TWI353845B - Process for preparing peptide products for promoti - Google Patents
Process for preparing peptide products for promoti Download PDFInfo
- Publication number
- TWI353845B TWI353845B TW097109668A TW97109668A TWI353845B TW I353845 B TWI353845 B TW I353845B TW 097109668 A TW097109668 A TW 097109668A TW 97109668 A TW97109668 A TW 97109668A TW I353845 B TWI353845 B TW I353845B
- Authority
- TW
- Taiwan
- Prior art keywords
- peptide
- soybean
- protease
- leu
- peptide product
- Prior art date
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Description
1353845 九、發明說明: 【發明所屬之技術領域】 進 本發明係關於一種製備胜肽產物的新穎方法。詳言之 本發明係關於一種利用蛋白酶將大豆殘逢製備成具:促 膽囊收縮素分泌效桌之大豆胜肽的方法。 【先前技術】
肥胖已經是全球化相當嚴重的問題,不論是醫療或是營 養體系均相當重視,也都試圖提出因應的對策。咸信降低 食愁與食物攝取並維持攝食量為解決肥胖問題之有效方 法,因此,許多能產生飽足感之減重保健食品因應而生。 動物的飽足感受到很多因素的調控,其中由消化道所分泌 的神經胜肽(neuropeptide)扮演著相當重要的角色。 膽囊收縮素(cholecystokinin,CCK)是由腸道黏膜細胞分 泌的一群神經胜肽,其已被證實可以促進膽囊與胰臟分泌 相關消化酵素至腸道中’同時也能提供腦部「飽足」的訊 息’以抑制動物攝食的慾望。CCK是由腸道黏膜上的第 一型内分泌細胞所產生的胜肽(Buchan et al.,Gut,1978 19: 403-407),第一型細胞大致上為三角形,其細胞頂端 面向腸腔’而含有CCK之分泌性顆粒則位於細胞底部。此 種排列方式,令其能夠接受攝食入動物腸道中的營養素的 刺激而於細胞底端釋放出CCK至血液及/周圍的組織中 (Moran and Kinzig, Am. J. Physiol. Gastrointest. Liver Physiol., 2004, 286: G1 83-G188)。CCK必須結合到接受器 上才能誘發一連串的生理作用,而這些接受器係位於腸道 122283.doc 1353845 底端和胃壁周邊的迷走神經上(Moran et al.,Am. J. Physiol. Regul. Integr. Comp. Physiol., 1997, 272: R1245-R1251)’甚至在中枢神經中也可發現CCK的接受器。因 此,CCK的釋出不僅影響了消化道的消化作用,同時也會 影響其它神經荷爾蒙的分泌,一起協同控制動物的攝食與
否(Woods,Am. J. Physiol. Gastrointest. Liver Physiol., 2004, 286: G7-G13)。例如 ’ Gutzwiller 等人(Gutzwiller et al.,Am. J. Physiol. Regul. Integr. Comp. Physiol·,2004, 287: 562-567)就發現 CCK-33和高血糖素樣肽」 (glucagons-like peptide-1; GLP-1)具有交互作用;而CCK 是屬於提供飽足訊息的神經胜肽,其被發現和提供肥胖訊 息的瘦體素(leptin)或騰島素(insulin)間也有明顯的相關 性(Bado et al.,Nature, 1998, 394·· 790-793; Brzozowski et
al., Eur. J. Pharmacol., 1999, 374: 363-376; Gallmann et al., Peptides, 2005, 26: 437-445; Morton et al., J. Clin. Invest” 2005,1 15: 703-710)。因此 ’ CCK的分泌在動物的 攝食行為上的確扮演相當重要的角色。 許多的食品成分都已經被發現具有誘發CCK分泌的效 果’特別是蛋白質和油脂類的成分。歐美國家近年來流行 的低碳水化合物飲食或是阿金式飲食(Atkins diet)都是基 於此種原理。不過’許多研究均發現,蛋白質和油脂類的 成分若是以胜肽和脂肪酸的型態提供給動物,其誘發CCK 分泌的功能更佳。例如:Nishi等人(Nishi et al., Exp. Biol. Med·,2001,226: 1031-1036)就發現蛋白質經胃液素 122283.doc 1353845 (pepsin)水解後的胜肽液具有促進CCK分泌的效果,且將 數種蛋白質水解胜肽比較後,發現大豆蛋白水解物的效果 最佳,而其中最有效的胜肽組成分係來自大豆蛋白β_ conglycinin的 β51-63 片段(Nishi et al.,J. Nutr·,2003,133: 352-357)。而胜肽促進CCK分泌的效應,可能與其胺基 酸組成(Furuse et al·,Comp. Biochem. Physiol·,1991, 99A: 449-451)和疏水性(Backus et al·,Regul. Pept.,1997,72:
3 1 -40)有關,且可能是透過c-AMP的途徑調控(Bernar(j et al.,Endocrinology,2001, 142: 721-729)。 JP2004010569揭示一種可促膽囊收縮素分泌且含有精胺 西夂之胜狀’其係將經純化的大丑蛋白質以胃 蛋白酶(pepsin)處理後所獲得,且該胜肽片段即為大豆卜 conglycinin之第5 i -63胺基酸殘基片段。惟該方法中原料之 獲得必須經過繁雜的純化過程,相對降低其於工業上的經
濟效益。故仍須更精簡的製備方法,以降低製造成本進而 提升工業上的經濟效益。 本發明驚人地發現,將大豆殘渣於無須純化該殘涪之過 程下,以蛋自酶處理即可獲得具有促膽囊㈣素分泌效果 之機能性胜肽,且該胜肽可以描斗 徒升動物攝食前的飽足感, 進而降低攝食量而達到體 遐菫控制之效果。故相較於 JP2004010569,本發明的方法 ,+ 、業利用上更能符合低成 本之要求。 【發明内容】 本發明所揭示的 疋種利用大豆殘渣來製造大豆胜肽 (S-) 122283.doc 1353845 產物的方法,而此大豆胜肽產物具有誘發CCK分泌的良好 效果,可以提升動物飽足感,進而降低攝食量而達到體重 控制之效果。 本發明另一方面揭示一種具有誘發CCK分泌效果的大豆 胜肽產物。 本發明又另一方面揭示一種組合物,其包含具有誘發 CCK分泌效果的大豆胜肽產物。 【實施方式】 本發明係提供一種製備具有促膽囊收縮素分泌效果之胜 月太產物的方法’該方法包含以下步驟: Ο)將大豆殘渣與水混合均勻; (b)將(a)之混合物與蛋白酶於適當溫度下進行水解; 0)將(b)所得之水解物中的酵素失活性;及 (d)將(c)中之固體與液體分離,並收集該經分離之液體 以獲得該胜肽產物。 根據本發明,「大豆殘渣」意指將大豆去除油脂後所剩 餘之脫脂大豆粉,經由製作大豆營養胜肽之製程後所剩餘 之固體殘渣。大豆營養胜肽之製程為將脫脂大豆粉與水混 合後’加入進行發酵及水解反應。將經發 酵及水解之產物去除液體部分後即可得到大豆殘渣。一般 而吕,該大豆殘渣含有約30%(w/w)之固形物及約 15%(w/w)之粗蛋白質。 根據本發明,大豆殘渣係與約0.5至約10倍量(w/w)之水 混。,較佳係與約!至約5倍量(w/w)之水混合,更佳係與 l222S3.doc 1353845 約3倍量(w/w)之水混合。 根據本發明,「蛋白酶」意指任意市售商品,或源自微 生物、動物或植物且具有水解蛋白質功效之酵素,其可為 内蛋白酶(endoprotease)、外蛋白酶(ex〇peptidase)或其組 合。該蛋白酶可為,但不限定為嗜熱菌蛋白酶、胃蛋白 酶、胰蛋白酶、bromelain、Alcalase(N〇v〇zyme 公司)、
Flavorzyme ^ Esperase > PTN 6.0 S ^ Acid Protease ^
Protease A ^ Protease M ^ Protease N ^ Protease NL ^ Protease P、Protease S、Pr〇tin SD、The_ase、
Flav〇rpro及Promod。較佳之蛋白酶為鹼性蛋白酶 Alcalase 。 根據本發明,水解反應所添加之蛋白酶的量視其所選用 之蛋白酶種類而定,其以每公斤大豆殘渣計可為約1〇〇〇〇 ι.υ.至約 125,000 LU_,較佳為約1〇 〇〇〇 Lu 至約 8〇 〇〇〇 I.U. ’更佳為約10,000 LU.至約4〇 〇〇〇 j u。於本發明之一 實施態樣中,當蛋白酶為Alcalase時,每公斤大豆殘渣中 加入約20,000至約125,000 LU.,較佳為約2〇〇〇〇 Lu至約 80,000 I.U.,更佳為約 20,000 I.U.至約 30,〇〇〇 LU.。 根據本發明,水解反應所需之時間及溫度係視其所選用 之蛋白酶種類而定,其水解反應的時間可為約i至約24小 時,較佳為約1至約8小時,最佳為約2至約4小時;且水解 反應的溫度可為約30至約60。〇,較佳為約4〇至約55<>c,更 佳為約45至約55。(:。於本發明之一實施態樣中,當蛋白酶 為Alcalase時,水解反應之時間為約1至約16小時,較佳約 122283.doc • 10· 1353845 2至約8小時,最佳為約2小時;水解反應之溫度為約30至 約55C ’較佳為約5〇至約55°C,更佳為約5〇〇c β 根據本發明之製備方法,本發明所屬技術領域中具有通 常知識者可利用該領域中所f遍知悉之操作方法,將水解 物中之酵素失活性4用於本發明之方法包括,但不限定 為加熱法、酸鹼調整法、加壓法或其組合❶於本發明之一 實施態樣中,係將水解物加熱至約8〇至約i〇〇£>c,較佳為 加熱至約85至約95。(:,更佳為加熱至約9〇t:。 根據本發明之製備方法,本發明所屬技術領域中具有通 常知識者可利用該領域中所普遍知悉之操作方法,將水解 物中之液體與殘渣分離,並收集該經分離之液體以獲得本 發明之胜肽產物。適用於本發明之分離方法包括,但不限 疋為過濾法、離心法、壓濾法、沉降法或其組合。於本發 明之一實施惡樣中’係利用壓遽分離之方式以板框式壓濾 機將水解物中之殘渣與液體分離。 根據本發明之製備方法,本發明所屬技術領域中具有通 常知識者可利用該領域中所普遍知悉之操作方法,將該液 體進步濃縮及乾燥,以製得經濃縮或乾燥的胜肽產物。 適用於本發明之濃縮、乾燥方法包括,但不限定為冷凍乾 燥喷霧乾燥、鼓風乾燥機乾燥、流動床乾燥及其組合。 本發明之較佳乾燥方法為喷霧乾燥。 本發明之另一方面係提供一種大豆胜肽產物,其係經由 上述方法製得。該胜肽產物具有效促進CCK之分泌,提升 動物飽足感,進而降低攝食量而達到體重控制之效果。 122283.doc 1353845 本發明之另一方面係提供一 々丄 供種組合物,其係含有本發明 之大豆胜肽產物。本發明 - 々人 月之組合物可為醫藥組合物或食品 組合物。本發明之醫藥組合 0物執& °σ組合物可藉由本發明 所屬技術領域中所普遍知来链你 磉知悉之刼作方法,將本發明之胜肽
產物與習知之載劑、稀釋劑、溶劑1色劑、抗氧化劑、 惰性物質及/或其他添加劑調配成錠劑、膠囊、粉劑、粒 劑、沖劑、飲料、健康食品、食品添加劑或飼料。 舉出下述實施例係為輔 述或所請求專利之發明。 書或前案資料而對於本發 助了解本發明,絕非限制此處所 熟習本發明之技藝人士基於說明 明所作之任何變化或修飾(包括 所有今日已知或未來將發展出的相當之取代),均應落入 本發明之範圍中。 實例1大豆殘渣之取得 大豆殘渣是依據製作營養胜肽製程獲得。於25公斤之脫 脂大豆粉(中聯油脂公司)中加入一半量(w/w)的水以100 # t蒸氣蒸煮45分鐘後冷卻至45它,加入4.2克含心 •so_/ae之麴粉(日本樋口公司)混合均勻,於相對濕度95%、 •室溫27°C下發酵48小時。而後於此發酵產物中加入三倍量 (w/w)的水,於4 5 C水解8小時後,以隔膜泵浦將固液混合 之水解液打入板框式壓濾機(德國Klinkau GmbH & Co·產 品)中,每一板框濾室外覆濾布,濾布孔徑1〇〇〇 mesh,利 用高壓(200-250 kg/cm2)讓液體流出,固體部分停留於滤 室中形成泥餅狀。上述板框式壓濾機壓榨所得之液體部分 即為大豆營養胜肽’殘餘在板框式壓濾機内的固體部分即 122283.doc , η - 1353845 為大豆殘渣。以上述方法獲得的大豆殘渣含有約3 0°/〇(w/w) 之固形物,且粗蛋白質之含量約為15%(w/w)。
實例2以Alca丨ase製作飽足感機能胜肽粉末 取實例1中所得之大豆殘潰以三倍量(w/w)的水混合均 勻,並調整混合物之pH值至9.0。於每公斤大豆殘渣加入 24 A.U.(約 27,360 I.U.)的鹼性蛋白酶(Alcalase)(Novozyme 公司),並於50°C下水解2小時。再將水解混合物加熱至90 °C並維持10分鐘使酵素失活。以上述板框式壓濾機壓榨收 取液體部分,所得液體部分再經減壓濃縮機(MT-3型;茅 台機械有限公司,台灣)濃縮至40 brix。再用喷霧乾燥機 (DA360-100H型;宏坂科技有限公司,台灣)進行乾燥,所 得的粉末即為飽足感機能胜肽粉末。
實例3飽足感機能胜肽之組成分析 將由實例2所獲得之胜肽粉末配製成0.5%(w/w)水溶液, 以等電點電泳設備(Rotofor; Bio-Rad Laboratories, CA, USA)依照設備廠商的指示方法,將胜肽溶液等分成20個 餾分。每一個餾分經冷凍乾燥後,再配製成適當濃度,以 高效率液相層析質譜儀鑑定胜肽溶液t的胜肽組成。共鑑 定出43種胜肽(表1)。 高效率液相層析質譜儀鑑定法所使用的裝置及條件: 高效率液相層析儀:Waters 600幫浦(Milford, MA, USA); 管柱:LiChroCART 125-2 Superspher 100 RP-18 管柱 (Merck, Darmstadt, Germany); 122283.doc -13 - 1353845 質譜儀:Micromass Quattro LC 質譜儀(Wythenshawe, UK);及 溶離溶劑:A液為水,B液為氰甲烧(acetonitril),溶離 條件為:15% B液3分鐘,15%至85% B液線性溶離60分 鐘,85% B液7分鐘。 表 1:飽足感機能胜肽中被鑑定出的胜肽組成
留間in) 滞時(m 列
序列 質核比 (m/z) 滯留 質核比 時間 (m/z) (min) SEQ ID NO: 203 6.61 Leu-Ala 1 281 5.49 Val-Tyr 23 204 5.27 Ala-Asn 2 288 16.91 Val-Gly-Leu 24 205 12.09 Ser-Val 3 295 6.92 Phe-Glu 25 231 8.15 Val-Leu 4 295 9.12 Leu-Tyr 26 232 7.27 Val-Asn 5 295 12.36 Tyr-Leu 27 232 8.82 Val-Gly-Gly 6 296 12.18 Tyr-Asn 28 233 5.42 Ser-Leu 7 304 7.56 Ala-Ser-Leu 29 233 6.26 Thr-Leu 8 304 8.70 Ala-Thr-Leu 30 234 5.29 Thr-Asn 9 318 6.83 Leu-Gly-Glu 31 237 7.16 Ala-Phe 10 329 14.75 Phe-Tyr 32 245 15.00 Leu-Leu 11 329 19.15 Tyr-Phe 33 246 5.97 Asa-Leu 12 330 18.96 Val-Val-Leu 34 246 6.37 Val-Gly-Ala 13 332 21.93 Ser-Ile-Leu 35 260 3.89 Leu-Gin 14 332 25.12 Leu-Ser-Leu 36 260 6.39 Gin-Leu 15 345 15.96 Val-Gly-Gly-Leu 37 263 4.59 Met-Leu 16 348 6.57 Leu-Ser-Glu 38 264 4.32 Ser-Gly-Ser 17 352 16.93 Val-Ala-Tyr 39 265 9.12 Phe-Val 18 352 23.09 Val-Ser-Phe 40 265 12.49 Val-Phe 19 366 10.48 Tyr-Ile-Ala 41 267 7.74 Ser-Phe 20 374 3.91 Lys-Val-Gln 42 276 6.11 Ser-Gly-Leu 21 380 11.25 Phe-Val-Asp 43 279 24.23 Phe-Leu 22 實例4飽足感機能胜肽促進腸道黏膜細胞分泌CCK之效果 根據 Nishi et al. (2003)描述之方法擷取 Sprague-Dawley (SD)鼠之腸道黏膜細胞。擷取黏膜細胞前一天,先令動物 122283.doc -14- 禁食12小時。於將動物犧牲後,取下靠近胃幽門處約20公 分長的小腸,立刻浸潰於生理食鹽水中,並以生理食鹽水 清洗腸道内外。而後剪開腸道,置於不含鈣的Krebs-Henseleit bicarbonate緩衝溶液(含 2.5 mmol/L EDTA,pH 7.4)中,於37°C下靜置5分鐘。接著以鐵氟龍刮杓輕輕刮取 腸道内面,所得之懸浮液先行過濾去除大顆粒的腸道組 織,而後離心去除上清液。沈澱的腸道黏膜細胞回溶於適 當的HEPES缓衝溶液中,使用Phoenix Pharmaceuticals公 司(Belmont, CA,USA)之酵素連結免疫吸附分析(ELISA)模 組進行CCK之分析。 結果發現,實例2之胜肽產物溶於生理食鹽水中,在 CCK之ELISA分析時的最終濃度為1,000 ppm情形下,其誘 發SD鼠之腸道黏膜細胞分泌CCK的效果,是生理食鹽水的 10倍左右(圖1)。 實例5飽足感機能胜肽的灌食對動物攝食量之抑制效果 雄性SD鼠飼養於各自獨立的籠中,室溫維持在 23±2°C,光照12小時。提供以酪蛋白為蛋白質來源的基礎 飲食 TestDiet 5800 (PMI Nutrition International, Richmond, IN, USA),令動物任意攝食。灌食試驗前先予動物禁食4 小時。接著將實例2之胜肽產物配成水溶液(10g/L)以胃管 灌食,每隻灌食量為體重的l%(v/w)。對照組灌食水。於 灌食胜肽溶液或水後10分鐘,給予動物30克基礎飲食,令 其任意攝食,於每一小時後測定飲食攝食量直至第3小 時。 122283.doc -15- 1353845 結采發S,在灌食本發明胜肽產物後,動物在!小時内 的攝食量即明顯低於灌食水的動物攝食量,顯示本發明胜 肽產物具有提升動物飽足感的效果,造成其攝食量的降 低,且此效應在灌食3小時後仍可維持(圖2)。 實例6飽足感機能胜肽的灌食對動物體重之影響 雄性四週齡的SD鼠係飼養於各自獨立的籠中,室溫維 持在23±2 C ’光照12小時’並令動物自由攝食,適應以酪 蛋白為蛋白質來源的基礎飲食TestDiet 58〇〇 NWritiQn
International,Richm〇nd,IN,USA)。於試驗前一週開始, :日降低動物可以接觸到飼料的時間,最後令動物習慣於 每曰僅有3小時攝食時間。試驗期間,將實例2之胜肽產物 配成溶液(20 g/L)並於每日不提供光照前15分鐘以胃管對 動物進行灌食,每隻動物灌食量為其體重的〇 5%(v/w)。 對照組則灌食水。於不提供光照開始時,給予動物足夠之 基礎飼料,令其任意攝食’並測定3小時内之攝食量,於 第3小時後取走飼料。每日重複相同的操作,共進行13週 之試驗,每週記錄動物的體重變化。 結果在13週的試驗後,灌食本發明胜肽產物的動物,其 平均體重較對照組動物的平均體重來得低(圖3)。推測是因 為灌食本發明胜肽產物後’降低了動物的攝食量,而於每 日相同的操作之後’ 13週下來所累積的結果。 實例7飽足感機能胜肽對人體攝食量之影響 以般市售之調味粉包調配成1.6°/〇(w/v)之鹹式熱湯, 試驗組則於熱湯中另加入3%(w/v)之實例2之胜肽產物。自 122283.doc 1353845 願參與本試驗之受試者有18_24人。試驗週期為兩天 裱,一天給予受試者含有本發明胜肽產物的熱湯,另一天 2為不3本發明胜肽產物的熱湯。受試者於試驗當日中 别半小時飲用80 mL湯物’中餐則統—給予市售便當, 天便當菜色相同。經—個循環的熟悉I,共進行五個循= 的試驗。試驗當日發予受試者記錄表,填寫喝湯前、喝= 後15刀鐘、喝湯後3〇分鐘的飽足感咖uness)與食煞 (hunger)感覺(採7分制的尺規),並記錄攝食量與飯後 鐘(喝湯後90分鐘)的飽足感與食慾。 結果顯示’受試者在飲用含有本發明胜肽產物熱湯後, 在各個時間點的飽足感評分均較喝—般湯時要高(圖 叫,其中以喝湯後15_3()分鐘之差異最為明顯(尤其η分鐘 之差異更為明顯);飲用含有本發明胜肽產物熱湯後的食 慾評分,在各個時間7皆卜卜s 门點也白比喝一般湯時來得低,且在喝 湯後的!5與30分鐘產生顯著差異(圖4b)。此結果說明,益 論從飽足或是食您的角度來觀察,喝含有本發明胜狀產物 的熱湯均比喝-般湯更能達到降低食您或促進飽足感的效 果。而喝湯後15-30分鐘感覺最為明顯,推測是飽足感機 能胜肽在體内發揮作用與持續的時間有關。因此,在喝湯 後30分鐘令受試者吃飯,在有飲用本發明胜狀產物的情形 下’許多人的攝食量因而降低’在五個循環的試驗中,每 次約有60德的受試者有攝食量降低的情形(表 122283.doc •17· 1353845 表2 :餐前攝食飽足感機能胜肽對當次進餐攝食量之影響
循環 1 2 3 4 5 -81.6 -105.5 -117.5 -91 -70 -80.1 -84.3 -93.6 -141 -52 -55.3 -69.3 57 -112.7 -51 -45.8 -45.6 -38.7 -76 -48.9 -35.7 -45.4 -17 -67.2 -48.8 -34.7 -31.1 -16 -54 -47 -33.9 -24.3 -7.4 -48.6 -38.7 -31.8 -23.3 -3.5 -46 -37.5 -30 -15 -3.4 -42 -35.4 -24.8 -13.6 -0.7 -29.5 -26.1 -16 -7.5 -0.2 -28 -25 攝食量差異(g)^ -8 -6.8 3 -21.2 -10.8 -3.6 -6.8 4.4 -13.5 -3.8 -3.3 -2 5.4 -12.7 -2.1 7.9 1.6 11.1 -7 15 16.4 2.1 12.7 -2.6 19.5 26.1 23 38.7 -2 45.9 28 25.3 43 0.9 47 37.8 27 7.4 84.5 46 34.5 26 54.1 46.4 29 91.8 70
105.5 107.9 平均值±S.D.(g) -8.0±43.7 -1.5±52.2 -13.2±41.0 -34.8±44.1 -15.0±41.2 攝食量(降低,增 加)之受試者數目 (-,+) 14-, 8+ 14-, 10+ 11-, 7+ 17-, 4+ 14-, 5+ 攝食量降低之受 試者數目/受試者 總數(%) 63.60% 58.30% 61.10% 80.90% 73.70% 1)攝食量差異=飲用機能性胜肽之攝食量-不飲用機能性胜肽之攝 食量 122283.doc -18 - 1353845 【圖式簡單說明】 圖1顯示飽足感機能胜肽促進腸道黏膜細胞分泌CCK之 效果。 圖2顯示飽足感機能胜肽的灌食對Sd大鼠3小時内攝食 里影響。圖上每一點為8隻動物攝食量的平均值±95%信任 區間。*表示具有顯著差異(P<〇.05)。 圖3顯示飽足感機能胜肽的灌食對sd大鼠體重之影響。 圖上每一點為6隻動物體重的平均值±95%信任區間。 圖4(a)及4(b)顯示飽足感機能胜肽添加至湯物中對於飲 用後之飽足感⑷與食您(b)之影響。圖上每—長條為5個試 驗循環總共104個問卷的平均值±95%信任區間。 122283.doc 19· 1353845 序列表 <110>財團法人食品工業發展研究所 <120>製備促進腺囊收縮素分泌之胜肽產物之方法及該胜肽產物之用途 <160> 43 <170> Patentln version 3.3 <210> 1 <211> 2 <212> PRT <213〉妓 <400> 1
Leu Ala <210> <211> <212> <213> <400> 2
Ala Asn 1 <210〉 <211> <212> <213> <400〉 3
Ser Val 42s <210> <211> <212> <213〉 <400> 4 Val Leu <210> 5 <211> 2 <212> PRT <213>大豆 <400> 5
Val Asn
T豆 R ^ 6 3 p IS <210> <211〉 <212> <213> <400> 6 Val Gly Gly 122283-序列表.doc 1353845 ^Λ > > Q 1 2 3 11 Ί1 1 11 <4(
Ser Leu <210> 8 <211> 2 <212> PRT <213>大豆 <400> 8 Thr Leu 1 <210〉 9 <211> 2 <212> PRT <213>大豆 <400> 9 Thr Asn 1 <210> 10 <211> 2 <212> PRT <213>大豆 <400> 10 Ala Phe <210> 11 <211> 2 <212> PRT <213>大豆 <400> 11 Leu Leu 1 <210> 12 <211> 2 <212> PRT <213>大豆 <400> 12 Asn Leu 1 <210> 13 <211> 3 <212> PRT <213>大豆 <400> 13 122283-序列表.doc 1353845
Val Gly Ala <210> 14 <211> 2 <212> PRT <213>大豆 <400> 14 Leu Gin <210> 15 <211> 2 <212> PRT <213>大豆 <400> 15 Gin Leu 1
<210〉 16 <211> 2 <212> PRT <213>大豆 <400〉 16 Met Leu 1 <210> 17 <211> 3 <212〉 PRT <213>大豆 <400〉 17 Ser Gly Ser
<210> 18 <211> 2 <212> PRT <213>大豆 <400> 18 Phe Val <210> 19 <211> 2 <212> PRT <213>大豆 <400〉 19 Val Phe <210> 20 <211> 2 <212> PRT <213>大豆 122283-序列表.doc 1353845 <400> 20 Ser Phe <210> 21 <211> 3 <212> PRT <213>大豆 <400> 21 Ser Gly Leu 1
<210> 22 <211> 2 <212> PRT <213>大豆 <400> 22 Phe Leu <210> 23 <211> 2 <212> PRT <213>大豆 <400> 23 Val Tyr <210> 24 <211> 3 <212> PRT <213>大豆 <400> 24 Val Gly Leu
<210> 25 <211> 2 <212> PRT <213>大豆 <400> 25 Phe Glu 1 <210> 26 <211> 2 <212> PRT <213>大豆 <400> 26 Leu Tyr
<210> 27 <211> 2 <212> PRT c s ) 122283-序列表.doc -4- 1353845 <213>大豆 <400> 1Ί Tyr Leu <210> 28 <211> 2 <212> PRT <213> <400> 28 Tyr Asn <210> 29 <211> 3 <212> PRT <213>大豆
<400> 29 Ala Ser Leu 1 <210> 30 <211> 3 <212> PRT <213>大豆 <400> 30 Ala Thr Leu
<210> 31 <211> 3 <212> PRT <213>大豆 <400> 31 Leu Gly Glu <210> 32 <211> 2 <212> PRT <213>大豆 <400> 32 Phe Tyr 1 <210> 33 <211> 2 <212> PRT <213>大豆 <400> 33 Tyr Phe 1 <210> 34 (S ) 122283-序列表.doc 1353845 <211> 3 <212> PRT <213> 大豆 <400> 34 Val Val Leu 5-3 3 p T\ >>>>> Q 1 2 3 n 11 11 11 2 2 2 2 < V < < <400> 35 Ser lie Leu <210> 36 <211> 3 <212> PRT <213>大豆 <400> 36 Leu Ser Leu <210> 37 <211> 4 <212> PRT <213>大豆 <400> 37 Val Gly Gly Leu <210> 38 <211> 3 <212> PRT <213>大豆 <400> 38 Leu Ser Glu <210> 39 <211> 3 <212> PRT <213>大豆 <400> 39 Val Ala Tyr <210> 40 <211> 3 <212> PRT <213>大豆 <400〉 40 Val Ser Phe (£ ) 122283-序列表.doc 1353845 <210> 41 <211> 3 <212> PRT <213>大豆 <400> 41
Tyr lie Ala <210> 42 <211〉 3 <212> PRT <213>大豆 <400> 42
Lys Val Gin 1
<210〉 43 <211> 3 <212> PRT <213>大豆 <400> 43
Phe Val Asp 1 122283·序列表.doc
Claims (1)
1353845 第097109668號專利申請案 ,中文申請專利範圍替換本(、100年4月)正本十、申请專利範圍: ---------
1· 一種製備具有促膽囊收縮素(CCK)分泌活性之胜肽產物 的方法,其包括下列步驟: (a) 將大豆殘渣與水混合均勻; (b) 將(a)之混合物與蛋白酶於適當溫度下進行水解; (c) 將(b)所得之水解物中的酵素失活性;及 (d) 將(c)中之固體與液體分離,並收集該經分離之液體 以獲得該胜肽產物, 其中該大豆殘渣係藉由將脫脂大豆粉與水混合後,加入 進行發酵及水解反應,及將經發酵及水 解之產物去除液體部分後所得到者。 2. 如凊求項丨之方法,其中水的體積為大豆殘渣重量之約 0.5至約10倍。 其中水的體積為大豆殘渣重量之約1 3. 如請求項2之方法 至約5倍。 其t水的體積為大豆殘渣重量之約3 4. 如請求項3之方法 倍。 月长項1之方法’其中該蛋白酶係為内蛋白酶 (endopr〇tease)、外蛋白酶(ex〇peptidase)或其組合。 6. 如請求項5之方法,其中該蛋白酶為Alcalase。 7. 如吻求項5之方法,其中該蛋白酶之量以每公斤大豆殘 潰計為約 1〇,〇〇〇 I.U.至約 125,000 I.u.。 8·如請求項6之方法,其中該AleaUse之量以每公斤大豆殘 產計為約20,000至約125,000 I.U.。 122283-1000418.d〇« 1353845 9· 如5月求項1之方,土 去’其中該混合物係在約3〇至約60°C下 以蛋白酶水解約1至約24小時。 10如請求項, 法’其中該混合物係在約3 0至約5 5 °c以 Alcalase水解約1至約16小時。 月长項1之方法,其中該胜肽產物包含seq D N〇s μ· 43所示之胜肽。 月求項1至11中任一項之方法其另包含步驟⑷其 係將(d)之該經分離液體濃縮及/或乾燥,以獲得經濃縮 或乾燥之胜肽產物。 13.如請求項12之方法,其中該乾㈣為喷霧乾燥。 14· 一種胜肽產物,其係以如請求項丨至丨^中任一項之方法 所製得,該胜肽具有促進CCK分泌之活性。 15. 如請求们4之胜肽錢,其包含卿⑴NQs卜 之胜肽》 16. -種用於促進CCK分泌之醫藥組合物,其包含請求们4 或15項之胜肽產物。 如請求項16之醫藥組合物,其係用於抑制人類或 食慾。 &如請求項16或17之醫藥組合物,其係用於減輕人 物之體重。 & -種用於促進CCK分泌之食品组合物,其包含請求項^ 或15項之胜肽產物。 2〇.如請求項丨9之食品組合物,其係為保健食品或食品添加 物。 21.如請求項19或20之食品組合物’纟係用於抑制人類或動 122283-I0〇〇4i8.d〇c 1353845 • 物之食慾。 22.如請求項19或20之食品組合物,其係用於減輕人類或動 物之體重。 122283-1000418.doc
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| US9238793B2 (en) | 2011-04-28 | 2016-01-19 | Lifecell Corporation | Method for enzymatic treatment of tissue products |
| US8673862B1 (en) | 2012-09-06 | 2014-03-18 | Food Industry Research And Development Institute | Peptides and use thereof in the inhibition of angiotensin converting enzyme |
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| TW200940080A (en) | 2009-10-01 |
| US8501685B2 (en) | 2013-08-06 |
| US8063017B2 (en) | 2011-11-22 |
| US20120270813A1 (en) | 2012-10-25 |
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