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研究生:劉棠晉
研究生(外文):Tang-Chin Liu
論文名稱:開發商業化乳酸菌配方應用於降低飲酒不適
論文名稱(外文):Development of commercially available lactic acid bacteria formula to apply in rsolving drinking discomfort
指導教授:葉娟美
指導教授(外文):Chunan-Mei Yeh
口試委員:陳勁初蔡稼光
口試委員(外文):Chin-Chu Chen
口試日期:2021-07-30
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:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:103
中文關鍵詞:酒精乙醛乙醛脫氫酶反應曲面法飲酒不適Levilactobacillus brevis YW108Levilactobacillus brevis YW21
外文關鍵詞:alcoholacetaldehydeacetaldehyde dehydrogenaseresponse surface methodologydrinking discomfortLactobacillus brevis YW108Levilactobacillus brevis YW21
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飲酒為全球文化,也是常見的社交行為,飲酒過量會造成許多問題,影響健康以及產生社會問題,增加了健康風險以及社會成本,酒精與代謝過程產生的乙醛,皆為毒性物質,特別是乙醛,乙醛已被認為是一種致癌物質。部分東方人,因為基因型影響,導致代謝乙醛酵素(acetaldehyde dehydrogenase , ALDH )活性低落,容易發生乙醛蓄積體內,影響健康,乙醛也被認為是飲酒不適(臉紅、噁心等)的主要成因之一。
先前研究室自酸白菜發酵液中,篩選出兩株具有代謝酒精與乙醛潛力乳桿菌株,分別為YW108與YW21。計畫以商業化角度,進行產品配方開發,應用於減緩飲酒不適用途,本論文將分成兩個部分來進行研究:
第一部分_開發商業化培養基用以生產Levilactobacillus brevis YW21。實驗室階段常以MRS培養基培養Lactobacillus spp,但MRS培養基所需成本花費高,並不適合未來在進行商業化量產所使用,因此本部分希望透過統計分析方法Plackett-Burman design (PBD)進行因子篩選後;再利用Response Surface Methodology ( RSM )找到最佳化培養基比例配方,結果顯示以35.9g/L高果糖糖漿、20g/L大豆蛋白腖A3SC、1.5g /L酵母萃取物、4.5g/L磷酸胺,做為配方,在24小時OD600預測值為3.64。以桌上型5L發酵槽進行實驗驗證,實際OD600值為4.10,落於95%預測值範圍內,結果與預測值結果相符,此配方培養基成本僅需MRS成本之6%,優於以MRS培養基結果。
第二部分_以乳酸菌配方開發降低飲酒不適產品。具代謝酒精潛力菌株Levilactobacillus brevis YW108,在先前人體試驗結果發現YW108因為其快速代謝酒精產生乙醛,反而容易導致不適感增加;因此需要透過具有代謝乙醛能力之YW21進行混和,同時降低酒精與乙醛。SGC結果顯示60分鐘內,YW108(1010CFU/mL)可降低酒精10000ppb(316uM),同時生成900 ppb乙醛;YW21(2 x109CFU/mL),可降低乙醛約3000ppb(68uM)含量,兩株菌混合使用,YW21預期可以降解來自於YW108代謝過程中所產生的乙醛,以及體內本身飲酒後產生的乙醛,藉由降低乙醇、乙醛含量來達到降低飲酒不適效果。
在商業化乳酸菌配方將預計會推出兩種配方,針對於飲酒前使用與飲酒後使用,飲酒前配方為混合使用YW108與YW21用以快速代謝酒精與乙醛;飲酒後配方為使用YW21單一菌株,快速降低體內乙醛含量,配方的成分會加入其他輔助成分,加強肝臟代謝能力,預期可以應用於降低飲酒不適症狀。
Drinking is a global culture also a common social behavior. Excessive drinking causes many problems, such as health problem and social problems. Thus, increasing health risks and social costs. Both alcohol and acetaldehyde are all toxic substances, especially acetaldehyde, it has regarded as a type of carcinogens. Due to the influence of genotype, the activity of metabolic acetaldehyde enzyme (Acetaldehyde dehydrogenase, ALDH) is low. Acetaldehyde is prone to accumulate in the body, which affects health. Considered to be one of the main causes of drinking discomfort (blushing, nausea, etc.)
Previously, the laboratory screened two strains of lactobacillus with the ability to metabolize alcohol and the potential for acetaldehyde from the fermentation broth of sauerkraut. They are YW108 and YW21 respectively. Our study plan to develop product formulas from a commercial perspective and apply this formulas to reduce the uncomfortable during on drinking or after drinking. Our Study will be divided into two parts for research:
Part I: Development of commercial culture medium For Levilactobacillus brevis YW21: In the laboratory stage, Lactobacillus spp is often culture with MRS medium, but the cost of MRS medium is too high to suitable for commercial production. Therefore, this section hopes to use the statistical analysis method Plackett-Burman design (PB design). for factor screening, then using Response Surface Methodology (RSM) to find the optimal medium ratio formula. Results show that The Optimal formula is 35.9g/L high fructose syrup, 20g/L soy peptone A3SC, 5.5g/L yeast extract . 4.5g/L amino phosphate , predicted value of OD600 in 24 hours is 3.64. Using 5-liter fermenters verification, The actual OD600 value is 4.10, falling within the range of 95% predicted value, and the result is consistent with the predicted value result .which is higher than the result of MRS culture and the cost of this culture medium is only 6% of the cost of MRS.
Part 2: Development of products to reduce drinking discomfort with lactic acid bacteria formula。Previously, IRB test showed that, the strain YW108 exhibited rapid alcohol metabolism capability, therefore, consuming YW108 cause production of acetaldehyde and that lead to discomfort. As consequence, it is necessary to mix YW108 with YW21 which has the ability to metabolize acetaldehyde. The mixed formula are expected to reduce alcohol and acetaldehyde. SGC results show that YW108 (1010CFU/mL) can reduce alcohol by 10000ppb (316uM) and produce 900 ppb of acetaldehyde within 60 minutes; YW21 (2 x109CFU/mL) can reduce the content of acetaldehyde about 3000ppb (68uM). The YW21 in mixed formula is expected to completely degrade the acetaldehyde produced by YW108 and lower the acetaldehyde content after drinking. In the commercial LAB formula, two formulas were designed for use before and after drinking, the pre-drinking formula is a mixture of YW108 and YW21 to quickly metabolize alcohol and acetaldehyde; the Post-drinking formula is to use a single strain of YW21 that can quickly reduce the content of acetaldehyde in the body. Other ingredients will be used in the formula as an auxiliary component to strengthen the metabolism of the liver. Formulas designed in this study are expected to reduce the symptoms of drinking discomfort.
摘要 i
ABSTRACT ii
目錄 iv
表目錄 vii
圖目錄 ix
壹、 前言 1
一、 飲酒文化與危害 1
(一) 酒精成癮與健康危害 1
(二) 酒精對人體的影響與疾病 1
(三) 乙醛對人體的影響與疾病 2
二、 宿醉狀況與成因 2
(一) 宿醉定義 2
(二) 宿醉成因 3
(三) 解宿醉產品介紹 3
三、 酒精吸收 4
(一) 影響酒精吸收與代謝的因素 4
(二) 飲酒後呼氣乙醛變化 5
四、 體內酒精代謝相關機制 5
(一) 乙醇去氫酶(Alcohol dehydrogenase , ADH) 5
(二) 微粒體乙醇氧化系統(Microsomal ethanol oxidizing system , MEOS) 5
(三) 過氧化氫酶(catalase) 6
五、 酒精代謝相關基因與酵素 6
(一) 酒精去氫酶基因 6
(二) 乙醛去氫酶基因 6
六、 酒精代謝基因之單一核苷酸多形性 7
(一) 乙醇去氫酶(ADH)單一核苷酸多形性 7
(二) 乙醛去氫酶(ALDH)單一核苷酸多型性 8
七、 具有酒精代謝潛力之微生物 8
(一) 釀酒酵母(Saccharomyces cerevisiae, yeast) 8
(二) 乳酸菌(Lactic acid bacteria, LAB) 9
(三) 鏈球菌( Streptococcus) 9
八、 乳酸菌簡介 9
(一) 乳酸菌應用 10
(二) 短毛乳酸桿菌(Levilactobacillus brevis, L.brevis) 10
九、 實驗設計用以開發最適化培養基 11
(一) Plackett Burman Design (PBD) 11
(二) Response surface methodology (RSM) 11
十、 商業化產品開發 12
貳、 研究起源與實驗目的 13
參、 研究架構 14
肆、 材料與方法 15
一、 菌種 15
(一) Levilactobacillus brevis YW108 15
(二) Levilactobacillus brevis YW21 15
二、 實驗中使用之引子 15
(一) 27F 15
(二) 1492R 15
三、 實驗中使用之儀器、藥品與試劑 15
四、 菌株鑑定相關分生技術 15
(一) 短乳桿菌(L.brevis)染色體 DNA 純化 15
(二) 聚合酶連鎖反應(Polymerase Chain Reaction, PCR) 16
(三) PCR 產物定序確認 16
五、 菌株基本特性探討 16
(一) YW21與YW108生長試驗 16
(二) YW21腸胃耐受性測試 16
(三) 菌落平板計數 17
(四) 吸光值與乾物重標準曲線繪製 17
六、 感應式氣相層析儀(Sensor Gas Chromatograph, SGC) 17
七、 YW21菌株誘導/共培養測試 18
(一) 以X物質誘導 18
(二) 以Y物質共培養 18
八、 YW108與YW21實驗菌株製備 18
(一) YW108誘導菌株製備 18
(二) YW21 實驗菌株製備 18
九、 菌株劑量對於降低乙醇、乙醛能力測試 19
(一) 降低乙醛能力測試 19
(二) 降低酒精能力測試 19
十、 實驗設計用於最適培養基開發 19
(一) Plackett Burman Design(PBD)實驗設計 19
(二) 生長最適碳、氮源分析 20
(三) Response Surface Methodology 20
十一、 5L 桌上型發酵槽試驗 21
伍、 結果與討論 22
一、 YW21 基本特性探討 22
(一) 菌株鑑定 22
(二) YW21生長特性分析 22
(三) 腸胃道耐受性結果 23
二、 最適培養基配方開發 23
(一) Plackett-Burman Design (PBD)分析結果 23
(二) 不同種類碳、氮來源對YW21生長影響 24
(三) 反應曲面法(RSM)結果分析 26
三、 發酵成本估算 27
四、 5L桌上型發酵槽結果 27
五、 YW21誘導/共培養條件測試 28
(一) 以X物質進行誘導 28
(二) 以Y物質加入培養基中共培養 29
六、 YW21不同菌量對於降低乙醛效果比較 29
(一) YW21代謝方式推測 29
七、 YW108 不同菌量對於降低酒精效果比較 30
八、 乳酸菌產品配方開發 30
陸、 結論 32
柒、 附錄一:表格 33
捌、 附錄二:圖片 55
玖、 附錄三:藥品與儀器 97
壹拾、 參考文獻 100
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