臺灣博碩士論文加值系統

我國飲茶風氣盛行，以致茶飲料工廠每年有大量的廢棄茶渣產生。本論文將探討自茶飲料工廠的廢棄茶渣回收酯型兒茶素類物質及二次茶渣再用以製作堆肥之可行性。
首先將茶葉經兩次沸水萃取，濾液以HPLC分析酯型兒茶素類物質和咖啡因含量。此自製茶渣再以 100 ℃、121 ℃ 沸水及 47.5 % 乙醇水溶液以不同時間進行萃取。以 HPLC 分析各萃取液中沒食子酸、酯化型兒茶素類物質（包括EGCG、GCG、ECG及CG）及咖啡因之含量變化，以探討不同條件對茶渣的萃取效率。結果顯示，自製茶渣經 121 ℃ 沸水萃取 10 分鐘得到的總酯化型兒茶素類物質含量（7.80 mg）（以每克茶葉計）與 47.5 % 乙醇水溶液萃取 40 分鐘（7.90 mg）大致相當。而如單以 EGCG 的萃取量來看，47.5 % 乙醇水溶液萃取 40 分鐘所得到的量卻顯著地高於 121 ℃ 沸水所能得到的萃取量。其主要原因是以 121℃ 沸水萃取時，會導致部分 EGCG 異構化為 GCG，萃取時間越長越明顯。基於回收酯化型兒茶素-EGCG 為主要目標。自統一公司新市廠所取得的廢棄茶渣，利用 47.5 % 乙醇水溶液萃取 40 分鐘，先將兒茶素類物質萃取出來。接著將萃取液濃縮凍乾再回溶，經由 Sephadex G-25 膠體管柱進行吸附，去除非酯化型兒茶素類物質與咖啡因及色素後，用 47.5 % 乙醇水溶液溶出。溶出物進行濃縮並凍乾後再回溶，最後通過 C18 管柱層析程序，即可得到純度達 90 % 的 EGCG 精製成品。
將上述自製茶渣經 121 ℃ 沸水或乙醇萃取所得之凍乾粉末及茶飲料工廠廢棄茶渣之乙醇萃取液凍乾粉末進行清除 DPPH 自由基能力及 TEAC 值的檢測。結果顯示，三種樣品的抗氧化能力確有不同，而是以茶飲料工廠的廢棄茶渣用 47.5 % 乙醇水溶液萃取的抗氧化能力最好。可能原因包括：兒茶素物質等殘留量較多，47.5 % 乙醇水溶液萃取率較好，EGCG不會發生異構化現象，極性較低的物質（特別是茶黃質- theaflavins 等）溶出量較多等。
而二次茶渣則進一步進行製作堆肥之發酵試驗，包括以接種外生菌和額外添加蔗渣、豆渣的情況下，藉由分析水份、pH 值、導電度、及有機質和灰份含量的變化來評估堆肥之肥效。堆肥試驗結果顯示，在 107 天堆肥化後，所有的堆肥試驗組皆呈現腐熟現象。在種子發芽率及進行種植小白菜的盆栽試驗來看，其每組的發芽率皆大於 90 %。另外，單純使用茶渣並添加纖維素分解菌之堆肥土壤，和其它以不同處理的堆肥土壤以種植小白菜結果做比較，則顯示前者的效果較佳。
綜合以上的結果顯示，本研究不但可以使用簡單的方式萃取出兒茶素類物質，進而回收並加以純化出主要的保健成分EGCG，而在萃取後的二次茶渣也可用於製作堆肥，藉此可明顯增進茶葉之經濟價值。

This study is based on the prevalence of tea drinking in our country, and there are thousands tons of tea wastes are produced from the factories each year. In this study, tea waste was collected from a tea drink factory located in Tainan County, and was employed for recovering of the residual esterified catechin-EGCG and further application of second waste in composting process.
Firstly, the tea was flushed twice with 100 ℃ boiled water for 20 min. The tea residues thus obtained (Self-prepared tea wastes) were then used for further extraction with different conditions, including extraction solvents, extraction temperature and duration of extraction time. The constituents of tea residue extracts, such as GA, EGCG, GCG, ECG, CG, and caffeine were examined by HPLC.
The data obtained by HPLC showed that the efficacy for recovering of total esterified catechins and the compositions were different significantly with different processes. The results showed that total esterified catechins obtained by 121 ℃boiling water for 10 min (7.80 mg) was almost the as 47.5 % aqueous ethanol at room temperature for 40 min (7.90 mg). However, EGCG content was much higher in aqueous ethanol extract, because overheating (121 ℃) might induce epimerization of EGCG to form GCG.
For the goal of EGCG recovery, the tea wastes from a beverage factory was extracted with 47.5 % aqueous ethanol for 40 minutes. The extracts were vacuum dried followed by freeze-drying. And the EGCG in it was further adsorbed by a Sephedex G-25 column, and fractionated by a preparative chromatography of C18 column. The EGCG product isolated was about 90 % pure according HPLC analysis.
Three freeze-dried powder samples: Self-prepared tea wastes treated with 121 ℃boiling water and 47.5 % aqueous ethanol and the tea wastes from factory treated with 47.5% aqueous ethanol were checked for their anti-oxidative activity by DPPH scavenging ability and TEAC evaluation. The results indicated that 47.5 % aqueous ethanol extracted factory’s sample shown as the best antioxidant on a basis of dry mass. The possible reasons included: higher residual catechins in the waste, better extraction efficiency of aqueous ethanol, no epimerization and higher content of low polarity compounds (e.g. theaflavins) in the extract.
The retreated tea wastes were fermented with or without exogenous bacteria and in the presence or absence of bagasse or soybean dregs, to see if can increase its digestibility and fertilizing activities. The composting experiment was continued for 107 days, all of tested compost groups were completed. The water content, pH, organic compounds, electro-conductivity, and ash content were analyzed during this experiment. The efficiency of composts from different combination was tested by seed germination assay and leafy vegetables growth of plate test. Germination percentage of all composts was greater than 90 %. However, compost obtained from retreated tea residues alone inoculated with cellulase-producing bacterium was demonstrated as the best one for growing leafy vegetables.
In conclusion, this study showed that EGCG remained in tea residues can be extracted and further purified by simple chromatographic processes mainly with eatable solvent- ethanol. Furthermore, a leafy-vegetables growth-promoting compost can be prepared from the retreated tea residues. These results were very significant in increasing the economical benefit of tea.

中文摘要………………………………………………………………………I
英文摘要………………………………………………………………..…...III
本文目錄…………………………………………………………………….VI

第一章 前言…………………………………………………………….……1
1.1 研究緣起…………………………………………………………….…...1
1.2 研究目的…………………………………………………………….…...3
圖1.1、實驗架構……………………..………………………..……………..4

第二章 文獻回顧…………………………………………………….………5
2.1、茶的介紹…………………………………………...……...…………....5
2.1.1、茶的認識………………………………………...……….……….5
2.1.2、茶葉中的化學成份及含量………………………………………5
表2.1、茶葉中化學成份及其含量………………………………………6
2.1.3、茶的種類…………………………………………….………….10
圖2.1、不發酵茶及發酵茶的製成…………………….……………….10
圖2.2、部分發酵茶的製成…………..………………………..…….....11
表2.2、不同發酵程度的茶………………………………….…..…….11
2.2、茶的保健弁遄K………………………………………………..…..….13
表2.3、茶葉中的保健成分及幼纂K………………………………..…13
2.2.1、兒茶素類物質的保健幼纂K…………………………….….…14
表2.4、兒茶素類物質的機能性………………………………….……14
2.2.2、兒茶素的應用發展………………….…………………………..16
表2.5、兒茶素類物質應用商品例及作用……………………………16
2.2.3、兒茶素抗氧化能力的檢測……………………………………...18
2.3、茶飲料簡介…………………………………….……………………….19
2.3.1、茶飲料製程的介紹……………………………………………...20
圖2.3、茶飲料之製造流程及廢棄物產生源..……………..…………..21
2.3.2、茶渣產生的問題及處理方式……………….…………………..22
2.4、茶渣萃取及兒茶素純化技術……………………………………….….23
2.4.1、影響茶渣萃取的因素…………………….……………………23
2.4.2、兒茶素的純化技術…………………………………………….25
表2.6、目前已商業化之吸附性填充物質………………………..…26
2.5、堆肥化技術介紹…………………………………………………….….28
2.5.1、堆肥化過程之變化…………………………………………….28
2.5.2、堆肥與微生物之間的關係……………….……………………31
2.5.3、影響堆肥化之因素…………………………………………….32
表2.7、茶渣成份分析………………………………………………..37
第三章 材料與試藥、菌種及儀器設備…………….………………..…….38

第四章 實驗方法…………………………………………………………...40
4.1、茶渣萃取條件測試………………………………..……………………40
4.1.1、原料茶葉前處理………………………….……..……………..40
4.1.2、茶渣之萃取………………………………...…………………..40
4.1.3、標準品及茶葉與茶渣萃取液之成分分析…………………….41
圖4.1、為標準品之HPLC分析結果………………………...….……42
表4.1、兒茶素類物質及咖啡因標準品之濃度值………….…….…42
4.1.4、茶飲料工廠廢棄茶渣的萃取………………………………….43
4.2、茶渣中EGCG之純化…………………………………..………………43
4.3、茶葉與茶渣萃取物抗氧化能力之比較……………………………….45
4.3.1、原料茶葉的前處理及茶渣的再萃取 ………….……………..45
4.3.2、DPPH 自由基清除效應 …………………….………………47
4.3.3、TEAC的測定…………………………………………………47
4.4、二次茶渣之利用-堆肥製作………………………………………….....48
4.4.1、堆肥原料分析……………………………….…………………48
4.4.2、纖維素分解菌之篩選及培養……………………………..…...49
圖4.2、纖維素分解菌篩選及培養流程………………………..….....50
表4.2、纖維素及茶渣培養基…………………………………..….....50
4.4.3、堆肥材料的前處理…………………………………………….51
圖4.3、堆肥材料的前處理……………..…………………………….51
4.4.4、堆肥化試驗…………………………………………………….52
圖4.4、碳氮比之調整與外生菌的添加之實驗流程圖………….….53
表4.3、堆肥資材調配比例………………………………………..….53
4.4.5、堆肥分析項目………………………….……………………....54
4.4.6、種子發芽試驗……………………………………………….....54
4.4.7、盆栽試驗…………………………………………………….....55

第五章 結果與討論 .…………………………………………………........57
5.1、茶渣萃取分析結果………………………………………..………........57
5.1.1、茶葉萃取次數與酯型兒茶素含量……………....……….........57
5.1.2、萃取條件和時間對酯化型兒茶素及咖啡因溶出的影響…....57
5.1.3、萃取條件和時間對個別酯型兒茶素及GA變化之探討…....58
5.1.4、茶飲料工廠的茶渣萃取……………………………………....59
5.2、EGCG 純化結果………………………………………………...........60
5.3、茶葉與茶渣萃取物抗氧化能力之比較…………………………..........61
5.3.1、茶葉與茶渣萃取物之凍乾物與組成…………………………61
5.3.2、DPPH 自由基清除效應………………………………………61
5.3.3、總抗氧化力（TEAC）的比較…………………………….…63
5.4、二次茶渣之利用-堆肥製作………………………………….……........64
5.4.1、纖維素分解菌之生理特性……………………….……..….....64
5.4.2、纖維素分解菌之生長最適 pH 值…………………………...65
5.4.3、堆肥實驗之腐熟程度探討………………………………….…65

第六章 圖表……………………..………………………..………………...68
圖5.1、原料茶葉及茶渣經過不同萃取條件下的圖譜變化………...……...68
圖5.2、不同萃取條件和時間對總酯化型兒茶素萃取量的影響……..……69
圖5.3、不同的萃取條件和時間對咖啡因萃取量的影響…………...…….69
圖5.4a、121℃高壓沸水組的兒茶素類物質及咖啡因的變化圖……….....70
圖5.4b、47.5% 乙醇萃取的兒茶素類物質及咖啡因的變化圖……….......70
圖5.5、茶飲料工廠廢棄茶渣經乙醇水溶液萃取之層析圖譜……..……..71
圖5.6、茶渣抽出液之C18管柱層析圖…………………………..……...72
圖5.7a、表5.3中的I區進行HPLC分析結果……..………………………73
圖5.7b、吸收光譜之比較圖……..…………………………………………73
圖5.8、檢測清除DPPH能力之標準曲線…..………………………...….....74
圖5.9、檢測TEAC值之標準曲線…..……………………………….….…74
圖5.10、茶飲料工廠茶葉萃取物經層析後之280nm圖譜及光譜……… 75圖5.11、茶渣萃取物及標準品層析光譜………………………………..…76
圖5.12、自行篩選出的纖維素分解菌的菌落型態………………..…….….77
圖5.13、自行篩選出的纖維素分解菌的革蘭式染色鏡檢圖…..………....78
圖5.14、自行篩選出的纖維素分解菌生長週期及培養環境pH變化…..…79
圖5.15、堆肥過程中pH變化…..………………….…………………………80
圖5.16、堆肥過程中EC變化…..………………….…………………………81
圖5.17、堆肥過程中水分含量變化…………………………………………82
圖5.18、堆肥過程中有機碳變化………………….………………………...83
圖5.19、堆肥過程中灰分變化………………………………………………84
圖5.20、盆栽試驗Ι……………………………….…………………………..85
圖5.21、盆栽試驗Π…………………………….……………………………85
圖5.22、不同堆肥處理後其種植小白菜所測得的乾重差異比較…….…...86
表5.1、茶葉與茶渣樣品於不同條件下的萃取液之成分變化…………….87
表5.2、茶飲料工廠茶渣之乙醇萃取液中兒茶素類及咖啡因含量……..88
表5.3、經Sephadex G-25及C18管柱處理後之茶渣萃取凍乾物重量….88
表5.4、茶葉及茶渣萃取物凍乾粉之兒茶素類及咖啡因物質…………..89
表5.5、茶葉及茶渣萃取物凍乾粉末抗氧化能力之比較………………..…90
表5.6、纖維素分解菌生長最適 pH ……………………………………….91
表5.7、堆肥前後碳氮比(C/N)變化…………………………………………91
表5.8、發芽率測試………………………………………………………….92
表5.9、各種處理後腐熟堆肥之金屬含量………………………………….92
表5.10、各種處理後腐熟堆肥之總磷含量………………………………...93

第七章 結論……………………..………………………..………………...94

第八章 參考文獻………………..………………………..……………..….96

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