器重量或脂肪墊重量除以小鼠體重,以相對臟器重量(relative size of organs/100g-體重)表示,結果請見表四。實驗第一週剛分籠時,小鼠體重並無顯著差異(p>0.05,數據未顯示),實驗進行八週後,ALC組別小鼠之平均體重為23.86g,跟CON組想比有增高之趨勢;然而在補充PA水解物之組別,其小鼠的最終體重相較於ALC組小鼠卻顯著地下降(p<0.05),平均為19.73g,並且與CON組小鼠間沒有顯著差異(p>0.05);相對臟器重量的分析中,三組間小鼠的心臟與腎臟之相對重量並無顯著差異(p>0.05),但ALC組小鼠的肝臟與腹部脂肪墊卻明顯較CON組小鼠重(p<0.05),而補充PA水解物後,與ALC組相比有減少肝臟重量的趨勢,亦顯著地減少腹部脂肪墊之相對重量(p<0.05)。
肝臟中富含天門冬胺基轉移酶(asparate aminotransferase,AST)、丙胺酸胺基轉移酶(alanine aminotransferase,ALT)以及鹼性磷酸酶
(alkaline phosphatase,ALKP),在肝臟細胞受損時,AST、ALT以及ALKP便會釋放進入血液當中,因此分析血清中三者之數值是肝臟受損的通用指標,可用以初步判斷肝臟受損的情況。慢性酒精攝取除了造成脂質恆定失調外,通常亦伴隨血脂異常的現象,因此亦檢測血清中脂質含量之變化,以分析PA水解物對於慢性酒精攝取小鼠血液生化指數的影響。本研究使用專業分析試紙(SpotechemTM II,Arkray Inc.,Kyoto,Japan)並搭配全自動乾式生化分析儀,分析小鼠血清中三酸甘油酯(TG)以及總膽固醇(TC)的濃度。
如表五,ALC組小鼠之AST、ALT及ALKP皆顯著高於CON組的小鼠(p<0.05),然而在補充PA水解物後,該些數值皆恢復成至與CON組間無差異(p>0.05)之水準,故推論八週慢性酒精的攝取確實造成小鼠肝臟受損,而補充PA水解物能減緩、甚至改善慢性酒精攝取下所造成的肝臟受損;此外,ALC組小鼠之TG與TC濃度顯著高於CON組小鼠(p<0.05),而補充PA水解物亦能有效降低血清中之TG與TC之含量(p<0.05),並恢復至與CON組同樣之水準(p>0.05)。
慢性酒精攝取易造成肝臟產生大囊泡性脂肪肝(maerovesicular fatty liver),可以使用蘇木素-伊紅染色(hematoxylin-eosin stain,H&E
stain)觀察肝臟的型態變化。參見第六圖,ALC組的小鼠肝臟切片上,在中央靜脈(central vein,CV)的周圍有許多膨大的脂肪小滴堆積,為典型大囊泡性脂肪肝的特徵,但給予PA水解物的小鼠,其肝臟切片形態與CON組相比並無顯著差異,表示PA水解物的確能緩解慢性酒精攝取造成肝臟脂肪堆積的情形。
為進一步了解肝臟脂肪堆積的情形,犧牲小鼠後取其肝臟進行萃取並分析其中TG與TC的含量,參見第七圖,ALC組小鼠的肝臟中,TG與TC含量顯著高於CON組(p<0.05),而在給予PA水解物組,小鼠肝臟中的TG相較於ALC組小鼠有顯著下降(p<0.05)的情形,下降後的數值也與CON組之間無顯著差異(p>0.05);另外,PA水解物組小鼠肝臟中TC的含量相較於ALC組亦有減少之趨勢。此外,檢測小鼠糞便中的脂質含量,發現補充PA水解物之小鼠糞便中,三酸甘油酯以及總膽固醇的含量皆顯著高於CON組以及ALC組(p<0.05),暗示PA水解物具有抑制脂質吸收或是促進脂質排出之功效。
為了解PA水解物對於肝臟脂質恆定相關基因的影響,自犧牲後小鼠的肝臟組織中抽取mRNA,並以即時定量反轉錄聚合酶鏈鎖反應(qRT-PCR)檢測其中脂肪酸β氧化相關基因以及脂質合成相關基因的表現,其中脂肪β氧化相關基因包含過氧化物酶體增殖物活化受體α(Peroxisome proliferator-activated receptor α,Ppar α)、視網酸X受體α(Retinoid X receptor α,Rxr-α)、肉鹼棕櫚醯基轉移酶(Carnitine palmitoyltransferase I,Cpt1)、以及去偶合蛋白
2(Uncoupling protein 2,Ucp2);脂質合成相關基因包含固醇調節元件結合蛋白(Sterol regulatory element-binding protein,Srbp1-c)、肝異受體(Liver X receptor alpha,Lxr-α)、脂肪酸合成酶(Fatty acid synthase,Fas)以及乙醯輔酶A羧化酶(Acetyl-CoA carboxylase,Acc)。參見第八圖,ALC組的Ppar α的表現量相較於CON組顯著下降(p<0.05),Rxr-α與Cpt1的表現量也有下降之趨勢,但在給予補充PA水解物後,小鼠肝臟中與β氧化有關的Ppar α、Rxr-α、Cpt1、以及Ucp2的表現量均顯著上升(p<0.05);此外,ALC組別之Srebp-1c以及Fas之基因表現相較於CON組均顯著上升(p<0.05),另外Lxr-α與ACC的基因表現量則無顯著提升的現象(p>0.05),而給予PA水解物後,小鼠肝臟中的Srebp-1c、Lxr-α與Acc的基因表現量與ALC組有下降之趨勢,而Fas的表現則有顯著下降(p<0.05)。
以ABTS自由基清除之情形,作為小鼠血清或肝臟樣品抗氧化能力(trolox equivalent antioxidant capacity,TEAC)的評估,單位以每重量單位樣品中含有多少trolox當量表示(TE);另以硫巴比妥酸反應物質(thiobarbituric acid reactive substances,TBARS)分析法量測小鼠體內脂質過氧化情形;最後量測小鼠肝臟中抗氧化防禦系統,如超氧化物歧化酶(superoxide dismutase,SOD)、過氧化氫酶(catalase,CAT)、榖胱甘肽過氧化氫酶(glutathione peroxidase,GPx)以及榖胱甘肽(glutathione,GSH)含量的變化。
參見第九圖,在八週的慢性酒精攝取下(ALC組),小鼠血清中的TEAC數值顯著低於CON組(p<0.05),且TBARS數值有上升的趨勢,但補充PA水解物之後,小鼠血清中的TEAC數值顯著上升
(p<0.05),甚至高於CON組,且TBARS數值也有恢復之趨勢。此外,慢性酒精攝取(ALC組)下,小鼠肝臟的TEAC數值為0.11μmole TE/mg-蛋白質,顯著低於CON組的0.14μmole TE/mg-蛋白質(p<0.05),而ALC組小鼠肝臟之TBARS值為0.55nmole MDA eq./mg-蛋白質,也顯著高於CON組之0.40nmole MDA eq./mg-蛋白質,表示酒精攝取會降低肝臟組織的抗氧化能力與提高脂質過氧化程度;補充PA水解物後,肝臟中TEAC上升為0.18μmole TE/mg-蛋白質,顯著高於其餘兩組(p<0.05),而肝臟TBARS值也顯著低於ALC組(p<0.05),並恢復至與CON組同樣之水平;故在酒精攝取下給予小鼠PA水解物能提升血清或肝臟抗氧化能力且抑制血清或肝臟中脂質過氧化程度。
參閱第十圖,小鼠肝臟中的SOD活性在三組之間並無顯著差異(p>0.05),CAT活性測試中,CON組之CAT活性為96.17unit/mg-蛋白質,而ALC組中CAT的活性顯著上升為129.16unit/mg-蛋白質(p<0.05),但補充PA水解物會將小鼠肝臟的CAT活性提升為162.20unit/mg-蛋白質,顯著高過其餘二組(p<0.05);GPx的活性分析中,CON組的GPx活性為1.01unit/mg-蛋白質,而ALC組為0.98unit/mg-蛋白質,二組別並無顯著差異(p>0.05),但補充PA水解物後,肝臟中GPx的活性為顯著提高,為1.36unit/mg-蛋白質(p<0.05);還原態GSH含量分析結果,ALC組之還原態GSH含量與CON組相比有減少的趨勢,而補充PA水解物後,肝臟中還原態GSH的含量顯著提升且高於其餘二組(p<0.05)。
長期飲酒會導致肝臟慢性發炎,因此透過分析常見發炎因子,包括腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)、介白素-1β(interleukin-1β,IL-1β)以及介白素-6(interleukin-6,IL-6)的濃度變化,評估補充PA水解物對肝臟發炎程度之影響。參見第十一圖,ALC組小鼠肝臟之TNF-α、IL-1β、以及IL-6含量均顯著高於CON組(p<0.05),而在給予小鼠補充PA水解物後,雖然TNF-α沒有降低的情況(p>0.05),但IL-1β以及IL-6的含量與ALC組相比有顯著下降之情形(p<0.05),表示PA水解物能降低慢性酒精攝取引起的發炎情形。
由上述之實施說明可知,本發明與現有技術相較之下,本發明具有以下優點:
1.本發明之卵繫帶水解物,與未經水解的卵繫帶相比,含有豐富的游離胺基酸、肌肽與甲肌肽。
2.本發明之卵繫帶水解物具有良好的體外抗氧化活性,包含良好的自由基清除率以及亞鐵離子螯合力。
3.本發明之卵繫帶水解物具有降低血脂之功效,能有效降低血液中的三酸甘油酯以及總膽固醇。
4.本發明之卵繫帶水解物能減緩肝臟脂肪堆積以及降低肝臟的氧化壓力。
5.本發明之卵繫帶水解物具有抑制肝臟發炎之功效,能有效降低肝臟發炎相關因子,如IL-1β與IL-6之分泌。
綜上所述,本發明卵繫帶水解物及其製備方法與用途,的確能藉由上述所揭露之實施例,達到所預期之使用功效,且本發明亦未
曾公開於申請前,誠已完全符合專利法之規定與要求。爰依法提出發明專利之申請,懇請惠予審查,並賜准專利,則實感德便。
惟,上述所揭之說明,僅為本發明之較佳實施例,非為限定本發明之保護範圍;其;大凡熟悉該項技藝之人士,其所依本發明之特徵範疇,所作之其它等效變化或修飾,皆應視為不脫離本發明之設計範疇。
Claims (8)
- 一種卵繫帶水解物,其係以蛋白酶A(Protease A),於酵素受質比1:100(w/w)之條件水解而得,該卵繫帶水解物包含90~120毫克/克的游離胺基酸,其中該游離胺基酸係包含10~20wt%白胺酸(leucine)、7~14wt%精氨酸(arginine)、8~10wt%苯丙胺酸(phenylalanine)、8~10wt%纈胺酸(valine)以及8~10wt%離胺酸(lysine)。
- 如申請專利範圍第1項所述之卵繫帶水解物,其中該白胺酸係為15.47wt%,該精氨酸係為10.89wt%,該苯丙胺酸係為9.74wt%,該纈胺酸係為9.64wt%,以及該離胺酸係為9.09wt%。
- 如申請專利範圍第1項所述之卵繫帶水解物,其係進一步包含肌肽(carnosine)以及甲肌肽(anserine)其中之一或兩種以上之組合。
- 如申請專利範圍第3項所述之卵繫帶水解物,其中該肌肽係為20~60毫克/100克或該甲肌肽係為95~150毫克/100克。
- 如申請專利範圍第4項所述之卵繫帶水解物,其中該肌肽係為48.06毫克/100克或該甲肌肽係為106.84毫克/100克。
- 一種製備請求項1所述之卵繫帶水解物的方法,其係包含:步驟一:將一卵繫帶解凍後利用去離子水洗去雜質,並離心以保留下層之一第一產物; 步驟二:將該第一產物以95℃恆溫加熱10~30分鐘並冷卻,再加入去離子水均質以獲得一卵繫帶均質液;步驟三:取100~200克之該卵繫帶均質液,與蛋白酶A(Protease A)以一酵素受質比1:100(w/w)混合,水解一作用時間之後獲得一第一水解液;步驟四:將該第一水解液於95℃恆溫加熱10~30分鐘並冷卻,離心後保留上層之一第二水解液;以及步驟五:過濾並凍乾該第二水解液以獲得該卵繫帶水解物。
- 一種以申請專利範圍第1項之卵繫帶水解物於製備保護慢性酒精攝取引起之肝損傷藥物之用途,係以一有效劑量施予一所需個體,以減少脂肪堆積於肝臟或降低肝臟氧化壓力,達到肝臟保護之功效;其中該卵繫帶水解物係包含90~120毫克/克游離胺基酸,其中該游離胺基酸係包含10~20wt%白胺酸(leucine)、7~14wt%精氨酸(arginine)、8~10wt%苯丙胺酸(phenylalanine)、8~10wt%纈胺酸(valine)以及8~10wt%離胺酸(lysine)。
- 如申請專利範圍第7項所述之用途,其中該脂肪係為三酸甘油酯(triglyceride)或總膽固醇(total cholesterol)。
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