臺灣博碩士論文加值系統

本研究係探討含N-乙醯幾丁寡醣之發酵豆奶對雄性Wistar大白鼠在高油高膽固醇的飲食模式 (6%的玉米油或10%豬油與0.5%的膽固醇)下的腸道生理與免疫活性之影響，佐以體外細胞實驗探討發酵產品之免疫功效。實驗共分為四組：（1）含幾丁寡醣豆奶組 (oligomer soymilk)、（2）含幾丁寡醣發酵豆奶組 (oligomer fermented soymilk)、（3）發酵豆奶組 (fermented soymilk)、及（4）豆奶組 (控制組) (soymilk, control)。豆奶依條件添加10%（v/v）的N-乙醯幾丁寡醣水解液，發酵菌種為Lactobacillus plantarum BCRC 12250與Streptococcus thermophilus BCRC 12268，於37℃下發酵約三天至pH4.6後進行噴霧乾燥，將乾燥的豆奶粉末依比例添加至動物飼料中，進行四週的動物飼養。飼養期間採自由進食與攝水的方式。體外細胞實驗則以各樣品之乾燥粉末水溶液與細胞共同培養，評估其對細胞株增生的影響。實驗結果發現，在以玉米油為油脂來源時，實驗各組間皆無明顯差異，而以豬油為油脂來源的飲食條件下，大白鼠攝食含N-乙醯幾丁寡醣之發酵豆奶，其肝臟與血漿中總膽固醇與三酸甘油酯皆較控制組低 (p<0.05)；GOT、GPT與LDH則與控制組無差異。在腸道生理與免疫方面，大白鼠在攝食含N -乙醯幾丁寡醣發酵豆奶後可增加盲腸中總厭氧菌及乳酸菌的數量，且脾臟淋巴細胞增生較控制組明顯增加 (p<0.05)，其腹腔巨噬細胞的吞噬能力也略強 (p>0.05)。體外細胞培養實驗方面，含N-乙醯幾丁寡醣的發酵豆奶組對於細胞株UMΦ及HB4C5均有較好的增生效果，且可刺激脾臟淋巴細胞分泌抗體，佐以刺激原後可使脾臟淋巴細胞增生略強於控制組。

This research was intended to investigate the effects of fermented soymilk containing N-acetylchitooligosaccharides on intestinal physiology and immuno-activity of male Wistar rats fed a high fat and a high cholesterol diet (6% corn oil or 10% lard and 0.5% cholesterol). The immuno-activity of fermented product also checked by in vitro experiments. The diets for rats used in the study were divided into four groups: (1) soymilk containing N-acetylchitooligosaccharides (oligomer soymilk), (2) fermented soymilk containing N-acetylchitooligosaccharides (oligomer fermented soymilk), (3) fermented soymilk, (4) soymilk (control). A 10% (v/v) chitin hydrolysate was added into soymilk according the design purposes. Lactobacillus plantarum BCRC 12250 and Streptococcus thermophilus BCRC 12268 were used as the starters of fermented soymilk. The fermentation was conducted at 37℃ for three days until the pH of the product revealed 4.6. The soymilk powder was added into the experimental diets after drying by spray-dry. The experiment was lasted for four weeks. During feeding, the ad libitum was carried out. The dried sample powder was used to co-incubate with cell line in in vitro experiment to assess the effects to cells.
Results showed that the total cholesterol and total triglyceride in plasma of rats fed the oligomer fermented soymilk were lower than those of control. There was no difference at GOT, GPT, and LDH in plasma among groups. The spleen lymphocytes proliferation and the phagocytosis of peritoneal exudates cells of rats fed on oligomer fermented soymilk were more than that of control group. In in vitro test, the oligomer fermented soymilk could not only enhance the proliferation of cell line UMΦ and HB4C5, but also stimulate the mice spleen lymphocytes to secrete more IgG and IgM.

中文摘要---------------------------------------------------------------------------1
英文摘要---------------------------------------------------------------------------3
壹、前言---------------------------------------------------------------------------5
貳、文獻回顧----------------------------------------------------------------------7
一、黃豆之營養組成及價值-------------------------------------------------7
(一)蛋白質---------------------------------------------------------------------7
(二)油脂------------------------------------------------------------------------7
(三)碳水化合物---------------------------------------------------------------8
(四)礦物質---------------------------------------------------------------------8
二、大豆的保健功效----------------------------------------------------------9
(一)異黃酮之生理機能------------------------------------------------------9
1. 抑制及預防癌症--------------------------------------------------------9
2. 預防心血管疾病-------------------------------------------------------10
3. 排除或緩和女性更年期之障礙-------------------------------------10
(二)生育酚的生理機能----------------------------------------------------11
(三)卵磷脂的生理機能----------------------------------------------------11
三、黃豆與黃豆製品在食用上之限制和改善--------------------------12
(一)減少豆臭味的方法----------------------------------------------------12
(二)改善脹氣的方法-------------------------------------------------------12
1. 全豆蒸煮----------------------------------------------------------------12
2. 發芽去除寡醣----------------------------------------------------------13
3. 酵素法分解-------------------------------------------------------------13
4. 乳酸菌發酵-------------------------------------------------------------13
四、乳酸菌--------------------------------------------------------------------14
(一)乳酸菌對糖類的代謝與利用----------------------------------------14
(二)乳酸菌的保健功能----------------------------------------------------15
1. 對腸道內有害菌有抑制功能----------------------------------------15
2. 抗癌性與免疫調節功能----------------------------------------------15
3. 降低膽固醇的能力----------------------------------------------------17
4. 緩和乳糖不耐症-------------------------------------------------------18
5. 提高營養價值與合成維生素----------------------------------------18
6. 抗致突變能力----------------------------------------------------------19
五、食品乾燥法的比較-----------------------------------------------------19
(一)冷凍乾燥----------------------------------------------------------------19
(二)噴霧乾燥----------------------------------------------------------------20
六、幾丁質與其衍生物之特性與利用-----------------------------------20
七、N-乙醯幾丁寡醣之製備------------------------------------------------23
(一)化學法-------------------------------------------------------------------24
(二)生物法-------------------------------------------------------------------25
八、幾丁聚醣與幾丁寡醣的生理活性------------------------------------26
(一)抑菌作用----------------------------------------------------------------26
(二)免疫活性----------------------------------------------------------------28
(三)降膽固醇功能----------------------------------------------------------28
(四)抗腫瘤功能-------------------------------------------------------------29
九、Cellulomonas flavigena NTOU 1之生化特性-----------------------30
十、實驗動物生理評估-----------------------------------------------------31
(一)飲食中添加膽固醇對脂質代謝的影響----------------------------31
(二)免疫功能的評估-------------------------------------------------------32
1. 細胞存活率 (MTT還原分析法)-----------------------------------32
2. 巨噬細胞釋出超氧陰離子之分析 (NBT reduction assay)-----33
3. 毒胞活性一氧化氮 (NO)產生量分析法--------------------------33
參、材料與方法------------------------------------------------------------------35
一、實驗設計------------------------------------------------------------------35
二、實驗材料------------------------------------------------------------------36
(一)菌株來源----------------------------------------------------------------36
(二)細胞株-------------------------------------------------------------------36
(三)大豆 (黃豆)-------------------------------------------------------------36
(四)培養基及藥品----------------------------------------------------------36
1. 發酵豆奶用之培養基-------------------------------------------------36
2. 化學試劑----------------------------------------------------------------37
(1) 有機溶劑-------------------------------------------------------------37
(2) 酸、鹼、鹽類--------------------------------------------------------37
(3) N-乙醯幾丁寡醣-----------------------------------------------------37
(4) 醣類-------------------------------------------------------------------37
(5) 動物細胞培養-------------------------------------------------------37
(6) 細胞增生測定 (MTT assay)--------------------------------------38
(7) 巨噬細胞分泌超氧陰離子分析(NBT assay)-------------------38
3. 儀器設備----------------------------------------------------------------38
4. 厭氧菌 (anarobes)培養基--------------------------------------------39
5. 厭氧稀釋液-------------------------------------------------------------39
6. 雙叉桿菌 (Bifidobacterium)計數用培養基-----------------------40
7. 乳酸菌 (Lactobacilli)計數用培養基-------------------------------40
8. Tryptose-sulfite-D-cycloserine (TSC) agar--------------------------40
(五)實驗動物----------------------------------------------------------------41
(六)菌種保存----------------------------------------------------------------41
(七)發酵菌株之製備-------------------------------------------------------41
(八)豆奶的製備-------------------------------------------------------------42
(九)幾丁質水解液的製備-------------------------------------------------42
(十)幾丁寡醣的測定-------------------------------------------------------42
(十一)醣類的測定----------------------------------------------------------43
(十二)噴霧乾燥方法-------------------------------------------------------44
(十三)一般成分分析-------------------------------------------------------44
1. 水分測定----------------------------------------------------------------44
2. 灰份測定----------------------------------------------------------------44
3. 粗脂肪含量-------------------------------------------------------------44
4. 粗蛋白質含量----------------------------------------------------------45
5. 碳水化合物含量-------------------------------------------------------45
(十四)動物實驗-------------------------------------------------------------46
1. 動物飼養與樣品取樣-------------------------------------------------46
2. 血漿分析----------------------------------------------------------------46
(1)總膽固醇 (Total cholesterol, TC)測定----------------------------47
(2)三酸甘油酯 (Triglyceride, TG)測定------------------------------47
(3)脂蛋白 (Lipoprotein)之測定---------------------------------------48
(4)GOT (Glutamic oxaloacetic transaminase)之測定---------------48
(5)GPT (Glutamic pyruvic transaminase)之測定--------------------48
(6)乳酸去氫酶(Lactate dehydrogenase, LDH)----------------------49
3. 肝臟脂質分析----------------------------------------------------------50
4. 糞便脂質分析----------------------------------------------------------50
5. 腸內菌相之計數-------------------------------------------------------51
(十五)細胞培養-------------------------------------------------------------51
1. 細胞存活率 (MTT assay)--------------------------------------------52
2. 老鼠脾臟細胞之取得-------------------------------------------------52
3. 脾臟細胞分泌抗體含量分析----------------------------------------53
4. 老鼠腹腔細胞之取得-------------------------------------------------53
5. 腹腔巨噬細胞吞噬能力分析----------------------------------------54
(十六)統計分析-------------------------------------------------------------54
肆、結果與討論------------------------------------------------------------------55
一、含N-乙醯幾丁寡醣之發酵豆奶成分分析---------------------------55
二、動物實驗結果------------------------------------------------------------57
(一)6%玉米油為油脂來源----------------------------------------------57
(二)6%豬油為油脂來源-------------------------------------------------60
三、體外細胞實驗------------------------------------------------------------64
伍、結論---------------------------------------------------------------------------67
陸、參考文獻---------------------------------------------------------------------68

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