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研究生:廖雯怜
研究生(外文):Wen-Ling Liao
論文名稱:以Lactobacilluscasei在批式及饋料批式方法下利用黃豆水解液產製乳酸
論文名稱(外文):The production of lactic acid by using Lactobacillus casei and soybean hydrolysate during batch and fed-batch fermentation
指導教授:傅以中
指導教授(外文):Yi-Chung Fu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:107
中文關鍵詞:乳酸菌批式發酵饋料批式發酵黃豆水解液
外文關鍵詞:Lactic acid bacteriabatch fermentationfed-batch fermentationsoybean hydrolysate
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乳酸是具有多種功能之化學物質,在立體結構上存在兩種不同光學活性之光學異構物,分別為L(+)及D(-)-Lactic acid,其製造方式大多運用微生物發酵或者化學合成。乳酸可合成之聚合物聚乳酸,具有高經濟價值,為生物可降解材料,相較於無法經自然分解的塑膠材質,較無廢棄物處理的問題,也由於低毒性,使得聚乳酸成為食品包材和其他消耗性產品的理想材料。乳酸的生產大多透過乳酸發酵方式,但由於發酵基質的氮源較昂貴,使得乳酸成本居高不下,因此有不少研究使用低成本氮源,取代常見的高價酵母萃取物,以降低乳酸的成本。
  本實驗採用批式方式針對Lactobacillus casei BCRC 10697在糖蜜或葡萄糖提供碳源、黃豆萃取液或水解液作為氮源條件下以發酵槽中進行發酵,觀察不同發酵基質發酵期間菌體生長情形、乳酸產量、產率、葡萄糖之消耗及轉化率等;另外進一步探討不同處理方式下所得之黃豆萃取液及水解液作為氮源、以葡萄糖作為碳源的情況下,批式及饋料批式產製乳酸的結果,作為初步加工應用之探討。
  結果顯示,在批式發酵條件下,以黃豆萃取液A及糖蜜作為發酵基質,能提供乳酸菌適當的養分產生乳酸,可降低發酵基質成本;而不同加工方式所獲得的氮源中,以黃豆水解液為最佳,能在葡萄糖濃度103 g/L的條件下,於72小時內產製100.88 g/L高量的乳酸。因此未來黃豆水解液可作為廉價之氮源,以應用在加工上。
  在饋料批式發酵方面,透過適當的饋料方式能改善基質抑制效應問題,使乳酸產量提升。未來或許可嘗試透過乳酸菌固定化技術,在適當發酵時間將乳酸菌及產物分離,並再次重新投入饋料,或許可提高乳酸的終產量,縮短乳酸菌在饋料期間受到稀釋後再次增殖的時間。
脫脂黃豆粉原料透過適當、簡易的加工方式所獲得的產物,可取代高單價之酵母萃取物氮源,在批式或饋料批式發酵下,將葡萄糖經由乳酸發酵產製乳酸,轉化率''皆可達到100%或超過100%,極具發展潛力,未來可進一步測試,應用在加工上。
Lactic acid is a multifunctional chemical substance that has a three-dimensional structure, and exists in two types of optical isomers with different optical activations, namely L(+) and D(-)-Lactic acid, respectively. They are mainly produced through the methods of utilization of microbial fermentation and chemical synthesis. The polylactic acid polymer that lactic acid is capable of synthesizing possesses a high economic value that should be treated as a biodegradable material. Compared with plastic materials that cannot be decomposed naturally, it generates less waste disposal problems, and due to its low toxicity, polylactic acid has become the ideal material to use in food packages and other consumer products. The production of lactic acid is mainly performed by means of lactic acid fermentation, but the expensive nitrogen source - the fermentation substrate, has been the culprit of high manufacturing costs. Therefore, this has prompted many studies to use a low-cost nitrogen source to replace the common high-priced yeast extraction method in order to reduce the cost of lactic acid production.
For this experiment we adopted the batch method utilizing Lactobacillus casei BCRC 10697 as a carbon source in molasses or glucose, using soybean extract or hydrolysate as the nitrogen source to perform fermentation in a fermentation tank. During the fermentation stage, the mycelia growing conditions of different fermented substrates, lactic acid production, production rate, glucose consumption and conversion rate, etc. were closely watched. On the other hand, the experiment also explored the different handling methods, using a soybean extract and hydrolysate as the nitrogen source, and glucose as the carbon source, batch and fed-batch methods were employed to obtain lactic acid yielding results.
The results indicate that under batch fermentation conditions using soybean extract solution A and molasses as the fermentation substrates it is possible to provide lactic acid bacteria with an appropriate amount of nutrition to generate lactic acid, and also capable of reducing fermentation substrate cost. Among the different processing methods to obtain the nitrogen source, the soybean hydrolysate ranks as the best one, being capable of yielding 100.88g/L of high volume lactic acid within 72 hours under the conditions of 103g/L glucose concentration. Therefore, soybean hydrolysate is able to serve as a cheap nitrogen source that can be applied to processing in the future.
In regard to the fed-batch fermentation method, we have shown that it is able to improve the inhibitory effect problem of substrate through appropriate feeding approach to increase lactic acid production. Perhaps in the future, we might attempt to separate lactic acid bacteria from the product at an appropriate fermentation time through lactic acid bacteria immobilization technique, and once again, to re-enter the feeding materials. This might be able to increase the final lactic acid production, and shorten the proliferation time while the lactic acid bacteria are being re-diluted during the feeding period.
The product obtained through the appropriate and simplified processing method of using defatted soybean powder as raw material is able to replace the high-priced nitrogen source from yeast extract. Using the batch or fed-batch method of fermentation to adopt lactic acid fermentation of glucose to yield lactic acid, the Yield'' is able to reach 100%, or even exceed 100%! This shows that there is indeed a great development potential. We ought to carry out further testing to apply it to processing methods.
目錄
中文摘要............................................................................................... 1
英文摘要............................................................................................... 2
第一章 前言....................................................................................... 4
第二章 文獻整理............................................................................... 6
一、乳酸之介紹................................................................................ 6
(一) 化學合成............................................................................. 6
(二) 微生物發酵......................................................................... 11
二、聚乳酸之介紹............................................................................. 12
(一) 聚乳酸之特性..................................................................... 12
(二) 聚乳酸之生合成................................................................. 13
三、乳酸菌之介紹............................................................................. 13
(一) 乳酸菌之簡介..................................................................... 13
(二) 乳酸菌的醣類代謝............................................................. 15
(三) 乳酸去氫酶......................................................................... 17
(四) 乳酸發酵之影響因子......................................................... 16
四、發酵之動力學型式………………………............................... 24
(一) 生長連動型......................................................................... 24
(二) 非生長連動型..................................................................... 22
(三) 混合型................................................................................. 23
五、饋料策略…………………………………............................... 27
(一) 前饋控制............................................................................. 27
(二) 回饋控制............................................................................. 28
六、黃豆蛋白之介紹…………………………............................... 29
(一) 黃豆的加工......................................................................... 30
(二) 黃豆蛋白的一般應用......................................................... 30
(三) 黃豆相關產品在乳酸菌上的應用..................................... 32
第三章 材料與方法........................................................................... 34
一、試驗材料..................................................................................... 34
(一) 發酵菌株............................................................................. 34
(二) 培養基................................................................................. 34
(三) 原料..................................................................................... 34
(四) 標準品................................................................................. 34
(五) 化學藥品............................................................................. 35
二、儀器設備..................................................................................... 35
三、實驗方法及條件......................................................................... 36
(一) 菌株之活化......................................................................... 36
(二) 菌種保存率......................................................................... 36
(三) 接種源之製備..................................................................... 36
(四) 發酵槽................................................................................. 36
四、實驗流程..................................................................................... 37
(一) 批式發酵............................................................................. 38
1. 不同碳源之乳酸發酵....................................................... 38
2. 不同氮源之乳酸發酵....................................................... 39
(二) 饋料批式發酵..................................................................... 41
1. 不同饋料方式之流程說明............................................... 41
五、分析方法..................................................................................... 42
(一) 乳酸菌數測定..................................................................... 42
(二) 醣類測定............................................................................. 42
(三) 乳酸含量測定..................................................................... 43
第四章 結果與討論........................................................................... 51
第一節 Lactobacillus casei在不同碳源下以批式 (Batch) 發酵方法進行乳酸發酵............................................................. 51
一、目的......................................................................................... 51
二、結果與討論............................................................................. 51
(一) 初始糖蜜濃度250 g/L..................................................... 51
(二) 初始葡萄糖濃度103 g/L................................................. 53
(三) 改變初始碳源糖蜜250 g/L及葡萄糖103.44 g/L之綜合比較.............................................................................. 55
(四) 結論.................................................................................. 57
第二節 Lactobacillus casei在不同氮源下以批式 (Batch) 發酵方法進行乳酸發酵............................................................. 67
一、目的......................................................................................... 67
二、結果與討論............................................................................. 67
(一) 氮源黃豆萃取液B........................................................... 67
(二) 氮源黃豆水解液.............................................................. 68
(三) 改變氮源黃豆萃取液A、黃豆萃取液B及黃豆水解液之綜合比較...................................................................... 69
(四) 結論.................................................................................. 72
第三節 Lactobacillus casei於起始葡萄糖濃度103.44 g/L 及黃豆水解液下以饋料批式發酵 (Fed-batch) 方法進行乳酸發酵................................................................................. 79
一、目的......................................................................................... 79
二、結果與討論............................................................................. 79
(一) 以葡萄糖103 g/L、黃豆水解液為基質,於不同饋料方法之試驗...................................................................... 79
(二) 不同饋料方法之綜合比較.............................................. 82
(三) 結論.................................................................................. 85
第四節Lactobacillus casei對葡萄糖消耗及產製乳酸之理論及實際值比較......................................................................... 93
一、目的......................................................................................... 93
二、結果與討論............................................................................. 93
(一) 批式發酵.......................................................................... 93
(二) 饋料批式發酵.................................................................. 94
第五章 總結 96
參考文獻 97

圖目錄
第二章
圖2-1 乳酸及其商業之使用及應用.............................................. 7
圖2-2 乳酸的光學異構物............................................................. 8
圖2-3 兩種主要之乳酸加工製造方法......................................... 10
圖2-4 以D、L-lactic acid合成聚乳酸 (PLA)............................... 14
圖2-5 乳酸菌的代謝機制............................................................. 16
圖2-6 發酵過程中,微生物生長(X)與產物生成(P)之動力學型式......................................................................................... 25
圖2-7 Gaden分類之發酵動力學.................................................... 26
圖2-8 黃豆加工流程圖................................................................. 31
第三章
圖3-1 Lactobacillus casei利用批式發酵方法之實驗架構............ 46
圖3-2 脫脂黃豆萃取流程圖.......................................................... 47
圖3-3 (a)脫脂黃豆粉 (b)黃豆萃取液............................................ 48
圖3-4 黃豆萃取液B及黃豆水解液............................................... 49
圖3-5 Lactobacillus casei利用饋料批式發酵方法之實驗架構. 50
第四章
第一節
圖4-1 以250 g/L 糖蜜、黃豆萃取液A作為發酵基質進行批式發酵,其過程中生菌數、乳酸、鹼液 (10N NaOH) 及各種醣類之參數變化. ............................................... 58
圖4-2 以103.44 g/L 葡萄糖、黃豆萃取液A作為發酵基質進行批式發酵,其過程中生菌數、乳酸、鹼液 (10N NaOH) 及各種醣類之參數............................................ 61
圖4-3 以103.44 g/L 葡萄糖、黃豆萃取液A作為發酵基質進行批式發酵之重覆實驗,其過程中生菌數、乳酸、鹼液 (10N NaOH) 及各種醣類之參數變化................ 62
圖4-4 以批式發酵方法在不同碳源發酵期間參數之變化 (a) 總乳酸產量 (b) 瞬間乳酸產率..................................... 63
圖4-5以批式發酵方法在不同碳源發酵期間轉化率之變化.... 65
第二節
圖4-6 以103.44 g/L 葡萄糖、黃豆萃取液B作為發酵基質進行批式發酵,其過程中生菌數、乳酸、鹼液 (10N NaOH) 及各種醣類之參數變化.................................... 73
圖4-7 以103.44 g/L 葡萄糖、黃豆水解液作為發酵基質進行批式發酵,其過程中生菌數、乳酸、鹼液 (10N NaOH) 及各種醣類之參數變化.................................... 74
圖4-8 以批式發酵方法在不同氮源發酵期間參數之變化 (a) 總乳酸產量 (b) 瞬間乳酸產率..................................... 76
圖4-9 以批式發酵方法在不同氮源發酵期間轉化率之變化.. 77
第三節
圖4-10 以103.44 g/L 葡萄糖、黃豆水解液作為發酵基質進行饋料批式發酵,待殘糖量低於30 g/L時加入饋料 (葡萄糖濃度800 g/L),使殘糖量達60 g/L,其過程中生菌數、乳酸、鹼液 (10N NaOH) 及各種醣類之參數變化....................................................................... 86
圖4-11 以103.44 g/L 葡萄糖、黃豆水解液作為發酵基質進行饋料批式發酵,待殘糖量低於30 g/L時第一次加入饋料 (葡萄糖濃度800 g/L),使殘糖量達60 g/L,待殘糖量再次低於30 g/L時第二次加入饋料 (葡萄糖濃度800 g/L),使殘糖量達60 g/L,其過程中生菌數、乳酸、鹼液 (10N NaOH) 及各種醣類之參數變化............................................................................... 87
圖4-12 以饋料批式發酵方法在初始葡萄糖濃度為103.44 g/L,待殘糖量降至70 g/L時加入饋料且維持在70 g/L,其發酵期間之 (a)菌體生長情形 (b)乳酸產量及殘糖量 變化........................................................... 88
圖4-13 以饋料批式發酵方法在不同饋料條件下,發酵期間乳酸濃度之變化........................................................... 89
圖4-14 以批式及不同饋料批式發酵方法在初始葡萄糖為103 g/L條件下之 (a)乳酸濃度 與(b)產率................. 90
圖4-15 以批式及不同饋料批式發酵方法在初始葡萄糖為103.44 g/L條件下之轉化率......................................... 91
圖4-16 以饋料批式發酵方法在初始葡萄糖濃度為130 g/L,待殘糖量降至90 g/L時加入饋料且維持在90 g/L,其發酵期間之 (a)菌體生長情形 (b)乳酸產量及殘糖量 變化........................................................... 92

表目錄
第二章
 表2-1 乳酸之物理性質................................................................... 9
 表2-2維生素B群與不同氮源在48小時之燒瓶培養對於乳酸產生之影響 (起始葡萄糖濃度100 g/L) ............................ 21
第三章
表3-1 MRS Broth 之組成............................................................... 44
表3-2 高蛋白黃豆粉之組成........................................................... 45
第四章
 第一節
表4-1 250 g/L糖蜜之醣類含量................................................... 59
表4-2 糖蜜組成份....................................................................... 60
表4-3 不同碳源之批式發酵對乳酸產量與產率之比較........... 64
表4-4不同碳源之批式發酵對菌體生長、碳源消耗率與轉化率之比較........................................................................... 66
 第二節
表4-5不同氮源之固形物含量、批式發酵對乳酸產量與產率之比較............................................................................... 75
表4-6不同氮源之批式發酵對菌體生長、葡萄糖消耗率與轉化率之比較....................................................................... 78
 第四節
表4-7批式發酵及不同饋料批式發酵方法在第28小時下實際葡萄糖消耗、乳酸產量及理論乳酸產量之比較......... 95
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