- 學位論文
農產品及副產品加工後活性成份之分析及探討
Exploring and profiling the biological components from agricultural products and byproducts after processing
摘要
來自農產品和農業副產物的生物活性化合物大多被歸類為具有健康益處的天然抗氧化劑。在過去的幾年中，消費者對健康食品的需求有所增加，因此食品產業一直朝開發機能性食品領域而努力。天然抗氧化劑常見的健康促進特性包括：抗發炎、抗高血壓、抗糖尿病等。如今，從農產品和副產物中尋找功能性食品已成為產業發展的主流之一。在食品加工過程中，抗氧化製劑或天然成分可與蛋白質和其他食品成分發生相互作用，導致某些活性成分產生變化。故已有許多研究報導了加工後產品和副產品的變化。本研究使用乾法加工（咖啡葉和魷魚）、酸性水解（魷魚）和酶促水解（木鱉果種子殘渣）來探索和分析加工後，農產品和副產品中的生物成分的變化與活性的改善。 第一項研究的重點是探討乾燥過程對農業副產品咖啡葉（Coffea arabica L.）的抗氧化活性的影響。咖啡葉含有眾多有益成分，例如：碳水化合物、胺基酸、蛋白質、有機酸、生物鹼、酚類化合物、萜烯、類胡蘿蔔素、植物甾醇及芳香性成分。咖啡葉含有豐富的酚類化合物，因此具開發成保健食品之潛力。這項研究的目的包括：i）鑑定乾燥後的未發酵咖啡葉提取物的酚類化合物；ii）評估不同乾燥溫度對其總酚含量、總原花青素含量和抗氧化劑活性的影響；以及iii）評估其相關性。結果顯示在乾燥過程中咖啡幼葉保有最高的總酚含量、總原花青素含量和DPPH自由基清除活性。因此，進一步研究嫩葉在不同乾燥溫度（30、40和50°C）對活性成分的影響。隨著乾燥溫度的升高，DPPH自由基清除活性、總酚含量和總原花青素均增加。在50℃觀察到最高的活性。本研究利用高效液相層析（HPLC）和液相層析-質譜（LC-MS）進行活性成分的定量與定性。數據顯示在加工後的咖啡葉中，可同時發現了兒茶素（或表兒茶素）、芒果苷（或異血管生成素）、原花青素B、咖啡酰奎尼酸（CQA）、咖啡因、槲皮素-3-O-葡萄糖苷、原花青素C、蘆丁和3.4-diCQA等天然物。對於乾燥加工和成熟咖啡葉的相關性方面，葉片中總酚含量和總花青素之間存在明顯關係，總酚含量與DPPH自由基清除活性高度相關。故由加工過咖啡葉製成的茶中含有大量的抗氧化天然物，可被視作一種具潛力的機能性食品。 第二個研究主題在探討酸性水解條件下，藉由不同的乾燥方法（間歇乾燥，曬乾和市售乾燥）製備的魷魚產品，其加工條件和特性之關聯。分別藉由SDS-PAGE和DPPH自由基清除法，監測蛋白質含量和抗氧化活性的變化。使用液相層析-串聯式質譜（LC-MS.MS）分析了不同加工過程，在酸性水解所生成的胜肽。利用所鑑定到的胜肽與T1R2 / T1R3味覺受體之間的分子對接，來模擬這些胜肽具鮮味的潛力。來自乾燥樣品的魷魚蛋白水解物（SPH）比新鮮魷魚蛋白水解物樣品具有更多的小肽。間歇乾燥過程得到的SPH具有最高的抗氧化活性（87.71±0.40％），並包含8種胜肽。在這些鑑定出的胜肽中，VGDEAQSKRGIL、EEELKV和VITIGNE顯示出其結合T1R2 / T1R3受體（鮮味受體）的潛力，這意味著間歇乾燥加工的SPH具備開發成機能性食品和天然調味劑的潛力。 在第三部分中，我們研究了藉由酶促水解木鱉果種子的廢渣而產生的活性肽。 木鱉果（Momordica cochinchinensis Spreng）種子的殘渣被視為農業廢棄物。於本研究，利用酶促水解加工，自廢渣中萃取木鱉果種子蛋白（GSP）產生短鏈胜肽，並探討水解物是否具血管收縮素I轉化酶（ACE）抑制活性。首先，使用包括胃蛋白酶，胰蛋白酶和胰凝乳蛋白酶等多種酶將GSP水解。經測定，來自GSP水解產物的ACE半抑制濃度(IC50)為70.0±4 µg / mL；接著利用親水作用液相層析（HILIC）和逆相高效液相層析（RP-HPLC）進行聯繼生物活性指引分群來篩選ACE抑制肽；最後利用LC-MS / MS從最高活性分液中鑑定出胜肽。 HILIC的直接流過分液（FT）顯示出最高的ACE抑制活性（78.20±0.30％）。再利用RP-HPLC進一步分離該分液。結果表明分液8（HILIC-FT / RP-F8）顯示出最高的ACE抑制活性（25.63±0.57％），並利用LC-MS / MS結合從頭定序法，從此分液中鑑定出14種胜肽。同時，利用BIOPEP數據庫進行電腦分析分析，以輔助活性肽的篩選。BIOPEP得分最高的四個肽：ALVY（0.078）、LLVY（0.078）、LSTSTDVR（0.064）和LLAPHY（0.055）被認為是ACE抑制肽候選物。 ACE抑制試驗表明，ALVY具有最高的ACE抑制活性（7.03±0.09 µM），並由 Lineweaver-Burk圖顯示其為競爭性抑制劑。本研究也以分子對接模擬ALVY和ACE之間的相互作用。其有效的ACE抑制活性可能是由於其與ACE活性口袋S1（Ala354，Tyr523）和S2（His353，His 513）的殘基His353、His513和Tyr523產生三組氫鍵、及與Ala356和Tyr523產生四組凡得瓦力所致。因此，我們認為酶促加工有助於GSP水解產生ACE抑制肽，可助於開發降血壓保健食品。 因此，食品加工有望提高農產品（魷魚）和副產品（咖啡葉和木鱉果種子殘渣）中生物活性成分的含量、活性與可用性，可作為功能食品、天然食品提味劑和營養保健品的替代來源。
並列摘要
Bioactive compounds from agricultural products and byproducts are generally categorized as natural antioxidants with well-known health benefits. Additionally, consumers’ demand for healthier foodstuffs has increased over the least years and thus the food industry has strived to answer this challenge. The health-promoting characteristics of natural antioxidant include anti-inflammatory, anti-hypertension, anti-diabetic and the others. Nowadays, extensive research is being carried out to find functional foods from agricultural products and byproducts. During food processing, interaction of antioxidant either as antioxidant preparations or natural constituents with proteins and other food constituents take place and the activity of some antioxidant may change. Many researchers have studied variations in products and byproducts after processing. The main objective of this study was to report exploring and profiling the biological components from agricultural products and byproducts after processing by using dried-processing (coffee leaves and squid), acid hydrolysis (squid) and enzymatic hydrolysis (gac seeds residue). The first study focused on the drying process effect on the antioxidant activity of the agricultural byproducts, coffee leaves (Coffea arabica L.). They are source of valuable compounds such as carbohydrate, amino acid, protein, organic acid, alkaloid, phenolic compounds, terpene, carotenoid, phytosterol and flavor compounds. Some of them had the antioxidant activities. Their high phenolics content lead to their potential use as health food products. The aim of this study included i) to identify the phenolic compounds of unfermented coffee leaf extracts after drying, ii) to assess the effect of different drying temperatures on their total phenolic content, total procyanidins and antioxidant activity and iii) to evaluate their correlation. The young leaves showed to drying processes had the highest total phenolic content, total procyanidins and DPPH radical scavenging activity. Therefore, the young leaves were further analyzed and the effect of different drying temperatures (30, 40 and 50˚C) was also studied. The DPPH radical scavenging activity, total phenolic content and total procyanidins were increased by the increase of drying temperature. The highest activity was observed at 50˚C. The bioactive compounds were investigated by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). The data indicated that catechin (or epicatechin), mangiferin (or isomangiferin), procyanidin B, caffeoylquinic acids (CQA), caffeine, quercetin-3-O-glucoside, procyanidin C, rutin and 3.4-diCQA were simultaneously found in the processed coffee leaves. For the correlation of dried processing and maturity leaves were evident between total phenolic content and total procyanidins, and the total phenolic content is highly correlated with DPPH radical scavenging activity. According to this study, the tea made from processed coffee leaves which contain lots of antioxidant natural products can be regarded as a potential functional food. The second project was to study the process requirements and properties of the squid products prepared by different drying methods (intermitted-dried, sun-dried and commercially available dried) on acidic hydrolysis. The changes of protein content and antioxidant activities were monitored by SDS-PAGE and DPPH radical scavenging assay, respectively. The resulting peptides generated from different processes were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The potential umami taste was simulated using molecular docking between the identified peptides and the T1R2/T1R3 taste receptor. The squid protein hydrolysate (SPH) derived from dried samples had more small peptides than the sample of fresh squid protein hydrolysate. The SPH derived from intermitted-dried process had the highest antioxidant activity (87.71 ± 0.40%) and contained eight peptides. Among these identified peptides, VGDEAQSKRGIL, EEELKV and VITIGNE showed the potentials to bind T1R2/T1R3 receptor (umami receptor), which implied that the intermitted-dried processed SPH can be a promising functional food and food ingredient. In the third part, we studied the active peptides generated by enzymatic hydrolysis towards waste residues of gac arils. Gac (Momordica cochinchinensis Spreng.) arils contain a high proportion of oil. After oil extraction, the residues of gac seeds were regarded as agricultural waste. In this study, the enzymatic process was performed in gac seed proteins (GSPs) extracted from waste residues. The resulting hydrolysate was investigated for angiotensin I-converting enzyme (ACE) inhibitory activity. Firstly, GSPs were hydrolyzed using a combination of enzymes including pepsin, trypsin and chymotrypsin. The ACE inhibitory activity from GSPs hydrolysate was determined to give an IC50 value of 70.0±4 µg/mL. Then the ACE inhibitory peptides were screened using two sequential bioassay-guided fractionations by hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high-performance liquid chromatography (RP-HPLC). The peptides in the fraction with highest ACE inhibitory activity were identified by LC-MS/MS. The flow through (FT) fraction of HILIC showed the highest ACE inhibitory activity (78.20 ± 0.30%). This fraction was further separated by RP-HPLC. The result indicated that fraction 8 (HILIC-FT/RP-F8) showed the highest ACE inhibitory activity (25.63 ± 0.57%). Fourteen peptides were identified using LC-MS/MS coupled with de novo sequencing. The in silico analysis using BIOPEP database was performed to assist the active peptide screening. The peptides with the top four high BIOPEP scores, ALVY (0.078), LLVY (0.078), LSTSTDVR (0.064) and LLAPHY (0.055) were regarded as ACE inhibitory peptides candidates. The ACE inhibitory assay indicated that ALVY showed a potent ACE inhibitory activity (7.03 ± 0.09 µM). The Lineweaver-Burk plot inferred that ALVY is a competitive inhibitor. The interaction between ALVY and ACE was simulated using molecular docking. Its potent ACE inhibitory activity may be due to the interactions via three H-bonds with the residues His353, His513 and Tyr523, and four van der Waals at Ala356 and Tyr523, in the S1 (Ala354, Tyr523) and S2 (His353, His 513) pockets of ACE. Therefore, we can suggest that GSP hydrolysate can be used as a potential nutraceutical with inhibitory activity against ACE. Therefore, food processing is promising to promote the content, activity, availability of bioactive compounds from the agricultural product (squids), and the byproducts (coffee leaves and gac seed residues), as alternative sources for functional food, food ingredient, and nutraceuticals.