檸檬由於其特殊的風味及口感一直廣受消費者所喜愛，台灣主要以南部為主要栽種地區約占總體產量之70%，其中以優利卡檸檬品種[Citrus limon (L.) Burm]為最，此外由於檸檬富含多種生物活性成分如橙皮苷、柚苷等黃酮類化合物及多種多酚化合物，而使其被作為高營養價值水果代表之一，也因此被衍生用於多種農產加工品之開發應用上，近年來檸檬產業更利用發酵技術於檸檬加工製作上，然而其功效表現以及發酵前後生物活性物質之變化之科學研究數據甚少，因此本研究以屏東產之優利卡檸檬[Citrus limon (L.) Burm]為原料，遵循產業生產發酵檸檬液之製程，進行乳酸發酵製作成檸檬發酵物，並將其區分為殺菌與未殺菌處理兩組，分別探討檸檬發酵物發酵前後以及殺菌前後生理活性之變化，此外更進一步利用檸檬副產物萃取其檸檬精油並評估其應用於檸檬發酵物品質改善之可行性。
研究成果顯示，在抗氧化能力試驗中證實檸檬發酵物之抗氧化能力顯著高於檸檬汁，且在樣品濃度100%下以未殺菌之檸檬發酵物(NLFP)表現最佳，殺菌之檸檬發酵物(LFP)次之，而以檸檬汁(LJ)組別最差，且各組間均具有顯著之差異(p<0.05)，進一步以腸胃道模擬評估之結果顯示檸檬汁經腸胃道模擬系統後其抗氧化能力會降低約50%，而發酵物樣品則僅下降約30~40%，顯示製備成檸檬發酵物後經腸胃道可保留更多的生物活性成分。綜合上述結果，顯示檸檬汁經發酵後其生物活性會增強，但是加工步驟中的殺菌(80°C,1min)步驟仍會破壞部分生物活性物質，其中LFP之總多酚含量為25.8 (mgGAE)、抗壞血酸含量為55.2 (mg/mL)以及類黃酮含量為75.2 (mg/L)，均略差於未殺菌組但仍優於檸檬汁。進一步以Clone-9細胞評估其抗氧化壓力之能力，結果顯示各濃度下的NLFP與LFP樣品皆有效使GPx、SOD、CAT活性上升、減少細胞株內ROS含量，並增加細胞粒線體膜電位，其中以0.75mg/mL的NLFP有最佳效果，而在感官品評評價上LFP為獲最佳之評分，不論在甜味、風味以及外觀上均達5.5分以上，且整體接受度更獲6.2分，顯示殺菌後之商品較受消費者喜好，由於殺菌程序為該項產品之主要程序，因此本研究成果證實雖然殺菌會影響少量的生物活性表現，但對於風味的喜好上卻更被消費者所接受，基於食品安全之基礎，仍需維持此加工方式以確保產品之安全性。檸檬經榨汁後會存留大量的帶皮果肉的副產物，過去該副產物僅可丟棄或做成農業堆肥使用，因此本研究進一步將副產物回收並進行蒸餾萃取精油(Essential oil)，經氣相層析分析發現精油中以檸檬桸(Limonene)為主要成分約占(41%)，將其依檸檬發酵物為計算基礎添加不同濃度(0%、1%、2%、3%、5%)之精油，且隨添加濃度增加其抗氧化能力亦隨之增加，此外於腸胃道模擬系統中顯示添加精油之發酵檸檬液其抗氧化能力僅降低35-40%，相較於未添加組降低約50%有顯著的增加(P<0.05)，於GC/MS分析結果證實添加Limonene之發酵檸檬液其Limonene含量亦有增加，此外發酵檸檬液中Limonene的增加於抗褐變試驗中可減緩發酵檸檬液儲存褐變反應以及產品穩定性，可有效改善產品之保存品質並減少副產物之浪費造成環境汙染問題，綜上所述檸檬汁經發酵後可增強其生物活性之能力，將副產物經蒸餾萃取後應用於發酵製程除可增加產品之生物活性成分外，更可提高產品穩定性減緩產品褐變問題，有效改善生產製程問題。

Lemon is a popular fruit by consumers due to its special flavor and taste. Taiwan mainly uses southern areas as the main planting area, accounting for about 70% of the total output, of which the Citrus limon (L.) Burm is the most. In addition, lemon is rich in a variety of biologically active ingredients such as hesperidin, naringin and other flavonoid compounds and a variety of polyphenol compounds, it is regarded as one of the representatives of high nutritional value fruits, and therefore it is derived for a variety of agricultural products in terms of development and application. In recent years, the lemon industry has made more use of fermentation technology in the processing of lemons. However, there are few scientific research on its performance and changes in biologically active substances before and after fermentation.
In this study, Citrus limon (L.) Burm was used as a raw material, following the process of industrial production of lemon fermentation, lactic acid fermentation is made into lemon fermented product (LFP), which is divided into two groups of sterilized and non-sterilized treatments (NLFP).
In addition, lemon by-products are used to extract essential oil (EO) and evaluate its application in lemon fermentation process improvement.
The results show that the antioxidant capacity of lemon fermented product is confirmed to be significantly higher than the lemon juice in the antioxidant capacity test, and the best performance is non-sterilized lemon fermented product (NLFP) at a sample concentration of 100%. LFP followed by the lemon juice (LJ) group, which had the most significant difference (p <0.05). Further evaluation by gastrointestinal simulation showed that lemon juice through the gastrointestinal simulation system and its antioxidant capacity will be reduced by about 50%, while the LFP is only reduced by about 30-40%, showing that after preparation of lemon fermened product, more bioactive compounds can be retained after through the gastrointestinal digestion. As mentioned above, after fermentation, the LFP bioactivity will be enhanced, but the sterilization (80 ° C, 1min) in the processing will still undermine apart of biologically active substances, in which the total polyphenol content of LFP is 25.8 (mgGAE) and the ascorbic acid content is 55.2 (mg / mL), and the flavonoid content is 75.2 (mg / L), which are slightly worse than the non-sterilized group but still higher than lemon juice. The ability of Clone-9 cells to evaluate their antioxidant stress was further evaluated. The results showed that NLFP and LFP at different concentration of samples are effective to make GPx, SOD, CAT activity increased, reduced ROS content in cell lines, and increased mitochondrial membrane potential. And the 0.75 mg / mL NLFP has the best effect.
The LFP is the best score in sensory evaluation, including sweetness, flavor and appearance are above 5.5 points, and the overall acceptance score is 6.2 points, showing that LFP are more popular with consumers. Since the sterilization is the main process of this product, the results of this study confirm that sterilization will affect a small amount of biological activity performance, but the taste preference is more accepted by consumers. Based on food safety, this processing method still needs to be maintained to ensure product safety. After squeezing lemon juice, a large amount of by-products will remain. In the past, this by-product could only be discarded or used as agricultural compost. Therefore, in this study, the by-products were further recovered and subjected to distillation extraction (EO). Gas chromatography (GC-MS) analysis identify that limonene was the main component in the EO (41%), and it was added with different concentrations (0%, 1%, 2%, 3%, 5%) based on the lemon fermented product. In addition, in the gastrointestinal simulation, the antioxidant capacity of fermented lemon juice with EO is only reduced by 35-40%, compared to non-added, there was a significant increase in the reduction of about 50% in the group (P <0.05). In addition, the results of GC-MS analysis confirmed that the content of limonene in the fermented lemon product added with limonene also increased. The increase in limonene in the fermented lemon product was tested in the browning resistance It can slow down the browning reaction and product stability of the fermented lemon juice storage, also can effectively improve the preservation quality of the product and reduce the waste of by-products to cause environmental issues. In summary, the lemon juice can enhance the biological activity after fermentation. The lemon by-products can be used in the fermentation process after distillation and extraction to increase the bioactivity compounds, improve the stability of the product, solve the browning problem, and effectively improve the production process issues.

中文摘要I
英文摘要IV
誌謝VII
目錄IX
表目錄XIII
圖目錄XIV
頁次XIV
壹、前言1
貳、文獻回顧3
2.1檸檬簡介3
2.2 檸檬的生物活性成分8
2.3 檸檬的產品與發展趨勢12
2.4 乳酸發酵12
2.5檸檬的副產物及應用性15
參、材料與方法21
3.1實驗材料與設備21
3.2儀器設備21
3.3化學試藥22
3.4實驗方法26
3.4.1樣品製備26
3.4.1.1檸檬汁與檸檬發酵物製備26
3.4.1.2精油製備27
3.4.2 抗氧化試驗27
3.4.2.1 DPPH清除率試驗27
3.4.2.2 還原力試驗27
3.4.2.3 TEAC總抗氧化力試驗28
3.4.3生物活性成分分析28
3.4.3.1維生素C含量測定29
3.4.3.2 總多酚含量測定29
3.4.3.3 總黃酮類分析29
3.4.3.4 酚酸試驗30
3.4.3.5 黃酮類試驗30
3.4.4腸胃道模擬試驗31
3.4.4.1胃部模擬階段31
2.4.4.2 模擬腸道階段31
3.4.5 細胞抗氧化酵素活性分析31
3.4.5.1 細胞存活率試驗31
3.4.5.2 超氧化物岐化酶活性測定32
3.4.5.3麩胱甘肽過氧化物酶活性測定32
3.4.5.4過氧化氫酶活性測定33
3.4.5.6細胞粒線體膜電位測定34
3.4.6揮發性化合物分析35
3.4.7 抗褐變試驗35
3.4.8感官品評分析36
3.4.9統計分析36
肆、結果與討論39
4.1檸檬發酵物之抗氧化能力39
4.2 檸檬乳酸發酵後生物活性成分之變化51
4.3 NLFP與LFP對細胞抗氧化壓力之影響65
4.4添加不同濃度精油對檸檬發酵物抗氧化能力之影響74
4.5 添加精油於檸檬發酵物對其生物活性成分之影響76
4.6 添加精油對檸檬發酵物抗褐變能力之影響91
4.7 添加精油對檸檬發酵物揮發性化合物之影響96
4.8 檸檬發酵物及其添加精油後之感官分析106
伍、結論111
參考文獻112
作者簡介137

台灣行政院農委會，2010，臺灣柑橘產銷概況。
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