請參閱『第１圖～第１７圖』所示，係分別為本發明之方法流程示意圖、本發明在溫度18°C下培養紅隱藻之結果示意圖、本發明在溫度24°C下培養紅隱藻之結果示意圖、本發明在溫度30°C下培養紅隱藻之結果示意圖、本發明在不同硝酸鈉濃度培養紅隱藻之結果示意圖、本發明以反覆凍融法萃取藻紅蛋白之結果示意圖、本發明以一階段硫酸銨沉澱藻紅蛋白之結果示意圖、本發明以兩階段硫酸銨飽和濃度沉澱藻紅蛋白之結果示意圖、本發明藻紅蛋白之吸光值及濃度間之迴歸關係示意圖、本發明紅隱藻藻紅蛋白之吸收光譜圖、本發明紅隱藻藻紅蛋白之螢光放射光譜圖、本發明紅隱藻藻紅蛋白之十二烷基硫酸鈉聚丙烯醯胺凝膠電泳之結果示意圖、本發明之pH值對藻紅蛋白安定性之影響示意圖、本發明之溫度對藻紅蛋白安定性之影響示意圖、本發明之低光照時間對藻紅蛋白安定性之影響示意圖、本發明紅隱藻藻紅蛋白之DPPH自由基清除能力測定結果示意圖、及本發明紅隱藻藻紅蛋白之還原力結果示意圖。如圖所示：本發明係一種紅隱藻之培養以產製藻紅蛋白之方法，係包括： 紅隱藻培養步驟s11：取紅隱藻（Rhodomonas
sp.）置於一盛有PG培養液（PG medium）之容器中，並將該容器置入一培養箱進行培養，其培養條件為溫度介於18～30°C之間，以白光且光度介於30～120 μmol photons m−2
s−1
之間，及光週期為12:12（光:暗）環境下培養，令紅隱藻藻水中生物量可達106
cells/ml，其中該PG培養液係於海水中添加巨量元素與微量元素，該巨量元素含有濃度為882～1764 μmol/L之硝酸鈉（NaNO3
）、磷酸二氫鈉（NaH2
PO4
．2H2
O）、乙二胺四乙酸二鈉（Na2
EDTA）及三氯化鐵（FeCl3
．6H2
O），而該微量元素含有硫酸銅（CuSO4
．5H2
O）、硫酸鋅（ZnSO4
．7H2
O）、氯化鈷（CoCl2
．6H2
O）、氯化錳（MnCl2
．4H2
O）、鉬酸鈉（NaMoO4
．2H2
O）、生物素（Biotin）、維生素B12
（Vitamin B12
）、及噻胺鹽酸鹽/維生素B1
（Thiamin HCl/Vitamin B1
）；以及 藻紅蛋白萃取步驟s12：將上述培養至穩定期（Stationary phase）之紅隱藻藻水進行高速冷凍離心，收集沉澱之藻細胞並加入pH 6.8之磷酸緩衝溶液，經高速冷凍離心後，取其上清液並加入90%硫酸銨飽和度之硫酸銨粉末將其沉澱，經凍結乾燥製成紅隱藻藻粉，令每克乾重紅隱藻藻粉可萃取得到除鹽藻紅蛋白粉190.1 ± 92.0 mg/g（每克乾重）及無除鹽藻紅蛋白粉805.0 ± 80.0 mg/g。 如是，藉由上述揭露之流程構成一全新之紅隱藻之培養以產製藻紅蛋白之方法。 茲以下列實例予以詳細說明本發明，唯並不意謂本發明僅侷限於此等實例所揭示之內容。 [實施例1]藻種培養及培養液的配製 本實驗使用之紅隱藻為本實驗室微藻種原庫所提供，培養條件為光週期12:12（光:暗），光度40 μmol photons m−2
s−1
，溫度24°C。 本實驗使用之培養液係修改自Provasoli配方及Guillard and Kilham之f/2配方，將新鮮海水（鹽度35‰）以1號濾紙過濾，配製成PG培養液（表1）後，放入高壓滅菌釜中，在1.1atm、121°C下滅菌20分鐘，取出後冷卻備用。其中該PG培養液之製備過程係以每升海水加入Stock1 、2，各1 ml之巨量元素與微量元素。 表1
[實施例2]紅隱藻之最佳培養條件測試 2-1生物量測定及藻紅蛋白萃取 生物量測定：將400 ml之藻類培養組（即內含400 ml藻水之500 ml三角瓶藻類培養組），每次取3 ml藻水，再回補3 ml之新鮮PG培養液，於血球計數板（Hemocytometer）在光學顯微鏡下計算藻細胞數目。 藻紅蛋白萃取：將前述計算完後3 ml藻水置於15 ml離心管，於405 × g轉速及4°C之桌上型冷凍離心機離心10分鐘，倒掉上清液後，加入3 ml之pH 6.8磷酸緩衝溶液，並包覆鋁箔紙避光，置入-20°C冰箱中冷凍一天後，取出在室溫下使其融化，再以405 × g轉速及4°C之桌上型冷凍離心機離心10分鐘，取粉紅色上清液，最後於550 nm之分光光譜儀測量其吸光值。 2-2以不同溫度與光度培養紅隱藻 將紅隱藻培養於500 ml之三角瓶共九組（3個溫度 × 3個光度），將其初始細胞濃度調整為5.5 × 105
cells/ml，藻水量為400 ml，分別置於溫度18°C、24°C及30°C，光度30、60及120 μmol photons m−2
s−1
下培養，光週期為12:12（光:暗）。在這些條件下培養四週後，分別更新一半之藻水（取200 ml藻水，再回補200 ml之PG培養液）避免其進入衰老期（Decline phase），然後再依照前述2-1方式每隔兩天取3 ml藻水，並回補3 ml培養液，計算生物量並同時萃取藻紅蛋白，再培養15天，本實驗三重複。 本實驗以三種溫度18°C、24°C、30°C及三種光度30、60、120 μmol photons m−2
s−1
下培養紅隱藻結果如第２～４圖所示： 第２圖為溫度18°C下培養紅隱藻結果。圖中(1)表示紅隱藻在光度30、60及120 μmol photons m−2
s−1
下培養之藻紅蛋白濃度，(2)表示紅隱藻在光度30、60及120 μmol photons m−2
s−1
下培養之生物量。結果發現，在溫度18°C及光度30 μmol photons m−2
s−1
下，其藻紅蛋白濃度在第七天達到最高905.33 μg/ml，且高於另外兩個光度組，生物量亦在第七天後達到穩定期。相對地，在60 μmol photons m−2
s−1
下，藻紅蛋白濃度則是提早在第三天達到最高565.33 μg/ml，生物量在第五天後達到穩定期。在120 μmol photons m−2
s−1
下，其藻紅蛋白濃度亦提早在第三天達到最高432.00 μg/ml，其最高值為三個光度組中最低者，生物量在第五天後達到穩定期，第十一天後開始明顯下降。顯然地，在溫度18°C下，以低光度（即30 μmol photons m−2
s−1
）可獲得最高之藻紅蛋白濃度。 第３圖為溫度24°C下培養紅隱藻結果。圖中(1)表示紅隱藻在光度30、60及120 μmol photons m−2
s−1
下培養之藻紅蛋白濃度，(2)表示紅隱藻在光度30、60及120 μmol photons m−2
s−1
下培養之生物量。結果發現，在溫度24°C及光度30 μmol photons m−2
s−1
，其藻紅蛋白濃度在第五天達到最高1478.66 μg/ml，且高於另外兩個光度組者，生物量亦在第七天後達到穩定期。相對地，在60 μmol photons m−2
s−1
下，藻紅蛋白濃度則是提早在第三天達到最高928.66 μg/ml，生物量在第五天後達到穩定期，在120 μmol photons m−2
s−1
下，其藻紅蛋白濃度在第五天達到最高918.66 μg/ml，其最高值為三個光度組中最低，生物量在第五天後達到穩定期。顯然地，在溫度24°C下，低光度（即30 μmol photons m−2
s−1
）可獲得最高之藻紅蛋白濃度。 第４圖為溫度30°C下培養紅隱藻結果。圖中(1)表示紅隱藻在光度30、60及120 μmol photons m−2
s−1
下培養之藻紅蛋白濃度，(2)表示紅隱藻在光度30、60及120 μmol photons m−2
s−1
下培養之生物量。結果發現，在溫度30°C及光度30 μmol photons m−2
s−1
下，其生物量及藻紅蛋白濃度皆為最高，且高於另外兩個光度組，生物量是在第十三天達到最高2.15 × 106
cells/ml，而藻紅蛋白濃度則是在第三天達到最高872.00 μg/ml，在60、120 μmol photons m−2
s−1
下，藻紅蛋白濃度分別在第七、五天達到最高675.33 μg/ml、758.66 μg/ml，兩者之最高值均低於光度30 μmol photons m−2
s−1
組者，且生物量亦沒有明顯的對數期（Logarithmic phase）及穩定期。顯然地，在溫度30°C下，低光度（即30 μmol photons m−2
s−1
）可獲得最高之藻紅蛋白濃度。 綜合上述九組實驗之結果，發現培養紅隱藻在生物量最高的時候（天數），其藻紅蛋白濃度並不是最高值，扣除第一天之生物量後，在溫度30°C、光度60 μmol photons m−2
s−1
及120 μmol photons m−2
s−1
此兩組之培養組中，其生物量皆維持在6.5 × 105
cells/ml以下，相對地，其餘七組實驗生物量均可達到1.5× 106
cells/ml，因此本實驗之結果發現紅隱藻較適合培養在溫度18°C及24°C。但是在溫度30°C、光度30 μmol photons m−2
s−1
（低光度）時，亦可維持其生長量。 另外，以藻紅蛋白濃度之結果發現，在本實驗之不同溫度（18°C～30°C）培養組亦是以低光度（30 μmol photons m−2
s−1
）下其藻紅蛋白濃度最高。因此證明在低光度環境下，紅隱藻之藻紅蛋白濃度會增加。其中在溫度24°C、光度30 μmol photons m−2
s−1
培養組之藻紅蛋白濃度最高可達1308.66 μg/ml，其餘組別之最高藻紅蛋白濃度皆在835.33 μg/ml以下。因此，最適培養紅隱藻產生最高藻紅蛋白濃度之條件為：溫度24°C、光度30 μmol photons m−2
s−1
，其中以光度之影響較大。 2-3以不同硝酸鈉濃度培養紅隱藻 將紅隱藻培養於500 ml之三角瓶共四組（4種硝酸鈉濃度），將初始細胞濃度調整為2.7 × 105
cells/ml，藻水量為400 ml，硝酸鈉濃度分別為882 μmol/L（原始濃度）、1103 μmol/L（1.25倍）、1323 μmol/L（1.5倍）、1764 μmol/L（2倍）。培養條件為根據之前最佳培養條件下之實驗結果，光週期為12:12（光:暗），依照前述2-1方式每隔兩天取3 ml藻水，不回添PG培養液，計算生物量及萃取藻紅蛋白，共培養35天，本實驗三重複。 第５圖為不同硝酸鈉濃度培養紅隱藻結果。圖中(1)表示紅隱藻在882 μmol/L、1103 μmol/L、1323 μmol/L、及1764 μmol/L下培養之藻紅蛋白濃度，(2)表示紅隱藻在882 μmol/L、1103 μmol/L、1323 μmol/L、及1764 μmol/L下培養之生物量。結果發現，四組（原始濃度、1.25倍、1.5倍、及2倍）不同硝酸鈉濃度培養組均在第十一天達到最高濃度之藻紅蛋白，分別為1052.00 μg/ml、1062.00 μg/ml、1257.00 μg/ml、及1277.00 μg/ml，其藻紅蛋白濃度會隨著硝酸鈉濃度增加而增加，在培養的第十一天後，原始硝酸鈉濃度組之藻紅蛋白濃度逐漸降低，但是2倍硝酸鈉濃度組之藻紅蛋白濃度維持到培養的第三十一天後才降低。此外，1.25倍硝酸鈉濃度，其藻紅蛋白濃度增加0.01%，1.5倍硝酸鈉濃度，其藻紅蛋白濃度增加0.19%，2倍硝酸鈉濃度，其藻紅蛋白增加0.21%，由此可知增加培養液中之硝酸鈉濃度確實會增加紅隱藻藻紅蛋白之濃度。 在原始濃度、1.25倍、1.5倍及2倍硝酸鈉濃度下，每毫升藻水所萃取之藻紅蛋白約可得到台幣1009.92元、1019.52元、1206.72元及1225.92元，但其硝酸鈉每毫升成本卻只需台幣0.000024元、0.00003元、0.000036元及0.000048元，相對成本效益（cost-performance）高。因此，增加紅隱藻培養液中之硝酸鈉濃度，使其藻紅蛋白濃度增加，是非常經濟且有效增加收益之方式。 [實施例3]紅隱藻藻粉之製備及萃取其藻紅蛋白 3-1紅隱藻藻粉之製備 本實驗收集兩個不同成長階段之紅隱藻，即培養至穩定期之紅隱藻藻水，以及培養至衰老期之紅隱藻藻水各一公升，將其分別裝入250 ml之離心管，以450 × g轉速及4°C之高速冷凍離心機離心20分鐘，收集沉澱之藻細胞置入-20°C冰箱冷凍一天後，再以凍結乾燥機凍乾兩天，製備成紅隱藻藻粉，最後保存於-20°C冰箱中，以備後續分析使用。 3-2紅隱藻藻紅蛋白之萃取 取培養至穩定期之紅隱藻藻水一公升，將其分別裝入四支250 ml之離心管，以450 × g轉速及4°C之高速冷凍離心機離心20分鐘，倒掉上清液後，收集沉澱之藻細胞，再加入pH 6.8之磷酸緩衝溶液，接著用1810 × g轉速及4°C之高速冷凍離心機離心20分鐘後，取上清液並加入90%硫酸銨飽和度之硫酸銨粉末，以沉澱藻紅蛋白，再以上述相同條件離心20分鐘，取其沉澱物，以pH 6.8之磷酸緩衝溶液回溶後分為兩組，一組直接置入-20°C冰箱冷凍一天後，以凍結乾燥機凍乾兩天，製備成藻紅蛋白乾粉，並保存於-20°C冰箱中備用，此為無除鹽組。另一組則經緩衝溶液回溶後，再以透析濃縮機除去硫酸銨及鹽分，其方法為：將回溶後之含硫酸銨及鹽分之藻紅蛋白溶液倒入60 ml大針筒中，以流速為 75 ml/min之幫浦送液，使含硫酸銨及鹽分之藻紅蛋白溶液經過孔徑大小為3 kDa之中空纖維透析膜管柱，去除硫酸銨及鹽分而留下大小為3 kDa以上之藻紅蛋白，其再由上方橡膠管回流入大針筒中，達到透析濃縮之效果。經透析除去硫酸銨及鹽分之藻紅蛋白濃縮液置入-20°C冰箱冷凍一天後，再以凍結乾燥機凍乾兩天，最後保存於-20°C冰箱中備用，此為除鹽之藻紅蛋白乾粉。 本發明將不同階段之紅隱藻製成藻粉，結果發現一公升藻水（細胞數為2.0 × 106
cells/ml）約可得到0.621 ± 0.118 g藻粉，而每克乾重藻粉可分別得到除鹽及無除鹽藻紅蛋白粉約190.1 ± 92.0 mg/g（每克乾重）及805.0 ± 80.0 mg/g（每克乾重），證實本實驗之紅隱藻具有豐富之藻紅蛋白。 [實施例4] 紅隱藻藻紅蛋白萃取條件之研究 4-1以反覆凍融法萃取藻紅蛋白 取培養至穩定期之藻水於15 ml之離心管，每管裝12 ml藻液共三組，以405 × g轉速及4°C之桌上型冷凍離心機離心10分鐘後，倒掉上清液，每管分別各加入3 ml 之pH 6.8之磷酸緩衝溶液、海水及去離子蒸餾水（deionized distilled water, ddH2
O）共三管，將其反覆凍融各0～5次（-20°C冷凍一小時、25°C水浴20分鐘），同樣地以上述冷凍離心機條件離心10分鐘，最後，以550 nm之分光光譜儀測量上清液吸光值，本實驗三重複。其中反覆凍融0次即為無反覆凍融組。 第６圖係以反覆凍融法萃取藻紅蛋白結果。統計分析各組之間之顯著性，以相同萃取溶劑及不同反覆凍融次數之間進行比較，顯著水準訂為p ＜ 0.05，以a ＞ b ＞ c表示各組間之顯著差異，若相同字母表示兩組之間無顯著性差異。結果發現，以pH 6.8之磷酸緩衝溶液萃取之無反覆凍融組即可獲得高濃度之藻紅蛋白，且將其反覆凍融1～5次之結果差異不大。相對地，以海水萃取之無反覆凍融組獲得最低濃度之藻紅蛋白，再將其反覆凍融1～5次，可增加藻紅蛋白濃度，但效果不如pH 6.8磷酸緩衝溶液組。另外，以去離子蒸餾水進行反覆凍融之組別則是隨著反覆凍融次數增加，其藻紅蛋白濃度反而減少，為所有凍融方法中效果最差者。因此，以pH 6.8之磷酸緩衝溶液萃取（酸鹼緩衝液）之無反覆凍融組可獲得最高濃度之藻紅蛋白。 4-2以不同硫酸銨飽和度沉澱藻紅蛋白 進行硫酸銨沉澱藻紅蛋白時，通常會使用兩階段沉澱，以找出藻紅蛋白之最適沉澱濃度。本實驗先使用一階段沉澱找出適合之最終硫酸銨濃度，再以其作為兩階段沉澱之最終濃度，以找出適合之兩階段沉澱條件。 4-2-1萃取紅隱藻藻紅蛋白方法 本實驗萃取藻紅蛋白之步驟為：取裝有15 ml藻水之15 ml離心管，以405 × g轉速及4°C之桌上型冷凍離心機離心10分鐘，倒掉上清液後加入6 ml之pH 6.8磷酸緩衝溶液，再以2531 × g轉速及4°C之桌上型冷凍離心機離心10分鐘。 4-2-2一階段硫酸銨沉澱實驗 分別取上述4-2-1離心管內5 ml 上清液，置於冰浴上各加入飽和度為10%、20%、30%、40%、50%、60%、70%、80%及90%之硫酸銨粉末，均勻搖晃使粉末完全溶解後，接著再使用上述同樣條件之桌上型冷凍離心機，離心20分鐘取沉澱物，再以2.5 ml之pH 6.8磷酸緩衝溶液回溶，最後以550 nm之分光光譜儀測量其吸光值，找出一階段沉澱適合之最終硫酸銨飽和濃度。 第７圖為一階段硫酸銨沉澱藻紅蛋白結果。統計分析各組之間的顯著性，顯著水準訂為p ＜ 0.05，以a ＞ b ＞ c ＞ d ＞ e ＞ f ＞ g ＞ h ＞ i表示各組間之顯著差異，若相同字母表示兩組之間無顯著性差異。結果發現，一階段沉澱為在藻紅蛋白溶液中分別加入硫酸銨飽和度為10%、20%、30%、40%、50%、60%、70%、80%及90%之硫酸銨粉末，將其沉澱以2.5ml之pH6.8磷酸緩衝溶液回溶，其一階段沉澱藻紅蛋白之結果依序為352.00 ± 12.47μg/ml、768.67 ± 71.33μg/ml、1195.33 ± 33.99±μg/ml、1558.67 ± 96.72μg/ml、1868.67 ± 16.99μg/ml、2465.33 ± 4.71μg/ml、3108.67 ± 63.77μg/ml、4155.33 ± 116.14μg/ml、及4548.67 ± 30.00μg/ml，由上述結果發現，以90%硫酸銨飽和度可以沉澱出最高濃度之藻紅蛋白。 4-2-3兩階段硫酸銨沉澱實驗 取裝有15 ml藻水之15 ml離心管共四組，以4-2-1所述方式萃取藻紅蛋白，第一階段同樣取上述4-2-1方法之離心管內5 ml 上清液，置於冰浴上緩慢加入硫酸銨粉末使其成為10%、20%、30%、40%硫酸銨飽和度之溶液，均勻搖晃使粉末完全溶解後，再以405 × g轉速及4°C之桌上型冷凍離心機離心20分鐘，取上清液再加入硫酸銨粉末至最終飽和度（一階段實驗最適硫酸銨沉澱濃度）作為第二階段沉澱，均勻搖晃使粉末溶解後，再使用上述同樣條件之桌上型冷凍離心機離心20分鐘，取沉澱物加入2.5 ml之 pH 6.8磷酸緩衝溶液回溶，最後以550 nm之分光光譜儀測量其吸光值，藉此找出最適合之兩階段硫酸銨沉澱之飽和度。 第８圖為兩階段硫酸銨飽和濃度沉澱藻紅蛋白結果。統計分析各組之間的顯著性，顯著水準訂為p ＜ 0.05，以a ＞ b ＞ c ＞ d表示各組間之顯著差異，若相同字母表示兩組之間無顯著性差異。依據圖中所示之兩階段沉澱實驗結果，由第一階段分別加入10%、20%、30%、及40%硫酸銨濃度，最後可得到之藻紅蛋白濃度依序為3806.17 μg/ml、2923.67 μg/ml、2211.17 μg/ml、及1976.17 μg/ml。綜合上述結果發現，以10%硫酸銨飽和度進行第一階段沉澱，再將硫酸銨飽和度提升至90%進行第二階段沉澱，可得到兩階段沉澱實驗中較高濃度之藻紅蛋白。 總綜合上述4-2-2與4-2-3實驗結果發現，以一階段沉澱藻紅蛋白即可得到較高濃度之藻紅蛋白。 [實施例5] 紅隱藻藻紅蛋白特性分析 5-1藻紅蛋白定量之迴歸線 配製藻紅蛋白標準溶液濃度分別為0、62.5、125.0、250.0、500.0、750.0、1000.0、2000.0、3000.0及4000.0 μg/ml共十組，以550 nm之分光光譜儀測量其吸光值，如第９圖所示藻紅蛋白之吸光值及濃度間之迴歸關係，獲得藻紅蛋白之吸光值與濃度之迴歸線公式，為y = 0.0001x + 0.0038，其中y = 吸光值，x = 藻紅蛋白濃度，R2 = 0.999，自由度（degree of freedom, df） = 8，p = 0.01，有正相關。統計分析顯著水準訂為p ＜ 0.05。 5-2光譜圖特性分析 取除鹽之藻紅蛋白粉5 mg，以pH 6.8磷酸緩衝溶液10 ml回溶，分別以分光光譜儀及螢光光譜儀偵測其液體之吸收波長及螢光釋放強度。如第１０圖所示紅隱藻藻紅蛋白之吸收光譜圖結果，發現在550 nm處得到最高吸收峰，為PE-545型之藻紅蛋白。 使用前述分光光譜儀偵測紅隱藻藻紅蛋白之最高吸收峰為550 nm，代入上述迴歸公式後，計算出藻紅蛋白濃度，並使用其最高吸收峰做為激發光之波長，偵測藻紅蛋白560 nm至620 nm之螢光釋放強度。如第１１圖所示紅隱藻藻紅蛋白之螢光放射光譜圖結果，發現在582 nm處得到其最大螢光釋放強度。 5-3十二烷基硫酸鈉聚丙烯醯胺凝膠電泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE） 秤取除鹽之藻紅蛋白粉2 mg，以pH 6.8磷酸緩衝溶液1 ml回溶，取其10 μl與5倍之蛋白質緩衝溶液（5X protein buffer）2 μl混和均勻，至沸水中煮10分鐘後，置於冰浴中降溫，即可注入樣品槽中，並注入11-180 kDa之蛋白質分子量標準液（BlueRay Protein Ladder）作為對照標記，以 80 伏特（volt）進行電泳30分鐘再以150伏特進行電泳90分鐘，直到樣品泳動至膠體底部時終止電泳，即可將膠體從裝置上取出置於蛋白質膠體染色液（Protein Gel Stain Solution）中，並置於迴轉式振盪器上均勻搖晃染色約20分鐘，倒掉染液，再以去離子蒸餾水放置隔夜退染，以呈現蛋白質色帶，最後使用數位相機擷取影像。 第１２圖為紅隱藻藻紅蛋白之十二烷基硫酸鈉聚丙烯醯胺凝膠電泳結果。由結果發現，本實驗之藻紅蛋白在蛋白質電泳系統（SDS-PAGE）中共出現兩條色帶，第一條分子量約在20 kDa左右，為β亞基，第二條分子量約為10 kDa，為α亞基。 [實施例6] pH值、溫度及低光照時間對紅隱藻藻紅蛋白安定性之影響 6-1 pH值對紅隱藻藻紅蛋白安定性之影響 配製pH 4～10之溶液各10 ml，並分為無除鹽組及除鹽組共十四組，再加入藻紅蛋白粉1.0 mg，於4°C冰浴且避光之環境下，混合均勻後，以550 nm作為螢光光譜儀之激發波長，測定其最高螢光發射波長之螢光強度。 第１３圖(a)、(b)分別為pH值對無除鹽組及除鹽組之藻紅蛋白安定性之影響。圖中Ex為激發光波長，Em為放射光波長，Ex550與Em582表示以550 nm作為激發光波長，來偵測582 nm下之螢光強度。結果發現，除鹽組之藻紅蛋白溶液適合保存之條件為pH 5至pH 7，而無除鹽組之藻紅蛋白溶液適合保存之條件為pH 5至pH 8。同樣地，兩組藻紅蛋白溶液在pH 10時幾乎無螢光。 6-2溫度對紅隱藻藻紅蛋白安定性之影響 分別用無除鹽及除鹽之1.0 mg藻紅蛋白粉與10 ml之pH 6.8磷酸緩衝溶液，配製成濃度100 µg/ml之藻紅蛋白溶液共八組，置於4°C、25°C、50°C及75°C水浴中加熱1小時後，最後以550 nm作為螢光光譜儀之激發波長，測定其最高螢光發射波長之螢光強度。 第１４圖(a)、(b)分別為溫度對無除鹽組及除鹽組之藻紅蛋白安定性之影響。結果發現，除鹽組及無除鹽組之藻紅蛋白溶液在四種不同溫度水浴下，其穩定度有相同地趨勢：其皆在4°C及25°C時之差異不大，在50°C時螢光強度約減少50%，在75°C時則幾乎無螢光。因此藻紅蛋白對於低溫下較穩定且能維持其活性。 6-3低光照時間對紅隱藻藻紅蛋白安定性之影響 分別用無除鹽及除鹽之1.0 mg藻紅蛋白粉與10 ml之pH 6.8磷酸緩衝溶液，配製成濃度100 µg/ml之藻紅蛋白溶液共十二組，置於30 μmol photons m−2
s−1
之光度下，分別照光0、1、3、6、12及24小時後，最後以550 nm作為螢光光譜儀之激發波長，測定其最高螢光發射波長之螢光強度。其中0小時為對照組。 第１５圖(a)、(b)分別為低光照時間對無除鹽組及除鹽組之藻紅蛋白安定性之影響。結果發現，本實驗除鹽組之藻紅蛋白在低光照時間24小時內，其藻紅蛋白降低幅度較低（其螢光強度僅降低約20%，並沒有大幅度下降），因此本實驗之藻紅蛋白除鹽組對光照之穩定度較好，較適合將其保存。 [實施例7] 紅隱藻藻紅蛋白之抗氧化測試 7-1紅隱藻藻紅蛋白清除2, 2 -二苯基- 1 -苦味肼基游離基（2, 2-diphenyl-1-picrylhydrazyl, DPPH）自由基能力之測定 DPPH（C18
H12
N6
O5
）係一種含有奇數電子之較安定自由基，故對 DPPH有清除力之抗氧化劑，對其他自由基將有更佳之清除效果。本實驗根據 Shimada et al. (1992)之方法，首先配製濃度為0.5、1.0、1.5、2.0、4.0及5.0 mg/ml之藻紅蛋白溶液，並分為無除鹽組及除鹽組共十二組。分別取500 μl上述溶液，加入等體積新鮮配製溶於95 %酒精之0.1 mM之DPPH，均勻混合。靜置避光處反應30分鐘後，以517 nm分光光度計測其吸光值。對照組以pH 6.8磷酸緩衝溶液替代樣品。當DPPH 與抗氧化劑發生反應而被清除，則其吸光值就會降低，若清除DPPH自由基之能力越佳，代表其樣品抗氧化效果越好。 對照組之測試吸光值為A、實驗組之測試吸光值為B。則： DPPH清除率= [1- B/A ]× 100% 第１６圖為紅隱藻藻紅蛋白之DPPH自由基清除能力測定結果。統計分析各組之間的顯著性，以同組別及不同藻紅蛋白濃度之間進行比較 ，顯著水準訂為p ＜ 0.05，以a ＞ b ＞ c ＞ d ＞ e表示各組間之顯著差異，若相同字母表示兩組之間無顯著性差異。結果發現，除鹽組之藻紅蛋白濃度由0.5 mg/ml增加至4 mg/ml可以達到DPPH自由基清除率100% ，但在相同濃度下，無除鹽組之DPPH自由基清除率則皆低於除鹽組，其在藻紅蛋白濃度為5 mg/ml時才可達到DPPH自由基清除率100%。因此除鹽組藻紅蛋白之DPPH自由基清除能力較佳。 7-2紅隱藻藻紅蛋白之還原力 由於鐵氰化鉀溶入水中後，[Fe(CN)6
]3-
會與還原劑反應，將其還原成[Fe(CN)6
]4-
，此分子會再與Fe3+
反應生成普魯士藍（Prussian blue），於 700 nm 下有較強之吸光值。當吸光值越高表示樣品所含之還原物質越多，還原力越強。本實驗根據Senevirathne et al. (2006)之方法，首先配製濃度為1、2、4、5、10、50、100、150、200及250 mg/ml之藻紅蛋白溶液，並分為無除鹽組及除鹽組共二十組。分別取100 µl上述不同濃度之藻紅蛋白溶液，並以去離子蒸餾水作為對照組，加入100 µl pH6.6之磷酸鈉緩衝溶液及1% K3
Fe(CN)6
混合均勻後，於50°C水浴20分鐘，利用冰浴冷卻後，再加入100 µl 10% 三氯醋酸（Trichloroacetic acid），以900 × g轉速及4°C之高速微量冷凍離心機離心10分鐘，取 100 µl上清液加入100 µl 之去離子蒸餾水以及20 µl FeCl3
•6H2
O反應10分鐘，以700 nm之分光光度計測其吸光值。 第１７圖為紅隱藻藻紅蛋白之還原力結果。結果發現，不論是除鹽組或是無除鹽組，其還原力皆會因為藻紅蛋白濃度增加而上升，除鹽組之藻紅蛋白溶液在濃度200 mg/ml後還原力不再上升，其值在1.155 ± 0.049，無除鹽組之藻紅蛋白溶液則是在濃度150 mg/ml開始呈現持平，其值在0.849 ± 0.067。因此除鹽組之還原力比無除鹽組佳。 以上實驗數據利用IBM SPSS Statistics 進行單項變異數分析法（one-way ANOVA）進行統計分析，再以杜凱確實差異檢定（Tukey Honestly Significant Difference test , Tukey HSD）進行各組處理均值之比較其結果是否顯著，顯著水準設定為p ＜ 0.05。 本發明之紅隱藻藻藻粉每克乾重可萃取得到除鹽藻紅蛋白粉190.1 ± 92.0 mg/g（每克乾重）及無除鹽藻紅蛋白粉805.0 ± 80.0 mg/g，其含量豐富，比起從原核藍綠藻與大型紅藻萃取及純化者，紅隱藻可在室內快速生長，且其只具單一種藻紅蛋白，不須再加以分離其他色素，又因其不具細胞壁非常容易萃取，更能減少製備之時間及成本，極具商業化應用價值。 本發明從實驗結果發現紅隱藻適合較低溫生長，而低光度可以增加其藻紅蛋白累積，並在培養液中增加硝酸鈉濃度可提高其藻紅蛋白濃度 。在萃取紅隱藻藻紅蛋白時，可以pH 6.8磷酸緩衝溶液作為直接萃取溶液，不需經過反覆凍融，即可一次以90%飽和度硫酸銨將其沉澱，獲得高濃度藻紅蛋白。本發明之紅隱藻藻紅蛋白最高吸收峰在550 nm ，放射光波長為582 nm，其α亞基分子量約為10 kDa，而β亞基分子量約在20 kDa。在pH 5～8及溫度25°C下，紅隱藻藻紅蛋白較為穩定，雖然其藻紅蛋白對光線較不敏感，但持續光照依然會使其逐漸降解。況且，本發明紅隱藻藻紅蛋白具有清除DPPH自由基能力及還原力，又因本發明藻紅蛋白對光線較不敏感，可使螢光反應較持久，因此極適合應用於分子螢光標記。 綜上所述，本發明係一種紅隱藻之培養以產製藻紅蛋白之方法，可有效改善習用之種種缺點，紅隱藻最佳培養條件為白光且光度在30 μmol photons m−2
s−1
，溫度為24°C之環境，其生物量可達106
cells/ml，且增加培養液中硝酸鈉濃度可增加其藻紅蛋白濃度，而紅隱藻之藻紅蛋白在不需反覆凍融之pH 6.8磷酸緩衝溶液萃取後，再以90%飽和度硫酸銨將其沉澱，即可獲得最多量之藻紅蛋白，除鹽組約為190 mg/g（每克乾重），無除鹽組約為805 mg/g。另外，紅隱藻藻紅蛋白在pH 5～8及低溫（4°C～25°C）下較為穩定，經過光照24小時後僅有20%降解，屬於對光線較不敏感者，極適合應用於分子螢光標記，同時其具有清除DPPH自由基能力及還原能力，進而使本發明之産生能更進步、更實用、更符合使用者之所須，確已符合發明專利申請之要件，爰依法提出專利申請。 惟以上所述者，僅為本發明之較佳實施例而已，當不能以此限定本發明實施之範圍；故，凡依本發明申請專利範圍及發明說明書內容所作之簡單的等效變化與修飾，皆應仍屬本發明專利涵蓋之範圍內。Please refer to FIG. 1 to FIG. 17 , which are schematic diagrams showing the flow of the method of the present invention, the results of cultivating the red cryptophyta at a temperature of 18° C., and the present invention cultivating red at a temperature of 24° C. Schematic diagram of the results of cryptophyta, a schematic diagram of the results of culturing red cryptophyta at a temperature of 30 ° C, a schematic diagram of the results of culturing cryptophyta in different sodium nitrate concentrations of the present invention, and a result of extracting phycoerythrin by reverse freeze-thaw method of the present invention BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing the results of precipitating phycoerythrin by a one-stage ammonium sulfate, a schematic diagram of the result of precipitating phycoerythrin in a two-stage ammonium sulfate saturated concentration, and a regression relationship between the absorbance and concentration of the phycoerythrin of the present invention. Absorption spectrum of red cryptophycoerythrin of the present invention, fluorescence emission spectrum of cryptophycoerythrin of the present invention, and sodium dodecyl sulphate polyacrylamide gel electrophoresis of red cryptophycoerythrin of the present invention Summary of the results, the effect of the pH value of the present invention on the stability of phycoerythrin, the effect of the temperature of the present invention on the stability of phycoerythrin, and the low light illumination of the present invention Effects on stability of phycoerythrin schematic diagram of the present invention, the DPPH hidden red phycoerythrin free radical scavenging capacity of showing the results of measurement, and a schematic view of the present invention implicitly red algae Red algae proteins results reducing power. As shown in the figure: the present invention is a method for producing phycoerythrin by culturing a cryptophyta, comprising: a cryptophyta culture step s11: taking Rhodomonas sp. in a PG culture solution (PG medium) in a container, and the container is placed in an incubator for culture at a temperature between 18 and 30 ° C, with white light and a luminosity between 30 and 120 μmol photons m −2 s Between -1 and the photoperiod of 12:12 (light: dark), the biomass of the red algae can reach 10 6 cells/ml, and the PG medium is added to the seawater to add a large amount of elements. With trace elements, the macroelement contains sodium nitrite (NaNO 3 ) at a concentration of 882 to 1764 μmol/L, sodium dihydrogen phosphate (NaH 2 PO 4 .2H 2 O), disodium edetate (Na 2 ) EDTA) and ferric chloride (FeCl 3 .6H 2 O), and the trace elements contain copper sulfate (CuSO 4 .5H 2 O), zinc sulfate (ZnSO 4 .7H 2 O), cobalt chloride (CoCl 2 . 6H 2 O), manganese chloride (MnCl 2 .4H 2 O), sodium molybdate (NaMoO 4 .2H 2 O), biotin (Biotin), vitamin B 12 (Vitamin B 12 And thiazide hydrochloride/vitamin B 1 (Thiamin HCl/Vitamin B 1 ); and phycoerythrin extraction step s12: the above-mentioned stationary phase (Stationary phase) of red cryptophyta water is subjected to high-speed freeze centrifugation, The precipitated algae cells were collected and added with a phosphate buffer solution of pH 6.8, and after centrifugation at high speed, the supernatant was taken and precipitated by adding ammonium sulfate powder of 90% ammonium sulfate saturation, and freeze-dried to prepare red algae. Powder, so that each gram of dry red cryptophyta powder can be extracted to obtain desalinated phycoerythrin powder 190.1 ± 92.0 mg / g (dry weight per gram) and no desalinated phycoerythrin powder 805.0 ± 80.0 mg / g. If so, the method of the above disclosure constitutes a novel method of culturing erythrophyll to produce phycoerythrin. The invention is illustrated by the following examples, which are not intended to be limited to the scope of the invention. [Example 1] Algae culture and preparation of culture solution The red cryptophyta used in this experiment was provided by the laboratory microalgae original library under the conditions of photoperiod 12:12 (light: dark), luminosity 40 μmol photons m −2 s −1 , temperature 24°C. The culture medium used in this experiment was modified from the Provasoli formula and the formula of 2/2 of Guillard and Kilham. The fresh seawater (salt 35 ‰) was filtered on a No. 1 filter paper to prepare a PG medium (Table 1), and then placed. The autoclave was sterilized at 1.1 atm and 121 ° C for 20 minutes, taken out, and cooled for use. The preparation process of the PG culture solution is to add Stock1, 2, 1 ml of each element and trace element per liter of seawater. Table 1 [Example 2] Optimal culture conditions of Cryptophyta sinensis 2-1 Biomass determination and phycoerythrin extraction Biomass determination: 400 ml of algae culture group (i.e., 500 ml triangle flask containing 400 ml of algae water) In the culture group, 3 ml of algae water was taken each time, and 3 ml of fresh PG medium was replenished, and the number of algal cells was counted under a light microscope on a Hemocytometer. Phycoerythrin extraction: After the above calculation, 3 ml of algae water was placed in a 15 ml centrifuge tube, centrifuged at 405 × g and 4 °C in a tabletop refrigerated centrifuge for 10 minutes, and the supernatant was decanted and added. Mol pH 6.8 phosphate buffer solution, covered with aluminum foil paper, protected from light, placed in a refrigerator at -20 ° C for one day, then taken out at room temperature to melt, and then at 405 × g speed and 4 ° C table The centrifuge was centrifuged for 10 minutes, the pink supernatant was taken, and the absorbance was measured at 550 nm. 2-2 Incubation of cryptophyta with different temperature and luminosity The cryptophyta was cultured in a 500 ml triangular flask for a total of nine groups (3 temperatures × 3 luminosity), and the initial cell concentration was adjusted to 5.5 × 10 5 cells/ml. The amount of algae water is 400 ml, and the temperature is 18 ° C, 24 ° C and 30 ° C, respectively, and the luminosity is 30, 60 and 120 μmol photons m −2 s −1 , and the photoperiod is 12:12 (light: dark) ). After four weeks of culture under these conditions, renew half of the algae water (take 200 ml of algae water and then replenish 200 ml of PG medium) to prevent it from entering the Decline phase, and then follow the 2-1 method above. Take 3 ml of algae water every other two days, and replenish 3 ml of the culture solution, calculate the biomass and simultaneously extract the phycoerythrin, and then culture for 15 days. This experiment is repeated three times. In this experiment, the results of culturing red cryptic algae at three temperatures of 18 ° C, 24 ° C, 30 ° C and three luminosity 30, 60, 120 μmol photons m −2 s −1 are shown in Figures 2 to 4: Figure 2 The results of culturing red cryptophyta were carried out at a temperature of 18 °C. In the figure, (1) indicates the concentration of phycoerythrin cultured in cryptophotos at 30, 60 and 120 μmol photons m −2 s −1 , and ( 2 ) indicates cryptophyta in luminosity 30 , 60 and 120 μmol photons m − Biomass cultured at 2 s −1 . The results showed that the phycoerythrin concentration reached a maximum of 905.33 μg/ml on the seventh day at a temperature of 18 ° C and a photometric 30 μmol photons m −2 s −1 , which was higher than the other two luminosity groups. A stable period is reached after the seventh day. In contrast, at 60 μmol photons m −2 s −1 , the phycoerythrin concentration reached a maximum of 565.33 μg/ml on the third day earlier, and the biomass reached a stable phase after the fifth day. At 120 μmol photons m −2 s −1 , the phycoerythrin concentration reached a maximum of 432.00 μg/ml on the third day, the highest value being the lowest of the three luminosity groups, and the biomass reached after the fifth day. The stable period began to decline significantly after the eleventh day. Obviously, the highest phycoerythrin concentration can be obtained at low light (i.e., 30 μmol photons m −2 s −1 ) at a temperature of 18 °C. Figure 3 shows the results of culturing Red Cryptophyta at a temperature of 24 °C. In the figure, (1) indicates the concentration of phycoerythrin cultured in cryptophotos at 30, 60 and 120 μmol photons m −2 s −1 , and ( 2 ) indicates cryptophyta in luminosity 30 , 60 and 120 μmol photons m − Biomass cultured at 2 s −1 . It was found that at a temperature of 24 ° C and a luminosity of 30 μmol photons m −2 s −1 , the phycoerythrin concentration reached a maximum of 1476.66 μg/ml on the fifth day, and higher than the other two luminosity groups, the biomass was also A stable period is reached after the seventh day. In contrast, at 60 μmol photons m −2 s −1 , the phycoerythrin concentration reached a maximum of 928.66 μg/ml on the third day earlier, and the biomass reached a stable phase after the fifth day, at 120 μmol photons m − At 2 s −1 , the phycoerythrin concentration reached a maximum of 918.66 μg/ml on the fifth day, and the highest value was the lowest among the three luminosity groups, and the biomass reached a stable phase after the fifth day. Obviously, at a temperature of 24 ° C, the lowest luminosity (ie 30 μmol photons m −2 s −1 ) gives the highest phycoerythrin concentration. Figure 4 shows the results of culturing Red Cryptophyta at a temperature of 30 °C. In the figure, (1) indicates the concentration of phycoerythrin cultured in cryptophotos at 30, 60 and 120 μmol photons m −2 s −1 , and ( 2 ) indicates cryptophyta in luminosity 30 , 60 and 120 μmol photons m − Biomass cultured at 2 s −1 . The results showed that the biomass and phycoerythrin concentrations were highest at 30 ° C and 30 μmol photons m −2 s −1 , and higher than the other two luminosity groups. The biomass was on the thirteenth day. The highest concentration was 2.15 × 10 6 cells/ml, while the phycoerythrin concentration reached a maximum of 872.00 μg/ml on the third day. At 60, 120 μmol photons m −2 s −1 , the phycoerythrin concentration was seventh. The highest value was 675.33 μg/ml and 758.66 μg/ml in five days. The highest value of both was lower than the photoluminescence 30 μmol photons m −2 s −1 group, and the biomass did not have a significant logarithmic phase. stable period. Obviously, at a temperature of 30 ° C, the lowest luminosity (ie 30 μmol photons m −2 s −1 ) gives the highest phycoerythrin concentration. Based on the results of the above nine groups of experiments, it was found that the phycoerythrin concentration was not the highest when the biomass was the highest (days), after deducting the biomass of the first day, at a temperature of 30 ° C, luminosity 60 Μmol photons m −2 s −1 and 120 μmol photons m −2 s −1 The biomass of the two groups was maintained below 6.5 × 10 5 cells/ml, and the remaining seven groups of experimental biomass Both can reach 1.5 × 10 6 cells / ml, so the results of this experiment found that cryptophyta is more suitable for cultivation at temperatures of 18 ° C and 24 ° C. However, when the temperature is 30 ° C and the luminosity is 30 μmol photons m −2 s −1 (low luminosity), the growth amount can be maintained. In addition, as a result of phycoerythrin concentration, the phycoerythrin concentration in the low temperature (30 μmol photons m −2 s −1 ) was also observed in the culture temperature at different temperatures (18 ° C to 30 ° C). highest. Therefore, it is proved that the phycoerythrin concentration of cryptophyta is increased in a low light environment. The phycoerythrin concentration in the culture group at the temperature of 24 ° C and the luminosity of 30 μmol photons m −2 s −1 was up to 1308.66 μg/ml, and the highest phycoerythrin concentration in the other groups was below 835.33 μg/ml. Therefore, the optimum conditions for the highest concentration of phycoerythrin in the cultivation of red cryptophyta are: temperature 24 ° C, luminosity 30 μmol photons m −2 s −1 , which is greatly affected by luminosity. 2-3 cultivating cryptophyta with different concentrations of sodium nitrate. The cryptophyta was cultured in four groups of 500 ml flasks (four sodium nitrate concentrations), and the initial cell concentration was adjusted to 2.7 × 10 5 cells/ml. For 400 ml, the sodium nitrate concentrations were 882 μmol/L (original concentration), 1103 μmol/L (1.25 times), 1323 μmol/L (1.5 times), and 1764 μmol/L (2 times). The culture conditions are based on the experimental results under the optimal culture conditions, the photoperiod is 12:12 (light: dark), and 3 ml of algae water is taken every two days according to the above 2-1 method, and the PG culture solution is not added back, and the calculation is performed. Biomass and extraction of phycoerythrin, co-culture for 35 days, three replicates of this experiment. Figure 5 shows the results of culturing Red Cryptophyta with different concentrations of sodium nitrate. In the figure, (1) indicates the concentration of phycoerythrin cultured in cryptophyta algae at 882 μmol/L, 1103 μmol/L, 1323 μmol/L, and 1764 μmol/L, and (2) indicates that cryptophyta is 882 μmol/L. Biomass cultured at 1103 μmol/L, 1323 μmol/L, and 1764 μmol/L. The results showed that the four groups (original concentration, 1.25 times, 1.5 times, and 2 times) of different sodium nitrate concentration groups reached the highest concentration of phycoerythrin on the eleventh day, respectively, 1052.00 μg/ml, 1062.00 μg/ml. At 1257.00 μg/ml and 1277.00 μg/ml, the phycoerythrin concentration increased with the increase of sodium nitrate concentration. After the eleventh day of culture, the concentration of phycoerythrin in the original sodium nitrate concentration group gradually decreased, but The phycoerythrin concentration in the 2-fold sodium nitrate concentration group was maintained until the thirty-first day of culture. In addition, the 1.25 times sodium nitrate concentration, the phycoerythrin concentration increased by 0.01%, the 1.5 times sodium nitrate concentration, the phycoerythrin concentration increased by 0.19%, the 2-fold sodium nitrate concentration, and the phycoerythrin increased by 0.21%, thereby increasing the phycoerythrin The concentration of sodium nitrate in the culture solution does increase the concentration of erythrophycein. At the original concentration, 1.25 times, 1.5 times and 2 times sodium nitrate concentration, the phycoerythrin extracted per milliliter of algae water can get NT$1009.92, 1019.52 yuan, 1206.72 yuan and 1225.92 yuan, but its sodium nitrate cost per milliliter. However, it only costs NT$0.00024, 0.00003, 0.000036 and 0.000048, which is relatively cost-performance. Therefore, increasing the concentration of sodium nitrate in the culture solution of the cryptophyta to increase the concentration of phycoerythrin is a very economical and effective way to increase the income. [Example 3] Preparation of red cryptic algae powder and preparation of its phycoerythrin 3-1 red cryptophyta powder This experiment collects two different growth stages of cryptophyta, ie, cryptophyta Algae water, and one liter of each of the red cryptophyta water cultured to the senescence stage, respectively, were placed in a 250 ml centrifuge tube, centrifuged at 450 × g speed and 4 ° C high-speed refrigerated centrifuge for 20 minutes to collect the precipitate. The algae cells were placed in a refrigerator at -20 ° C for one day, and then freeze-dried for two days in a freeze dryer to prepare red algae algae powder, which was finally stored in a refrigerator at -20 ° C for later analysis. 3-2 extraction of red cryptophycophycoerythrin A liter of red cryptophyta water cultured to a stable period, which was separately charged into four 250 ml centrifuge tubes, and frozen at 450 × g speed and 4 ° C high speed. Centrifuge in a centrifuge for 20 minutes, pour off the supernatant, collect the precipitated algae cells, add a pH 6.8 phosphate buffer solution, and then centrifuge at 2010 × g speed and 4 ° C high-speed refrigerated centrifuge for 20 minutes, then take The supernatant was added with 90% ammonium sulfate saturation ammonium sulfate powder to precipitate phycoerythrin, and then centrifuged for 20 minutes under the same conditions as above, and the precipitate was taken and dissolved in phosphate buffer solution of pH 6.8 to be divided into two groups. One group was directly placed in a -20 ° C refrigerator for one day, and then freeze-dried in a freeze dryer for two days to prepare a dry powder of phycoerythrin, which was stored in a refrigerator at -20 ° C for use, which was a salt-free group. The other group is then dissolved in the buffer solution, and then the ammonium sulfate and the salt are removed by a dialysis concentrator by pouring the remelted ammonium sulphate and salt phycoerythrin solution into a 60 ml large syringe. The pump with a flow rate of 75 ml/min is used to feed the phycoerythrin solution containing ammonium sulfate and salt through a hollow fiber dialysis membrane column with a pore size of 3 kDa to remove ammonium sulfate and salt and leave a size of 3 kDa or more. The phycoerythrin is recirculated into the large syringe by the upper rubber tube to achieve the effect of dialysis concentration. The phycoerythrin concentrate which has been subjected to dialysis to remove ammonium sulfate and salt is placed in a refrigerator at -20 ° C for one day, and then freeze-dried in a freeze dryer for two days, and finally stored in a refrigerator at -20 ° C for use as a desalting solution. Dried phycoerythrin powder. The invention makes different stages of cryptophyta as algal flour, and finds that one liter of algae water (cell number is 2.0 × 10 6 cells/ml) can obtain about 0.621 ± 0.118 g of algal flour, and per gram of dry weight algae powder can be Desalination and non-desaled phycoerythrin powder were obtained, respectively, about 190.1 ± 92.0 mg / g (dry weight per gram) and 805.0 ± 80.0 mg / g (dry weight per gram), which confirmed that the red cryptophytes in this experiment are rich in algae. Red protein. [Example 4] Study on extraction conditions of phycoerythrin from red cryptophyllum 4-1 Extraction of phycoerythrin by reverse freeze-thaw method Take the algae water cultured to a stable period in a 15 ml centrifuge tube, each tube containing 12 ml of algae solution After three minutes of centrifugation at 405 × g and 4 °C in a tabletop refrigerated centrifuge, the supernatant was decanted, and 3 ml of pH 6.8 phosphate buffer solution, seawater and deionized distilled water were added to each tube. (deionized distilled water, ddH 2 O) a total of three tubes, which were repeatedly frozen and thawed 0 to 5 times (-20 °C for one hour, 25 ° C water bath for 20 minutes), and similarly centrifuged under the above-mentioned refrigerated centrifuge conditions. Minutes, finally, the absorbance of the supernatant was measured by a spectrometer at 550 nm, and the experiment was repeated three times. Among them, the freezing and thawing of 0 times is the no-freezing-freezing group. Figure 6 shows the results of extracting phycoerythrin by reverse freeze-thaw method. The significance of each group was statistically analyzed. The comparison between the same extraction solvent and the number of different freeze-thaw cycles was made. The significant level was set to p < 0.05, and a > b > c indicates the significant difference between the groups. Indicates no significant difference between the two groups. As a result, it was found that a high concentration of phycoerythrin was obtained by the non-reversed freeze-thaw group extracted with a phosphate buffer solution of pH 6.8, and the results were not significantly frozen and thawing 1 to 5 times. In contrast, the lowest concentration of phycoerythrin obtained by seawater extraction without re-freezing and thawing, and then freeze-thawed one to five times, can increase the phycoerythrin concentration, but the effect is not as good as the pH 6.8 phosphate buffer solution group. In addition, the combination of deionized distilled water for reverse freezing and thawing is accompanied by an increase in the number of repeated freeze-thaw cycles, and the phycoerythrin concentration is reduced, which is the worst among all freeze-thaw methods. Therefore, the highest concentration of phycoerythrin can be obtained by the non-reversed freeze-thaw group extracted with a pH 6.8 phosphate buffer solution (acid-base buffer). 4-2 When phycoerythrin is precipitated with different ammonium sulfate saturation to precipitate phycoerythrin by ammonium sulfate, a two-stage precipitation is usually used to find the optimum precipitation concentration of phycoerythrin. This experiment first uses a one-stage precipitation to find the appropriate final ammonium sulfate concentration, which is then used as the final concentration of the two-stage precipitation to find a suitable two-stage precipitation condition. 4-2-1 Extraction of cryptophyta phycoerythrin method The procedure of extracting phycoerythrin in this experiment is as follows: 15 ml centrifuge tube containing 15 ml of algae water, frozen at 405 × g and desktop at 4 °C Centrifuge for 10 minutes in a centrifuge, pour off the supernatant, add 6 ml of pH 6.8 phosphate buffer solution, and centrifuge at 1031 × g for 4 minutes in a tabletop refrigerated centrifuge at 4 °C. 4-2-2 One-stage ammonium sulfate precipitation experiment Take 5 ml of the supernatant in the above 4-2-1 centrifuge tube, and place the saturation on the ice bath for 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% ammonium sulfate powder, after uniformly shaking to completely dissolve the powder, and then using a tabletop refrigerated centrifuge of the same conditions as above, centrifugation for 20 minutes to take a precipitate, and then 2.5 ml of pH 6.8 phosphate buffer solution was dissolved, and finally the absorbance was measured by a 550 nm spectrometer to find the final ammonium sulfate saturation concentration suitable for the first stage precipitation. Figure 7 shows the results of a one-stage ammonium sulfate precipitation of phycoerythrin. Statistical analysis of the significance between the groups, the significant level is set to p < 0.05, with a > b > c > d > e > f > g > h > i indicates a significant difference between the groups, if the same letter indicates two groups There was no significant difference between the two. As a result, it was found that the first-stage precipitation was to add ammonium sulfate powder having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% ammonium sulfate saturation to the phycoerythrin solution. The precipitate was reconstituted with 2.5 ml of pH 6.8 phosphate buffer solution, and the results of the first stage precipitation of phycoerythrin were 352.00 ± 12.47 μg/ml, 768.67 ± 71.33 μg/ml, and 1193.33 ± 33.99 ± μg/ml. , 1558.67 ± 96.72μg/ml, 1186.67 ± 16.99μg/ml, 2465.33 ± 4.71μg/ml, 3108.67 ± 63.77μg/ml, 4145.33 ± 116.14μg/ml, and 4454.67 ± 30.00μg/ml, from the above results, 90% ammonium sulfate saturation can precipitate the highest concentration of phycoerythrin. 4-2-3 two-stage ammonium sulfate precipitation experiment Four groups of 15 ml centrifuge tubes containing 15 ml of algae water were used to extract phycoerythrin in the manner described in 4-2-1. The first stage also took the above 4-2 5 ml of the supernatant in the centrifuge tube of the -1 method, placed on an ice bath, slowly added ammonium sulfate powder to make a solution of 10%, 20%, 30%, 40% ammonium sulfate saturation, and evenly shake to completely dissolve the powder. After that, it was centrifuged at 405 × g and 4 °C in a tabletop refrigerated centrifuge for 20 minutes. The supernatant was added and then ammonium sulfate powder was added to the final saturation (the optimal concentration of ammonium sulfate in the first stage experiment) as the second stage. Precipitate, evenly shake to dissolve the powder, and then centrifuge for 20 minutes using the same type of table-top refrigerated centrifuge. The precipitate was added to 2.5 ml of pH 6.8 phosphate buffer solution for dissolution, and finally the absorbance was measured by a 550 nm spectrometer. The value is used to find the saturation of the two-stage ammonium sulfate precipitate that is most suitable. Figure 8 shows the results of precipitation of phycoerythrin at a two-stage ammonium sulfate saturation concentration. The significance of each group was statistically analyzed. The significant level was set at p < 0.05, and a > b > c > d indicates a significant difference between the groups. If the same letter indicates no significant difference between the two groups. According to the results of the two-stage precipitation experiment shown in the figure, 10%, 20%, 30%, and 40% ammonium sulfate concentrations were added from the first stage, and the final phycoerythrin concentration was 3806.17 μg/ml. 2923.67 μg/ml, 221.11 μg/ml, and 1976.17 μg/ml. Based on the above results, it was found that the first stage precipitation was carried out with 10% ammonium sulfate saturation, and the ammonium sulfate saturation was increased to 90% for the second stage precipitation, and a higher concentration of phycoerythrin in the two-stage precipitation experiment was obtained. The results of the above 4-2-2 and 4-2-3 experiments were combined to find that a higher concentration of phycoerythrin was obtained by precipitating phycoerythrin in one stage. [Example 5] Analysis of phycoerythrin characteristics of cryptophyta 5-1 The regression line of phycoerythrin quantification The concentration of phycoerythrin standard solution was 0, 62.5, 125.0, 250.0, 500.0, 750.0, 1000.0, 2000.0, 3000.0 and 4000.0, respectively. A total of ten groups of μg/ml were measured by the spectrometer at 550 nm. The regression relationship between the absorbance and concentration of phycoerythrin as shown in Fig. 9 was obtained, and the regression formula of the absorbance and concentration of phycoerythrin was obtained. For y = 0.0001x + 0.0038, where y = absorbance, x = phycoerythrin concentration, R2 = 0.999, degree of freedom, df = 8, p = 0.01, positive correlation. The statistical analysis showed a significant level of p < 0.05. 5-2 Spectrogram Character Analysis 5 mg of phycoerythrin powder was removed and dissolved in 10 ml of pH 6.8 phosphate buffer solution. The absorption wavelength and fluorescence emission intensity of the liquid were detected by spectrophotometer and fluorescence spectrometer, respectively. As shown in Fig. 10, the absorption spectrum of red cryptophycoerythroprotein was found to have the highest absorption peak at 550 nm, which is the phycoerythrin of PE-545 type. The highest absorption peak of phycoerythrin was detected by the above spectrophotometer to be 550 nm. After substituting the above regression formula, the phycoerythrin concentration was calculated, and the highest absorption peak was used as the wavelength of the excitation light to detect algae. Fluorescence release intensity of protein from 560 nm to 620 nm. As shown in Fig. 11, the fluorescence emission spectrum of red cryptophycoerythroprotein was found to have its maximum fluorescence emission intensity at 582 nm. 5-3 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Take 2 mg of desalinated phycoerythrin powder and dissolve it in 1 ml of pH 6.8 phosphate buffer solution. Mix 10 μl with 5 times protein buffer solution (2X protein buffer) 2 μl, mix in boiling water for 10 minutes, then cool in an ice bath, then inject into the sample tank and inject 11-180 kDa As a control marker, the BlueRay Protein Ladder was electrophoresed at 80 volts for 30 minutes and then electrophoresed at 150 volts for 90 minutes until the sample was moved to the bottom of the gel and the electrophoresis was terminated. The sample was taken out in Protein Gel Stain Solution and shaken on a rotary shaker for about 20 minutes. The dye solution was drained and then deionized with distilled water to give an overnight dyeing to reveal a protein ribbon. Finally, use a digital camera to capture images. Figure 12 shows the results of gel electrophoresis of sodium dodecyl sulfate polyacrylamide in cryptophyta. It was found that the phycoerythrin in this experiment showed two bands in the protein electrophoresis system (SDS-PAGE). The first molecular weight was about 20 kDa, which was β subunit, and the second molecular weight was about 10 kDa. Is the alpha subunit. [Example 6] Effect of pH value, temperature and low light time on the stability of phycoerythrin of red cryptophyte 6-1 Effect of pH value on the stability of phycoerythrin of red cryptophyll Preparing 10 ml of solution of pH 4-10 And divided into 14 groups without salt removal and desalting group, then add phycoerythrin powder 1.0 mg, in an ice bath at 4 ° C and protected from light, after mixing evenly, 550 nm as a fluorescence spectrometer The excitation wavelength is measured and the fluorescence intensity of the highest fluorescence emission wavelength is measured. Fig. 13 (a) and (b) show the effect of pH on the stability of phycoerythrin in the no-salt group and the desalting group, respectively. In the figure, Ex is the wavelength of the excitation light, Em is the wavelength of the emitted light, and Ex550 and Em582 indicate the wavelength of the excitation light at 550 nm to detect the fluorescence intensity at 582 nm. As a result, it was found that the phycoerythrin solution of the desalting group was suitable for storage under the conditions of pH 5 to pH 7, and the phycoerythrin solution without the desalting group was suitable for storage from pH 5 to pH 8. Similarly, the two groups of phycoerythrin solutions had almost no fluorescence at pH 10. 6-2 The effect of temperature on the stability of phycoerythrin of red cryptophyta was prepared with 1.0 mg phycoerythrin powder without salt removal and desalting and 10 ml pH 6.8 phosphate buffer solution, respectively, to prepare algae at a concentration of 100 μg/ml. A total of eight groups of red protein solution were placed in a water bath at 4 ° C, 25 ° C, 50 ° C and 75 ° C for 1 hour, and finally the highest fluorescence emission wavelength was determined with 550 nm as the excitation wavelength of the fluorescence spectrometer. Fluorescence intensity. Fig. 14 (a) and (b) show the effect of temperature on the stability of phycoerythrin in the no-salt group and the desalting group, respectively. The results showed that the stability of the phycoerythrin solution in the salt-removal group and the salt-free group had the same trend in four different temperature water baths: they all had little difference at 4 ° C and 25 ° C, at 50 At °C, the fluorescence intensity is reduced by about 50%, and at 75 °C, there is almost no fluorescence. Therefore, phycoerythrin is relatively stable at low temperatures and maintains its activity. 6-3 The effect of low light time on the stability of phycoerythrin of red cryptophyta was prepared by using 1.0 mg phycoerythrin powder without salt removal and desalting and 10 ml pH 6.8 phosphate buffer solution, respectively, to a concentration of 100 μg/ml. A total of twelve groups of phycoerythrin solution were placed under the luminosity of 30 μmol photons m −2 s −1 for 0, 1 , 3, 6, 12 and 24 hours respectively, and finally 550 nm as the fluorescence spectrometer. The excitation wavelength was measured and the fluorescence intensity of the highest fluorescence emission wavelength was measured. Among them, 0 hours was the control group. Figure 15 (a) and (b) show the effect of low light time on the stability of phycoerythrin in the salt-free and desalin-free groups. It was found that the phycoerythrin in the salt-removing group of this experiment had a lower decrease in phycoerythrin within 24 hours of low light time (the fluorescence intensity was only reduced by about 20% and did not decrease significantly), so the experiment The phycoerythrin desalting group has better stability to light and is more suitable for preservation. [Example 7] Antioxidant test of erythrococcal phycoerythrin 7-1 Red cryptophyta phycoerythrin clearance 2, 2 -diphenyl-1 -picryl group free radical (2, 2-diphenyl-1-picrylhydrazyl , DPPH) Determination of free radical capacity DPPH (C 18 H 12 N 6 O 5 ) is a relatively stable free radical containing genomic electrons, so it has a scavenging antioxidant for DPPH, which will have better scavenging effect on other free radicals. . According to the method of Shimada et al. (1992), the phycoerythrin solutions at concentrations of 0.5, 1.0, 1.5, 2.0, 4.0 and 5.0 mg/ml were first prepared and divided into no salt removal group and salt removal group. Two groups. 500 μl of the above solution was taken separately, and an equal volume of freshly prepared 0.1 mM DPPH dissolved in 95% alcohol was added and uniformly mixed. After standing for 30 minutes in the dark place, the absorbance was measured by a 517 nm spectrophotometer. The control group was replaced with a pH 6.8 phosphate buffer solution. When DPPH is removed by reaction with an antioxidant, its absorbance is reduced. The better the ability to remove DPPH free radicals, the better the antioxidant effect of the sample. The test absorbance value of the control group was A, and the test absorbance value of the experimental group was B. Then: DPPH clearance rate = [1- B/A ] × 100% Figure 16 shows the results of DPPH free radical scavenging ability of red cryptophycoerythroprotein. The significance of each group was statistically analyzed. The comparison between the same group and different phycoerythrin concentrations was made. The significant level was set at p < 0.05, and a > b > c > d > e indicates the significant difference between the groups. If the same letter indicates no significant difference between the two groups. The results showed that the phycoerythrin concentration in the desalting group increased from 0.5 mg/ml to 4 mg/ml, and the DPPH free radical scavenging rate was 100%. However, at the same concentration, the DPPH free radical scavenging rate in the non-salting group was Below the salt removal group, the DPPH free radical scavenging rate was 100% at a phycoerythrin concentration of 5 mg/ml. Therefore, the desalting group phycoerythrin has a better DPPH radical scavenging ability. Reducing power of 7-2 red cryptophycoerythroprotein [Fe(CN) 6 ] 3- will react with reducing agent to reduce it to [Fe(CN) 6 ] 4- This molecule will react with Fe 3+ to form Prussian blue, which has a strong absorbance at 700 nm. The higher the absorbance value, the more reducing substances are contained in the sample, and the stronger the reducing power. According to the method of Senevirathne et al. (2006), the phycoerythrin solution at concentrations of 1, 2, 4, 5, 10, 50, 100, 150, 200 and 250 mg/ml was first prepared and divided into no divisions. There were 20 groups in the salt group and the salt removal group. Take 100 μl of the above different concentrations of phycoerythrin solution, and use deionized distilled water as the control group, add 100 μl of sodium phosphate buffer solution of pH 6.6 and 1% K 3 Fe(CN) 6 and mix evenly at 50°. C water bath for 20 minutes, after cooling in an ice bath, add 100 μl of 10% trichloroacetic acid, centrifuge at a speed of 900 × g and a high-speed micro-refrigerated centrifuge at 4 ° C for 10 minutes, and take 100 μl of the supernatant. 100 μl of deionized distilled water and 20 μl of FeCl 3 •6H 2 O were added for 10 minutes, and the absorbance was measured by a spectrophotometer at 700 nm. Figure 17 shows the results of the reducing power of phycoerythrin. The results showed that the reducing power of the desalin group or the salt-free group increased due to the increase of phycoerythrin concentration. The reducing power of the phycoerythrin solution in the salt-removing group was no longer increased after the concentration of 200 mg/ml. The value was 1.155 ± 0.049, and the phycoerythrin solution without the salt removal group began to appear flat at a concentration of 150 mg/ml, with a value of 0.849 ± 0.067. Therefore, the reducing power of the desalting group is better than that of the salt-free group. The above experimental data was analyzed by IBM SPSS Statistics for one-way ANOVA, and the results of each group were compared by Tukey Honestly Significant Difference Test (Tukey HSD). Significantly, the significant level was set to p < 0.05. The red cryptophyta powder of the present invention can be extracted per gram of dry weight to obtain dehydrated phycoerythrin powder of 190.1 ± 92.0 mg/g (dry weight per gram) and no desalinated phycoerythrin powder of 805.0 ± 80.0 mg/g. Rich in content, compared with those extracted and purified from prokaryotic blue-green algae and large red algae, red cryptophytes can grow rapidly indoors, and they only have a single phycoerythrin, no need to separate other pigments, and because they do not The cell wall is very easy to extract, which reduces the time and cost of preparation and is of great commercial value. The experimental results show that the cryptophyta is suitable for lower temperature growth, and the low luminosity can increase the accumulation of phycoerythrin, and increasing the concentration of sodium nitrate in the culture solution can increase the phycoerythrin concentration. When extracting red cryptic acid phycoerythrin, the pH 6.8 phosphate buffer solution can be used as a direct extraction solution, and it can be precipitated once with 90% saturation ammonium sulfate without repeated freezing and thawing to obtain a high concentration of phycoerythrin. The highest absorption peak of phycoerythrin of the present invention is 550 nm, the wavelength of the emitted light is 582 nm, the molecular weight of the α subunit is about 10 kDa, and the molecular weight of the β subunit is about 20 kDa. At pH 5-8 and temperature 25 °C, phycoerythrin is more stable. Although its phycoerythrin is less sensitive to light, continuous illumination will gradually degrade it. Moreover, the red cryptophycoerythrin of the present invention has the ability to scavenge DPPH radicals and the reducing power, and the phycoerythrin of the present invention is less sensitive to light, and the fluorescence reaction is longer, so it is highly suitable for molecular fluorescent labeling. . In summary, the present invention is a method for producing phycoerythrin by culturing a cryptophyta, which can effectively improve various disadvantages. The optimal culture condition of erythrophyta is white light and the luminosity is 30 μmol photons m −2 s −1 , the environment with a temperature of 24 ° C, the biomass can reach 10 6 cells / ml, and increasing the concentration of sodium nitrate in the culture solution can increase the concentration of phycoerythrin, while the phycoerythrin of red cryptophyta does not need to be repeated After freezing and thawing pH 6.8 phosphate buffer solution, it is precipitated with 90% saturation ammonium sulfate to obtain the most amount of phycoerythrin. The salt removal group is about 190 mg/g (dry weight per gram), no The desalting group is approximately 805 mg/g. In addition, red cryptophycophytin is stable at pH 5-8 and low temperature (4 °C ~ 25 °C), only 20% degradation after 24 hours of illumination, which is less sensitive to light, and is very suitable for application. In the molecular fluorescent labeling, and at the same time it has the ability to scavenge DPPH free radicals and reduce the ability, so that the production of the present invention can be more advanced, more practical, more in line with the needs of the user, and indeed meets the requirements of the invention patent application, File a patent application. However, the above is only the preferred embodiment of the present invention, and the scope of the present invention is not limited thereto; therefore, the simple equivalent changes and modifications made in accordance with the scope of the present invention and the contents of the invention are modified. All should remain within the scope of the invention patent.