開發鳳梨莖中鳳梨蛋白酶的純化方法與鳳梨莖利用之探討

根據我國行政院農業委員會農糧署(Agriculture And Food Agency Council Of Agriculture, Executive Yuan)的統計，從民國102年開始至民國106年為止，台灣鳳梨的產量從41萬噸增長到了55萬噸。但隨著鳳梨產量的增長，其廢棄物的產生也隨之增加，一般鳳梨廢棄物是包含除了果肉之外的部分，如葉子、莖、果皮等等不可食用的部位，且根據研究指出，鳳梨莖中含有的鳳梨蛋白酶的量高於鳳梨果肉中所含有的量。因此為了進一步改善並減少生物資源的浪費，本研究將開發鳳梨莖中鳳梨蛋白酶之純化方法，同時以鳳梨莖作為原料加工製作成食品，最終減少生物資源的浪費並提升其農業產值。實驗結果顯示，利用離子交換層析純化鳳梨蛋白酶後，將得到的餾液收集並測其蛋白水解活性，得到的比活性為142.27 (U/mg)，而粗萃液的比活性為31.19 (U/mg)，可以發現經純化過後的餾液與粗萃液相比，純化倍數為4.56，而回收率則高達129%，緊接著利用濾膜為10 kDa的濃縮管進行超過濾離心以去除溶液中鹽類，可以得到比活性為130.69 (U/mg)且純化倍數及回收率分別為4.19及110.6%。將純化過後的鳳梨蛋白酶測定其最適合作用溫度及pH值，可以得到最適作用溫度約在50 ˚C、最適作用pH值在pH 6~8都有相當不錯的活性反應。緊接著利用冷凍乾燥將酵素液粉末化以利後續保存及應用，實驗結果得知，凍乾處理後與原先相比酵素活性保留了40%而且貯藏於-20 ˚C具有最好的穩定性。最後由SDS-PAGE結果顯示，萃取液經過離子交換及濃縮過濾的步驟後，可以發現在24 kDa處的鳳梨蛋白酶的保留。而利用高壓加熱法及沸水加熱法製作出的鳳梨莖蜜餞其水分、粗脂肪、粗蛋白及粗灰分的含量都不高，但色澤上從外觀可以看到差異，高壓加熱與沸水加熱的蜜餞成品的膳食纖維含量分別為8.66%及3.37%，根據衛生福利部食品藥物管理署的公告，能將高壓蜜餞成品標榜為具有高膳食纖維含量之食品，也因其具有的高糖度及低水活性，使得能達到抑制微生物生長的效果。綜合以上結果，透過離子交換層析及超濃縮過濾技術進行鳳梨蛋白酶的純化方法是可行的，最終得到的結果具有不錯的純化倍數及回收率，而且為了有效的利用鳳梨莖，將其加工製作為蜜餞是具有潛力的，既能對人體的健康產生益處又能減少生物資源的浪費。

關鍵字

鳳梨蛋白酶；酵素；離子交換層析；純化

並列摘要

According to the statistics of the Agriculture And Food Agency Council of Agriculture of the Executive Yuan, the output of Taiwan pineapples increased from 410,000 tons to 550,000 tons from the beginning of the Republic of China in 102 to the end of the Republic of China in 106. However, as the output of pineapples increases, the production of its wastes also increases. Generally, pineapple wastes contain parts other than the pulp, such as leaves, stems, peels and other inedible parts. According to research, pineapples The amount of pineapple protease contained in the stem is higher than that contained in the pineapple pulp. Therefore, in order to further improve and reduce the waste of biological resources, this research will develop a purification method for the pineapple protease in pineapple stems, and use the pineapple stems as raw materials to process them into food, ultimately reducing the waste of biological resources and increasing its agricultural output. The experimental results showed that after purifying ananas by ion exchange chromatography, the obtained distillate was collected and its proteolytic activity was measured. The specific activity obtained was 142.27 (U/mg), while the specific activity of the crude extract was 31.19 (U/mg), it can be found that compared with the crude extract, the purified distillate has a purification multiple of 4.56, and the recovery rate is as high as 129%. Then the concentrated tube with a 10 kDa membrane is used for ultrafiltration and centrifugation to remove the solution. For the medium salt, the specific activity is 130.69 (U/mg), and the purification factor and recovery rate are 4.19 and 110.6%, respectively. The most suitable temperature and pH value of the purified pineapple protease were determined, and the optimum temperature was about 50 ˚C, and the optimum pH value was at pH 6~8, which showed good activity. Then, freeze-drying was used to powder the enzyme solution for subsequent storage and application. The experimental results showed that the enzyme activity was retained by 40% compared with the original after freeze-drying treatment, and it has the best stability when stored at -20 ˚C. Finally, the results of SDS-PAGE showed that after the steps of ion exchange and concentration and filtration of the extract, the retention of ananasase at 24 kDa can be found. The content of water, crude fat, crude protein and crude ash in pineapple stem preserves made by autoclaving and boiling water heating methods is not high, but the color and luster can be seen from the appearance. Finished products heated by autoclaving and boiling water The dietary fiber content of edible candied fruit is 8.66% and 3.37% respectively. According to the announcement of the Food and Drug Administration of the Ministry of Health and Welfare, the high-pressure preserved product can be advertised as a food with high dietary fiber content. It is also due to its high sugar content and low water activity. Make it possible to achieve the effect of inhibiting the growth of microorganisms. Based on the above results, it is feasible to purify pineapple protease by ion exchange chromatography and ultra-concentration filtration technology, and the final result has a good purification multiple and recovery rate, and in order to effectively use the pineapple stem, it is processed into Candied fruit has potential, which can not only produce benefits to human health but also reduce the waste of biological resources.