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研究生:林文郁
研究生(外文):Wen Yuh Lin
論文名稱:乳酸菌和雙叉乳桿菌益生特性之探討
論文名稱(外文):Studies on the Probiotic Characteristics of Lactic Acid Bacteria and Bifidobacteria
指導教授:張勝善黃建榕黃建榕引用關係
指導教授(外文):Hsi-Shan ChangChien-Jung Huang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:畜產學系
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:143
中文關鍵詞:乳酸菌雙叉乳桿菌耐酸性耐膽鹽性抗氧化性抗致突變性
外文關鍵詞:lactic acid bacteriabifidobacteriaacid tolerancebile toleranceantioxidantantimutagen
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本研究收集由嬰兒糞便中分離純化、商業上販售及購自菌種保存及研究中心的乳酸菌和雙叉乳桿菌共計35株進行研究,由其中篩選出具備耐酸性和耐膽鹽性、對病原菌具抑制性、具抗氧化、抗致突變性及可水解乳糖合成機能性寡醣類等益生特性的菌株。
由醣類水解和合成變化之結果顯示,隨著混合菌元添加種類和比例的差異,乳糖水解強度也隨之改變。以HPLC分析醣類組成方面,X處理組(Bifidobacterium sp., Streptococcus thermophilus, Lactobacillus bulgaricus以2:2:1比例混合)中以X01和X06組的乳糖水解量最高,而寡醣合成量依序為X14、X04、X08及X24組。Y處理組(B. sp., S. thermophilus, L. acidophilus以2:2:1比例混合)中以Y08組的乳糖水解量最高,而Y01和Y08組的寡醣合成量較高。Z處理組(B. sp., S. thermophilus, L. bulgaricus, L. acidophilus以1:1:1:1比例混合)中以Z02、Z04、Z16、Z21、Z22組的乳糖水解量較高,Z02,Z06及Z21組則具較高的寡醣合成量。以薄層色層分析醣類組成得知,所有處理組皆有寡醣合成,但以三醣類為主;以活性碳層析法萃取出之寡醣經核磁共振光譜分析並比對其化學位移的結果後推測,純化出的醣類為β-galactose-(1→4)-β-galactose-(1→4)-glucose結構的半乳寡醣。
耐酸性試驗結果顯示,大部分乳酸菌和雙叉乳桿菌對於pH 2.0磷酸緩衝溶液不具耐受性,只有B. adolescentis CCRC 14609,B. bifidum CCRC 14630及B. catenulatum CCRC 14667等3株為耐低酸菌株。耐膽鹽性試驗中,共有10株具有膽鹽耐受性。依據耐酸性和耐膽鹽性試驗選出L. acidophilus CCRC 10695,L. bulgaricus CCRC 10696,L. brevis CCRC 12187,L. rhamnosus CCRC 14029,B. adolescentis CCRC 14607, 14609,L. reuteri CCRC 14625,B.catenulatum CCRC 14667及B. bifidum CCRC 14670等9株菌株接受模擬消化過程之測試,結果所有菌株存活率都能達到80%以上。
在抑制腸道有害菌方面,30株乳酸菌和雙叉乳桿菌皆能抑制Bacillus circus,但對Staphylococcus aureus的抑制性最差,僅有L. rhamnosus CCRC 10940,B. longum CCRC 11847及L. kefir CCRC 14011等3株具有抑制效果。有18株菌株具有抑制Escherichia coli的能力,以B. bifidum CCRC 12585抑菌性最佳;26株菌株可抑制Salmonella typhimurium,以B. breve CCRC 11846的抑菌性最佳;27株菌株可抑制Pseudomonas fluorescens,以B. breve CCRC 11846的抑菌性最佳。
在抗氧化性方面,測定20株乳酸菌和雙叉乳桿菌抑制亞麻油酸過氧化物的形成情形,有8株菌株於培養5天後仍具抗氧化力,以B. adolescentis CCRC 14609最佳。L. reuteri CCRC 14625在還原力部分表現最佳,而商業型乳酸菌株B03有最強的清除超氧陰離子能力。在清除DPPH自由基能力方面,有7株菌株清除率達80%以上,B. bifidum CCRC 14614表現最佳;螯合亞鐵能力較佳的是L. reuteri CCRC 14625和B. adolescentis CCRC 14609,其螯合能力可達50%以上。
在抗致突變性方面,乳酸菌的添加對Salmo. typhimurium TA 98和TA 100並不具有毒性和致突變性,而這兩個系統中致突變性最低的皆為B. adolescentis CCRC 14608。添加致突變劑NQNO誘發Salmo. typhimurium TA 98和TA 100的突變,發現所有菌株皆有輕微的抗致突變性,其中B. breve CCRC 11846和B. adolescentis CCRC 14608對Salmo. typhimurium TA 98的抗致突變性較佳,抑制比例達70%;而TA 100系統中乳酸菌株的抗致突變性較TA 98系統佳,抑制比例達85%的有B. adolescentis CCRC 14608,B. bifidum CCRC 14614及B. adolescentis CCRC 14609等3株,B. infantis CCRC 14661和B. catenulatum CCRC 14667亦達70%以上。
Lactic acid bacteria and bifidobacteria are widely used for human food. The basic requirements for lactic acid bacteria as probiotics are the capability for tolerance to gastric acid and bile salts, the ability to inhibit pathogenic bacteria, antioxidative activity, and antimutagen activity. In addition, the lactose hydrolysis and synthesis of functional oligo- saccharides of lactic acid bacteria were investigated in order to promote the probiotic value and be used for food. In this study, 35 probiotic strains of lactic acid bacteria and bifidobacteria were screened and selected from infant stool samples, some commercial dairy products, and Food Industry Research and Development Institute in Taiwan.
The results in the hydrolysis and synthesis of saccharides showed that the hydrolysis of lactose changes with variety and ratio of mixed strains. In X treatments (Bifidobacterium, S. thermophilus, L. bulgaricus mixed in 2:2:1), the lactose hydrolysis of X01 and X06 were higher than others, and oligosaccharides synthesis of X14, X04, X08, and X24 were higher than others. In Y treatments (Bifidobacterium, S. thermophilus, L. acidophilus mixed in 2:2:1), the lactose hydrolysis of Y08 was the highest, and oligosaccharides synthesis of Y01 and Y08 were higher than others. In Z treatments (Bifidobacterium, S. thermophilus, L. bulgaricus, L. acidophilus mixed in 1:1:1:1), the lactose hydrolysis of Z02, Z04, Z16, Z21, and Z22 were higher than others. The oligosaccharides synthesis of Z02, Z06, and Z21 were higher than others. The results of TLC showed that all treatments could synthesize oligosaccharides and trisaccharides were the highest. The chemical structures of oligosaccharides were identified by 13C-nuclear magnetic resonance, and the results showed that the oligosaccharides were galactooligo- saccharide which structure was β-galactose-(1→4)-β-galactose-(1→4)-glucose.
Except B. adolescentis CCRC 14609, B. bifidum CCRC 14630 and B. catenulatum CCRC 14667, most lactic acid bacteria and bifidobacteria didn’t have tolerance on pH 2.0 phosphate buffer. And 10 strains have the tolerance on bile salts. According to the acid and bile resistance tests, the selected strains of L. acidophilus CCRC 10695, L. bulgaricus CCRC 10696, L. brevis CCRC 12187, L. rhamnosus CCRC 14029, B. adolescentis CCRC 14607, 14609, L. reuteri CCRC 14625, B.catenulatum CCRC 14667, and B. bifidum CCRC 14670 had 80% viability in imitating digestive tract condition.
All the 30 strains of lactic acid bacteria and bifidobacteria had the inhibition for Bacillus circus, but only L. rhamnosus CCRC 10940, B. longum CCRC 11847, and L. kefir CCRC 14011 had the inhibition for Staphylococcus aureus. There were 18 strains that inhibit Escherichia coli in which B. bifidum CCRC 12585 had the highest efficiency. 26 strains inhibit Samonella typhimurium in which B. breve CCRC 11846 had the highest efficiency. 27 strains inhibit P. fluorescens in which B. breve CCRC 11846 has the highest efficiency.
20 strains of lactic acid bacteria and bifidobacteria were tested for antioxidative activity by the inhibition of linoleic acid oxidation. There were 8 strains have antioxidative activity after incubated for 5 days as measured by the thiocyanate method. Bifidobacterium adolescentis CCRC 14609 has the best antioxidative acitivity. Lactobacillus reuteri CCRC 14625 was the best reducing activity. Commercial lactic acid bacteria B03 had the best scavenging effects on superoxide anion. 7 strains had above 80% scavenging effects on α-diphenyl-2-picryl hydrazyl (DPPH‧) radical. Bifidobacterium bifidum CCRC 14614. Lactobacillus reuteri CCRC 14625 and Bifidobacterium adolescentis CCRC 14609 had more superior chelating effect on Fe2+, which achieve 50%.
In the experiment of antimutagen activity, the results showed that no mutagenicity or toxicity in S. typhimurium TA 98 and TA 100 with any lactic acid bacteria and bifidobacteria strains. B. adolescentis CCRC 14608 had the lowest mutagenicity toward S. typhimurium TA 98 and TA 100. Detected the antimutagen activity of lactic acid bacteria and bifidobacteria on the mutagenicity of S. typhimurium TA 98 and TA 100 induced by NQNO, and which presented light antimutagenity. B. breve CCRC 11846 and B. adolescentis CCRC 14608 had a higher antimutagenic ability for Salmonella typhimurium TA 98. And the percentage of inhibition was over 70%. The inhibition from S. typhimurium TA 100 was higher than from S. typhimurium TA 98. B. adolescentis CCRC 14608, B. bifidum CCRC 14614, and B. adolescentis CCRC 14609 had 85% inhibition rate and B. infantis CCRC 14661 and B. catenulatum CCRC 14667 had 70% inhibition rate.
壹、中文摘要-------------------------------------------------001
貳、前言-----------------------------------------------------003
參、文獻檢討-------------------------------------------------004
一、人體腸道微生物的生態-------------------------------------004
(一)人體腸道內的微生物---------------------------------------004
(二)消化過程中細菌的存活-------------------------------------004
(三)腸內菌叢的變動因素---------------------------------------005
(四)乳酸菌附著腸壁的機制-------------------------------------006
二、益生菌的特性---------------------------------------------006
(一)為人體常駐菌群-------------------------------------------006
(二)能產生抗菌物質,抑制其它雜菌生長-------------------------007
(三)耐胃酸和膽鹽的能力---------------------------------------008
(四)具吸附上皮細胞的特性-------------------------------------009
(五)於消化道中能快速增殖-------------------------------------010
三、乳酸菌和雙叉乳桿菌對人體產生的益處-----------------------010
(一)對腸道有害菌有抑制能力-----------------------------------010
(二)抗癌與免疫的效果-----------------------------------------011
(三)降低膽固醇的能力-----------------------------------------014
(四)緩和乳糖不耐症-------------------------------------------014
(五)改善營養價值和乳糖的消化性-------------------------------015
四、乳酸菌和雙叉乳桿菌對醣類的代謝與利用---------------------015
(一)醣類的代謝-----------------------------------------------016
(二)醣類的利用-----------------------------------------------018
五、乳酸菌和雙叉乳桿菌與寡醣的關係---------------------------018
(一)寡醣的種類-----------------------------------------------018
(二)寡醣的生理機能性-----------------------------------------020
(三)寡醣的合成機制-------------------------------------------021
(四)寡醣對雙叉乳酸桿菌生長的助益-----------------------------022
六、乳酸菌和雙叉乳桿菌的耐酸性和耐膽鹽性---------------------023
(一)耐酸性---------------------------------------------------023
(二)耐膽鹽性-------------------------------------------------023
七、乳酸菌和雙叉乳桿菌的抗氧化能力---------------------------024
(一)活性氧與疾病---------------------------------------------024
(二)抗氧化劑-------------------------------------------------025
(三)抗氧化劑的作用機制---------------------------------------026
(四)天然抗氧化劑的開發---------------------------------------027
(五)微生物來源的天然抗氧化劑---------------------------------028
(六)乳酸菌的抗氧化能力---------------------------------------033
八、乳酸菌和雙叉乳桿菌的抗致突變特性-------------------------034
肆、實驗部分-------------------------------------------------038
第一章 乳酸菌和雙叉乳桿菌混合菌元之酸酪乳中醣類變化之探討
壹、摘要-----------------------------------------------------039
貳、前言-----------------------------------------------------040
參、材料與方法----------------------------------------------041
肆、結果與討論-----------------------------------------------045
一、乳酸菌和雙叉乳桿菌混合菌元的酸酪乳中醣類分析---------------045
(一)高效能液相層析-------------------------------------------045
(二)薄層色層分析---------------------------------------------047
二、寡醣的鑑定--13C-NMR核磁光譜鑑定--------------------------047
第二章 雙叉乳酸桿菌和乳酸菌的耐酸耐膽汁特性探討
壹、摘要-----------------------------------------------------059
貳、前言-----------------------------------------------------060
參、材料與方法-----------------------------------------------061
肆、結果與討論-----------------------------------------------065
一、乳酸菌和雙叉乳桿菌對酸液、膽鹽的耐受性試驗---------------065
(一)耐酸性試驗-----------------------------------------------065
(二)耐膽鹽性試驗---------------------------------------------067
二、模擬腸道消化過程試驗-------------------------------------076
三、抑菌性的評估---------------------------------------------077
第三章 雙叉乳酸桿菌和乳酸菌的抗氧化和抗致突變性探討
壹、摘要-----------------------------------------------------089
貳、前言-----------------------------------------------------090
參、材料與方法-----------------------------------------------091
肆、結果與討論-----------------------------------------------098
一、乳酸菌和雙叉乳桿菌的抗氧化性-----------------------------098
(一)抗氧化性的測定-------------------------------------------098
(二)還原力的測定---------------------------------------------101
(三)清除超氧陰離子的測定-------------------------------------103
(四)清除DPPH自由基能力的測定---------------------------------106
(五)螯合亞鐵離子能力的測定-----------------------------------109
二、乳酸菌和雙叉乳桿菌抗致突變性-----------------------------112
(一)毒性試驗-------------------------------------------------112
(二)抗突變性-------------------------------------------------112
(三)抗致突變性-----------------------------------------------112
伍、結論-----------------------------------------------------119
陸、參考文獻-------------------------------------------------121
柒、英文摘要-------------------------------------------------140
捌、作者小傳-------------------------------------------------143
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