臺灣博碩士論文加值系統

摘 要
為探討溫度及pH 值與時間對EPS(exopolysaccharides)產量及性狀之影響，本研究取Lactobacillus helveticus BCRC14030培養於含脫脂乳之發酵槽中，在25 、37 、50 ℃及pH 5、6、7，培養48、72、96、108小時後進行採樣，並分析發酵液黏度、菌落數目、EPS產量、黏絲性、單醣成分、分子量及菌酛繼代培養次數對產黏之影響。
L. helveticus BCRC14030在不同培養條件下，各發酵時間黏度差異大多不顯著(p＞0.05)，介於1.54~5.88 cp；在三種pH值下，pH 5之發酵液黏度最高。在三種溫度下，L. helveticus BCRC14030在25及50 ℃下培養之發酵液黏度皆較高，而37 ℃因適合此菌酛生長，24小時之培養時間之菌落數最多，其營養成分需求量高，使酪蛋白比例下降，造成48小時以後測得之黏度也相對下降。
在25 ℃，不同pH值下，L. helveticus BCRC14030菌落生成高峰皆在72小時，而於96及108小時菌落數開始下降，而各發酵時間EPS產量差異不顯著(p＞0.05)。25 ℃、pH 6培養48~108小時為EPS生成量最佳培養條件，介於1.79~2.81 g/L間。黏絲性比較上，L. helveticus BCRC14030在25 ℃、pH 6培養條件下，經96及108小時發酵後EPS黏絲性可達4.78及5.86 cm之最大值。
單醣組成及其比例方面，L. helveticus BCRC14030所生成EPS在大多數處理組之單醣組成上含有galactose，其次為glucose 及N-acetyl-glucosamine，而含有rhamnose之比例為最低，而各單醣含量比例依培養條件不同而有所差異。
EPS含0~2種不同分子量之片段，其分子量分別為347~324168 kDa及3.309~84.733 kDa，比例範圍則分別介於35~100%及7~100%之間，黏絲性最佳為L. helveticus BCRC14030在25 ℃、pH 6所培養生成EPS之高分子片段分子量為1636 kDa；但不具黏絲性的處理組，如菌酛於37、50 ℃及pH 6之各時間採樣處理組下，所培養生成EPS亦呈現含有高分子量情形，其分子量介於103029~202752 kDa，比例為35~100％，亦呈現高分子量EPS比例較高的情形。
不同繼代培養次數之L. helveticus BCRC14030在25 ℃、pH 5培養下，經15次繼代培養菌落數於72小時達6.95x108 CFU/mL為其高峰，較經7次繼代培養菌落數達3.29x109 CFU/mL差。而不同繼代培養菌酛生成之EPS產量介於0.17~0.34 g/L。在EPS黏絲性比較，除了兩種不同繼代培養時間之菌酛在培養48小時生成之EPS黏絲性相同外，其餘發酵時間之EPS黏絲性明顯皆以經7次繼代培養菌酛之EPS黏絲性較佳，且經15次繼代培養菌酛於48及108小時生成之EPS黏絲性兩者呈差異顯著(p< 0.05)外，其餘發酵時間之EPS黏絲性則差異不顯著(p＞0.05)。所含單醣組成種類皆含有rhamnose、N-aceyl-glucosamine、galactose及glucose，依各發酵時間之單醣組成而有所不同，並皆有單醣組成比例之變動。分子量及其比例方面，經7次繼代培養菌酛在各發酵時間生成之EPS為高分子量，經15次繼代培養菌酛生成之EPS則為低分子量。
綜合所述， L. helveticus BCRC14030為黏質乳酸菌，將其培養於含脫脂乳之發酵槽中，以培養條件為25℃、pH 6經96小時所生成之EPS產量及黏絲長最佳。

關鍵詞：胞外多醣、乳酸菌

Abstract
The aim of this research was to determine the yield and examine the properties of exopolysaccharides (EPS) produced by Lactobacillus helveticus CCRC14030. L. helveticus CCRC14030 was inoculated in skim milk medium and incubated in a fermentor at 25, 37, and 50℃, pH 5, 6, and 7 for 0-108 h. The fermented samples were withdrawn at intervals of 48, 72, 96 and 108 h for determinations of viscosity, total viable counts, EPS yield, ropy condition, monomer, molar mass, and times of subculture on viscosity.
The viscosities of fermented samples ranged from 1.54 to 5.88 cp were not significant different (P>0.05) among incubation times. The highest viscosity was observed in the fermented sample of pH 5 among three pHs and 25 and 50℃ were found to be the better incubation temperatures for improving the viscosity. The viscosity of the fermented samples after 48 h of incubation decreased due to increase in total viable counts at 24 h. Higher viable counts resulted in the decrease in the amount of casein in the skim milk medium and the viscosity.
The highest total counts of L. helveticus CCRC14030 was observed at 72 h of incubation. The viable counts decreased after 96 and 108 h. Although EPS yields were not significant different (P>0.05) among incubation times, the highest EPS yields of 1.79-2.81 g/L were found in the incubation condition of 25℃, pH6 and 48-108 h.
The highest ropiness values were observed in the EPS produced by L. helveticus CCRC14030 incubated at 25℃, pH6 for 96 and 108 h, and the values were 4.78 and 5.86 cm, respectively.
Galactose was found in EPS produced by most of L. helveticus CCRC14030 treatments, and the content was in the descending order by glucose, N-acetyl-glucosamine, and rhamnose. The content of rhamnose was the lowest. The different content of each monomer was found at different inoculated condition.
Two fragments of different molar mass found in EPS were 347~324168 and 3.309~84.733 kDa, respectively and the contents were 35~100% and 7~100%. The highest ropiness value of EPS produced by L. helveticus CCRC14030 was inoculated at 25℃, pH 6, it was 1636 kDa. The non-ropy EPS produced by L. helveticus CCRC14030 was inoculated at 37、50 ℃, pH 6, they were also high molar mass, about 103029~202752 kDa, occupied in 35~100%.
L. helveticus CCRC14030 with different subculture times of was inoculated at 25 ℃, pH 5. The highest total viable counts of the culture strain with 15 subculture times was 6.95x108 CFU/mL, which was lower than 3.29x109 CFU/mL of the culture strain with 7 subculture times.The EPS yield of the culture strain with different subculture times produced ranged from 0.17 to 0.34 g/L. Camparing the EPS ropiness value , the ropiness value of EPS produced by all fermented times except 48 h of the culture strain with 7 subculture times were better. The ropiness value of EPS produced by 48 and 108 h fermented times of the culture strain with 15 subculture times were significant different (P＜0.05), others were not significant different (P>0.05). The monomer of the culture strain with different subculture times contained rhamnose, N-aceyl-glucosamine, galactose and glucose, which was different from the fermented times and also varied the composion of monomer. The molar mass of EPS and it's proportion of the culture strain with 7 subculture times were high molar mass EPS, the molar mass of EPS produced by the culture strain with 15 subculture times were low molar mass EPS.
In conclusion, L. helveticus CCRC14030 was the ropy strains. It was inoculated in fermentor filled with skim milk at 25 ℃, pH 6, after 96 h which produced the EPS yield and it's ropy condition of this condition were the best.

KEYWORD：Exopolysaccharides, Lactic acid bacteria.

目 錄
頁次
中文摘要 I
英文摘要 III
表目錄 V
圖目錄 VII
壹、前言 1
貳、文獻探討 2
一、胞外多醣(exopolysaccharide, EPS) 2
1.簡介 2
2.分類與組成 2
3.分子量 10
4.生合成 11
二、乳酸菌(Lactic acid bacteria, LAB) 12
1.定義 12
2.分類 13
3.乳酸菌之益生效果 15
三、乳酸菌胞外多醣之生產 18
1.遺傳特性 18
2.培養基組成之影響 19
3.培養條件之影響 22
4.胞外多醣(EPS)的產黏特性 27
四、胞外多醣的重要性 28
1.對乳製品的影響 28
2.相關製品的發展 30
五、發酵槽(fermenter) 31
1.優點 31
2.類型 31
3.發酵槽在EPS生產上之應用 37
參、材料與方法 39
一、菌酛 39
二、藥品 39
三、菌酛之保存與更新 39
四、生長條件對乳酸菌EPS產量與黏度之影響試驗 40
五、不同繼代培養次數對生成EPS之性狀影響 45
肆、結果與討論 46
伍、結論 71
陸、參考文獻 73
表 目 錄
目次
表一. 乳酸菌酛生成之同質胞外多醣組成份 3
表二. 乳酸菌酛生成之異質胞外多醣組成份 8
表三. 乳酸菌酛生產之EPS所含之六種不同重覆單位之單醣成分、比例及相關生產菌酛 9
表四. 以微生物生長速率隻快慢與對混合攪拌應力之承受壓，作為發酵槽形式之依據 32
表五. L. helveticus BCRC14030在25、37、50 ℃與pH 5、6、7 培養於牛奶培養基經48~108小時後之發酵液黏度比較 48
表六. L. helveticus BCRC14030在25、37、50 ℃與pH 5、6、7 培養於牛奶培養基經48~108小時後EPS之產量 55
表七. L. helveticus BCRC14030在25、37、50 ℃與pH 5、6、7 培養於牛奶培養基經48~108小時後EPS之黏絲性比較 58
表八. L. helveticus BCRC14030在25、37、50 ℃與pH 5、6、7 培養於牛奶培養基經48~108小時後所得EPS之單醣組成及其比例 60

表九. L. helveticus BCRC14030在25、37、50 ℃與pH 5、6、7 培養於牛奶培養基經48~108小時後所得EPS之滯留時間、分子量及比例 63
表十. 不同繼代培養次數之L. helveticus BCRC14030在25℃、pH 5培養於牛奶培養基經48~108小時後生成之EPS產量比較 66
表十一. 不同繼代培養次數之L. helveticus BCRC14030在25℃、pH 5培養於牛奶培養基經48~108小時後生成之EPS黏絲性比較 67
表十二. 不同繼代培養次數之L. helveticus BCRC14030在25℃、pH 5培養於牛奶培養基經48~108小時後生成之EPS單醣組成及其比例 69
表十三. 不同繼代培養次數之L. helveticus BCRC14030在25℃、pH 5培養於牛奶培養基經48~108小時後生成之EPS分子量及其比例 70






圖 目 錄
目次
圖一. 同質多醣之構造：聚葡萄糖 4
圖二. 同質多醣之構造：聚果糖 4
圖三. 胞外多醣生合成之途逕 12
圖四. 依據是否具有攪拌機構及通氣設備分類之發酵槽形式 33
圖五. 機械攪拌式發酵槽結構 33
圖六. 氣舉式發酵槽結構 34
圖七. 機械攪拌式發酵槽之攪拌翼形式 34
圖八. 5 L桌上型發酵槽(由左至右分別為充氣幫浦、恆溫水槽、發酵槽體、控制主機) 36
圖九. 槽體上蓋各孔洞位置及名稱 36
圖十. L. helveticus BCRC14030在25 ℃與pH 5、6、7培養於牛奶中經0~108小時之發酵液菌數 50
圖十一. L. helveticus BCRC14030在37 ℃與pH 5、6、7培養於牛奶中經0~108小時之發酵液菌數 51
圖十二. L. helveticus BCRC14030在50 ℃與pH 5、6、7培養於牛奶中經0~108小時之發酵液菌數 52
圖十三. 不同繼代培養次數之L. helveticus BCRC14030在25℃、pH 5培養於牛奶培養基經48~108小時後之菌數比較 65

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