比較不同方法由Sargassum polycystum萃取生物活性蛋白

Comparisons of Various Methods to Extract Bioactive Protein from Sargassum polycystum

白水晶(Daina Balan)

指導教授 ： 邱亞伯

國立屏東科技大學/國際學院/熱帶農業暨國際合作系/碩士(2021年)

https://doi.org/10.6346/NPUST202100433

摘要

海藻是許多抗癌、抗發炎和抗氧化特性生物活性化合物的重要來源。因此，研究海藻內生物活性化合物對食品科學和生物技術行業具重大意義。本次實驗中所研究的生物活性化合物為生物活性海藻蛋白。實驗用的海藻為台灣墾丁採集的匍枝馬尾藻Sargassum polycystum。海藻樣品保存在-20⁰ C保鮮，然後在-80⁰ C冷凍的海藻樣品進行預先處理，之後將等分試樣冷凍樣品在液態氮 (LN2)中進行研磨。剩餘的冷凍樣品將被冷凍乾燥並磨碎。接下來我們將對新鮮、液氮冷凍和凍乾樣品進行水分測定並根據水分含量結果計算樣品的乾重。隨後利用凱氏定氮法測定出匍枝馬尾藻樣品的粗蛋白百分比為 4.51%。通過計算我們可以得出乾燥樣品內可提取的粗蛋白之比重。此研究包括了五種萃取法。首先，冷凍乾燥的樣品提取方法為改良版三氯乙酸 (TCA)/丙酮沉澱法，標記為1a. 經液氮研磨所提取的海藻樣品則標記為1b。由鹽析（硫酸銨，（NH4）2SO4，分餾法）法從凍乾樣品中獲得蛋白質提取物標記為2a；由新鮮的樣品提取蛋白質的方法則標記為 2b。硫酸銨分餾法的樣本根據不同的飽和百分比分別標記為（20%-2a/2b20、40%-2a/2b40、60%-2a/2b60和80%-2a/2b80）。第三種從凍乾樣品中提取蛋白質的方法為經優化超音波震盪輔助蛋白質提取法，標記為 3a。第四種方法為混合萃取法, 採用了部分超音波震盪與鹽析法的萃取方式，標記為 4a。第五種方法同為混合萃取法，整合了鹽析、三氯乙酸/丙酮沉澱法和超音波震盪法的萃取方式，標記為4b。兩組混合萃取法所用的樣品均經液氮研磨處理。混合萃取法的飽和度百分比分別標記為(20%-4a/4b20、40%-4a/4b40、60%-4a/4b60和80%-4a/4b80)。我們用 Thermo Scientific™ NanoDrop 2000 對每種方法的所得蛋白質萃取物進行蛋白質的濃度測定。通過該數據計算蛋白質產率，並比較蛋白質濃度與可提取的總粗蛋白質。接著，我們利用ABTS法 (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid))和DPPH (1, 1-Diphenyl-2- Picrylhydrazyl) 來測定蛋白質的抗氧化活性。然後，經由計算得出樣本的半抑制值IC50 以用於ABTS檢定。最後，使用 SDS-PAGE（十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳）對蛋白質萃取物進行分子量測定。實驗後我們對所有結果進行統計分析與顯著性差異檢定。在這項研究中，第一種組合方法 4a和超音波震盪法3a所萃取物中的蛋白質濃度與其他方法相比有顯著提升。根據實驗結果， 4a20樣品中的蛋白質濃度為 28.9 mg/mL，3a 樣品中的蛋白質濃度則為 16.6 mg/mL。TCA/丙酮沉澱法、第一綜合萃取法和第二綜合萃取法皆呈現高蛋白質產率。1b、4a40和4b40 樣品的產量分別為51.6%、39.1%和36.3%，因此得出以上樣品所採用的方法能有效萃取蛋白質。此外，ABTS 和DPPH 測定結果顯示第二種混合萃取法的蛋白質萃取物與其他方法所獲得的萃取物相比擁有更高的抗氧化活性。 ABTS 測定中樣品1a、4b20、4b60及4b80的抑制率各為 98.2%、 99.1%、98.7%及98.5%。此外，4b80 樣品的IC50 ABTS 測定值0.067 mg/mL為測試組中最低，這表明該蛋白質萃取物樣品能有效清除ABTS 自由基。雖然抑制率不如4b80那麼顯著，但就總體而言樣品4b20、4b40、4b60 和1a均呈現低 IC50值。反之從其他方法獲得的樣品均呈現較高的IC50值。第二種混合萃取法的樣品4b40、4b60及4b80在 DPPH 測定中均呈現高抑制率，分別為87.2%、88.6%及93.0%。實驗中唯一經SDS-PAGE電泳分析的樣品為80% 硫酸銨飽和度下的萃取物，估算出分子量為43 kDa。本研究的宗旨為通過比較在五種萃取法中尋找溫和有效的生物活性蛋白萃取法。此研究成功證明了所萃取的蛋白質具有效抗氧化能力，並有希望在食品生物技術行業中進行實際應用。
關鍵字：ABTS、生物活性蛋白、DPPH、匍枝馬尾藻、SDS-PAGE

關鍵字

ABTS ； 生物活性蛋白 ； DPPH ； 匍枝馬尾藻 ； SDS-PAGE

並列摘要

Seaweed is a source of many important bioactive compounds that exhibit anti-cancer, anti-inflammatory, and antioxidant properties. Thus, the compounds responsible for these attributes are important to the Food Science and Biotechnology industries. The bioactive compounds of interest in this study are bioactive seaweed proteins. Therefore, samples of the brown seaweed species, Sargassum polycystum, were collected from Kenting, Taiwan. The seaweed sample was primarily kept fresh at -20⁰ C, and the seaweed sample frozen at -80⁰ C was later pretreated. An aliquot of the frozen sample was crushed in liquid nitrogen (LN2). The remaining frozen sample was freeze-dried and ground. Then, the moisture analysis of the fresh, liquid nitrogen frozen, and freeze-dried samples was conducted. The dry weight of the samples was calculated from the moisture content results. Subsequently, the Kjeldahl method determined the crude protein percent of the Sargassum polycystum sample, which was 4.51%. The total crude protein calculations revealed the crude protein available for extraction based on the dry weight of samples. Five distinct methods were employed to extract the available proteins. First, the modified trichloroacetic acid (TCA)/Acetone method was labeled 1a provided that the extraction sample was freeze-dried. If the seaweed sample for extraction was pretreated with LN2, then the method was labeled 1b. Second, the salting-out (ammonium sulfate, (NH₄)₂SO₄, fractionation) method attaining protein extracts from the freeze-dried samples was labeled 2a; if the samples were fresh, the method was labeled 2b. The ammonium sulfate fractionation method was additionally labeled according to the saturation percentages (20%-2a/2b20, 40%-2a/2b40, 60%-2a/2b60, and 80%-2a/2b80). Third, the optimized ultrasound-assisted method, labeled 3a, extracted protein from the freeze-dried samples. Fourth, the first combined method, which incorporated some processes from the ultrasound-assisted and salting-out methods, was labeled 4a. Fifth, the second combined method, labeled 4b, integrated different processes from the salting out, TCA/acetone, and ultrasound-assisted methods. The combined methods both extracted protein from samples pretreated with LN2. The combined methods were further labeled according to the saturation percentages (20%-4a/4b20, 40%-4a/4b40, 60%-4a/4b60, and 80%-4a/4b80). The resulting protein extracts derived from each respective method were quantified using a Thermo Scientific™ NanoDrop 2000 to determine the concentration of proteins within a sample. The protein yield was then calculated and indicated the concentration of protein extracted by each method compared to the total crude protein available for extraction. Subsequently, the antioxidant activity of the protein extracts produced by each method was investigated by ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging and DPPH (1, 1-Diphenyl-2-Picrylhydrazyl) assays. Then, the IC50 values of samples were calculated for the ABTS assay. Finally, the protein extracts were visualized using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to determine the molecular weight of extracted proteins. The statistical analysis for all the results was conducted, and the significant differences were determined. In this study, the first combined method, 4a, and the ultrasound-assisted method, 3a, resulted in higher protein concentrations in samples compared to the other protein extraction protocols. The results revealed protein concentrations of 28.9 mg/mL in 4a20 samples and 16.6 mg/mL in 3a samples. The TCA/acetone, first combined, and second combined methods exhibited high protein yield. Since these methods producing 1b, 4a40, and 4b40 samples revealed yields of 51.6%, 39.1%, and 36.3% respectively, then the methods employed were effective in protein extraction. Furthermore, results from the ABTS and DPPH assays conducted on the protein extracts from the second combined method indicated that these samples had high antioxidant activity compared to the samples derived from the other methods. The inhibition percentages of samples in the ABTS assay was 98.2% for 1a, 99.1% for 4b20, 98.7% for 4b60, and 98.5% for 4b80. Furthermore, the 4b80 sample displayed the lowest IC50 value for the ABTS assay, 0.067 mg/mL, which indicated that this protein extract sample can effectively scavenge the ABTS radicals. Similarly, however not at the same capacity, the 4b20, 4b40, 4b60, and 1a samples exhibited low IC50 values. Conversely, samples obtained from all other methods resulted in higher IC50 values. The samples of the second combined method also attained the highest inhibition percentage in the DPPH assay of 87.2% for 4b40, 88.6% for 4b60, and 93.0% for 4b80. The protein extracts derived from the combined method at 80% ammonium sulfate saturation was the only sample visualized in SDS-PAGE analysis, with an estimated molecular weight of 43 kDa. The premise of this study, to deduce the gentle and effective method for extracting bioactive protein among the five compared methods, has been achieved. The study revealed the capacity of the extracted proteins functioning as beneficial antioxidants, which has potential application in the Food Biotechnology industry.
Keywords: ABTS, bioactive proteins, DPPH, Sargassum polycystum, SDS-PAGE

並列關鍵字

ABTS ； bioactive proteins ； DPPH ； Sargassum polycystum ； SDS-PAGE

參考文獻

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