臺灣博碩士論文加值系統

工業製造幾丁質的方式常使用傳統化學法，會有大量酸鹼廢液造成環境汙染、去除的蛋白質無法再利用等缺點，因此，目前致力於開發綠色環保生產幾丁質之製程。以微生物法製備幾丁質為一種綠色製程，使用產蛋白酶的菌株去除蝦蟹殼中的蛋白質，而後使用產酸菌去除礦物質。故本研究藉由產蛋白酶菌株Bacillus subtilis BCRC 10255發酵蝦殼廢棄物，探討最佳發酵條件，並結合高密度培養，建立最佳去蛋白質率的合成培養基及發酵策略，再分別藉由產乳酸菌株Lactobacillus plantarum BCRC 10069及檸檬酸去除蝦殼廢棄物中的礦物質，以達到最佳去礦物質率，期望以實驗室規模之製程達到與化學法相同效率，以利後續工業化應用。結果顯示，B. subtilis於磷酸鹽種類為32 mM NaH2PO4·2H2O、29 mM K2HPO4的合成培養基中發酵12小時，發酵至6小時後，每兩小時調整發酵液pH值至pH 8，有最佳的去蛋白質率(92.23%)，去礦物質的部分，L. plantarum的最佳發酵條件為B. subtilis發酵蝦殼粉12小時後，靜置3小時，額外添加5%葡萄糖，再接入L. plantarum發酵72小時，最終去礦物質率為69.85%；然而，以L. plantarum去礦物質無法達到商用幾丁質之規格，因此，研究以較為環保且環境友善之檸檬酸進行去礦物質，蝦殼內碳酸鈣和檸檬酸莫耳比為3：8時，有最佳的去礦物質率(99.10%)，並且產物的物化性質和化學法製備的幾丁質相似。綜合上述，B. subtilis發酵接續檸檬酸處理，藉以去除蝦殼廢棄物中的蛋白質和礦物質並回收幾丁質為具有潛力、能替代傳統製程的綠色方法。

Current industrial methods employed for chitin production predominantly rely on conventional chemical processes, resulting in the generation of substantial acidic and alkaline wastewater, causing significant environmental pollution. Additionally, the drawbacks involve the wasting of extracted proteins. Consequently, there is a current imperative to develop environmentally sustainable processes for chitin production. Microbial approaches to chitin production are considered eco-friendly, employing strains capable of producing proteases to eliminate proteins from the shells of shrimp and crabs, and subsequently, acid-producing bacteria are utilized to remove minerals. This study aims to develop a green and environmentally sustainable processes for chitin production from shrimp shell waste, and investigate optimal fermentation conditions. The strategies was featured with high-density cultivation, designation of a defined medium, and mediating other crucial fermentation conditions to achieve the maximum protein removal rate. Following this, Lactobacillus plantarum BCRC 10069 and citric acid were applied to eliminate minerals from shrimp shell waste, with the aim of achieving the optimal mineral removal rate. The objective was to achieve efficiency comparable to conventional chemical methods on a laboratory scale, which the conditions could be a reference for subsequent industrial applications. The findings revealed that under defined medium conditions with phosphate concentrations of 32 mM NaH2PO4·2H2O and 29 mM K2HPO4, B. subtilis exhibited optimal protein removal at 92.23% after 12 hours of fermentation, with pH adjustments every two hours to maintain a pH of 8 after 6 hours of fermentation. For mineral removal, the optimal fermentation conditions for L. plantarum involved an additional 3-hour incubation after deproteinization process, additional 5% glucose supplementation, and subsequent L. plantarum fermentation for 72 hours, resulting in a final mineral removal rate of 69.85%. However, the use of L. plantarum alone for mineral removal did not meet the specifications for commercial chitin. Therefore, an environmentally friendly approach utilizing citric acid was explored, with an optimal molar ratio of calcium carbonate to citric acid (3:8) achieving a mineral removal rate of 99.10%. The physicochemical properties of the resulting product closely resembled chitin prepared using chemical methods. In summary, the fermentation of B. subtilis followed by citric acid treatment for shrimp shell waste, aiming to eliminate proteins and minerals and recover chitin, presents a promising and sustainable alternative to conventional processes.

摘要 i
目錄 iii
圖目錄 v
表目錄 vii
第一章、前言 1
第二章、文獻回顧 3
2.1. 蝦廢棄物 3
2.2. 幾丁質(Chitin) 3
2.2.1. 來源及結構 3
2.2.2. 特性及應用 4
2.2.3. 以化學法萃取幾丁質 4
2.3. 氣爆膨發(Explosive puffing)-多孔化結構 4
2.4. 以微生物法萃取幾丁質 5
2.4.1. 單一菌株發酵製備幾丁質 6
2.4.2. 複合菌株發酵製備幾丁質 7
2.5. 以檸檬酸去除蝦殼中礦物質 8
2.6. 工業生產 9
第三章、實驗架構 10
第四章、材料與儀器 12
4.1. 實驗材料 12
4.2. 實驗菌株 12
4.3. 實驗藥品 12
4.4. 儀器設備 12
第五章、實驗方法 14
5.1. 原料之前處理 14
5.2. 一般組成分析 14
5.2.1. 水分含量 14
5.2.2. 灰分含量 14
5.2.3. 蛋白質含量測定 14
5.2.4. 粗脂肪含量 15
5.2.5. 幾丁質含量分析 15
5.2.6. 去蛋白質率(DP%)分析 16
5.2.7. 去礦物質率(DM%)分析 16
5.2.8. 幾丁質產率(CY%)分析 16
5.3. 氣爆膨發多孔化蝦殼 16
5.4. 以芽孢桿菌發酵去除蛋白質 17
5.4.1. 芽孢桿菌接種液之培養 17
5.4.2. 芽孢桿菌之生長曲線 17
5.4.3. 芽孢桿菌發酵條件 17
5.4.4. 合成培養基之製備 17
5.4.5. 芽孢桿菌發酵 17
5.4.6. 擬定發酵策略 18
5.4.7. 收集樣品 18
5.5. 以乳酸菌發酵去除礦物質 18
5.5.1. 乳酸菌接種液之培養 18
5.5.3. pH值分析 18
5.5.4. 可滴定酸度分析(Total titratable acid, TTA) 19
5.5.5. 收集樣品 19
5.6. 檸檬酸去除礦物質 19
5.7. 化學法製備幾丁質 19
5.8. 掃描式電子顯微鏡(SEM)外觀型態及孔徑分析 20
5.9. 傅立葉紅外線光譜儀(FTIR)化學結構分析 20
5.10. X光繞射儀(XRD)結晶度測定 20
5.11. 效益分析 20
5.12. 統計分析 21
第六章、結果與討論 22
6.1. 一般成分分析 22
6.2. 芽孢桿菌發酵條件 22
6.2.1. 接菌量 22
6.2.2. 發酵容器 22
6.3. 合成培養基之製備 22
6.3.1. 蝦殼粉(SP)發酵液之組成 22
6.3.2. 不同組合合成培養基 23
6.4. 擬定優化芽孢桿菌發酵去蛋白質的策略 24
6.4.1. 調整發酵pH值 24
6.4.2. 探討菌量、溫度及膨發對芽孢桿菌發酵去蛋白質之效果 25
6.4.3. 調整發酵時間 26
6.5. 以乳酸菌發酵蝦殼去礦物質 27
6.5.1. 連續發酵時間 27
6.5.2. 補充碳源條件 28
6.6. 以檸檬酸去除礦物質 29
6.7. 產物之物化性質分析 30
6.7.1. 微觀結構(SEM)分析 30
6.7.2. FTIR分析 30
6.7.3. XRD分析 31
6.8. 效益分析 31
第七章、結論 32
第八章、參考文獻 33
第九章、圖 37
第十章、表 61

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