Investigation of interaction between lactic acid bacteria and milk fat globule membrane material isolated by a novel method using hydroxyapatite pretreatment and microfiltration

本研究利用氫氧磷灰石 (hydroxyapatite) 可解離與吸附乳蛋白質的特性，針對酪乳 (buttermilk) 原料進行前處理，藉以提升微過濾 (microfiltration) 對乳脂肪球膜 (milk fat globule membrane；MFGM) 的分離效果，同時探討由此製程所獲得之產物與乳酸菌的吸附現象以及是否可增進乳酸菌對耐酸與耐膽鹽之特性。試驗使用國立中興大學畜產試驗場生乳經乳脂分離 (cream separation) 以及攪打 (churning) 製成酪乳，隨後冷凍乾燥為酪乳粉並回溶至9% (w/v) 以供研究使用。經標準化的酪乳以1410 ×g離心5分鐘去除脂肪後分為5% 氫氧磷灰石處理組 (HA)、2%檸檬酸鈉處理組 (SC) 以及對照組進行前處理，其中氫氧磷灰石處理組於室溫攪拌2小時，再以1410 ×g離心5分鐘移除氫氧磷灰石顆粒；檸檬酸鈉處理組則於4℃反應至隔日。經前處理後，各處理組與控制組以逆滲透水4倍稀釋，隨後使用0.2 μm 孔徑之陶瓷濾膜以43 psi過膜壓力進行4次滲濾 (diafiltration) 操作之掃流式微過濾以獲得富含MFGM的產物。結果顯示以HA前處理之MFGM其總碳水化合物、膽鹼相關磷脂與粗蛋白質回收率分別為1.25 ± 0.51%、56.67 ± 9.85%與60.81 ± 5.07%，顯示HA前處理於微過濾後可有效截留乳脂肪球膜磷脂成分以及具有良好的醣類去除能力；SDS-PAGE結果中亦指出HA前處理組的微過濾分離物相較於對照組與SC前處理者具有較低之酪蛋白與乳清蛋白含量，且其乳脂肪球膜蛋白所占比例較高，顯示此類製程具有較佳之分離效率。於MFGM吸附乳酸菌特性部分，先以十六烷進行菌株表面疏水性之篩選並鑑定後獲得親水性與疏水性菌株各一，分別為 Lactobacillus plantarum ATCC 14917以及 Lactobacillus casei Y310，經螢光染色並以共軛焦雷射掃描顯微鏡 (confocal laser scanning microscopy; CLSM) 可觀察到其中親水性乳酸菌 (Lb. plantarum ATCC 14917) 具有吸附至乳脂肪球表面的能力，並於MFGM溶液中可觀察到菌體聚集的現象。進一步對此具吸附性的菌株進行耐酸與耐膽鹽特性分析，其結果指出由HA前處理配合微過濾所分離之乳脂肪球膜可分別於pH 3與0.3% 膽鹽濃度之模擬胃腸道環境中增進菌株的存活性，且此現象隨分離物添加量上升而呈現劑量依賴性。綜觀上述，HA前處理配合微過濾可有效分離酪乳中的乳脂肪球膜，相對現行之SC前處理者具有較佳的乳脂肪球膜蛋白分離性，同時此類製程所獲得之分離物具有結合聚集乳酸菌的特性以及增進其耐酸與耐膽鹽的能力。The aim of this study was to develop a novel milk fat globule membrane (MFGM) isolation method by using hydroxyapatite pretreatment and microfiltration. The interaction between lactic acid bacteria and the MFGM material was also evaluated. Raw milk was obtained from experimental farm of National Chung Hsing University and the cream was further separated following by churning into buttermilk. The freeze-dried buttermilk powder was reconstituted into 9% weight by volume and centrifuged in 1410 ×g for 5 minutes to remove the remaining fat globules before use. Reconstituted buttermilk was divided into three experiment groups: (1) 5% hydroxyapatite stirring for 2 hour in room temperature, (2) 2% sodium citrate standing overnight in 4℃ and (3) untreated control. All the pretreated samples were four folds diluted and conducted through a 0.2 μm crossflow ceramic microfiltration system under 43 psi transmembrane pressures with four diafiltration steps. The recovery rate of total carbohydrate, choline-contained phospholipids and crude protein were 1.25 ± 0.51%, 56.67 ± 9.85% and 60.81 ± 5.07% respectively. These results indicate that hydroxyapatite pretreatment combining with microfiltration is able to retain phospholipids and remove lactose in the buttermilk efficiently. The protein profiles of MFGM material in SDS-PAGE were shown that the distribution of caseins and whey proteins were obviously less in hydroxyapatite pretreatment group than others, which suggesting a higher MFGM separation efficiency was observed. To investigate the interaction between MFGM and lactic acid bacteria by confocal laser scanning microscopy, Lactobacillus plantarum ATCC 14917 and Lactobacillus casei were selected due to its low and high surface hydrophobicity respectively. The results showed that Lb. plantarum ATCC 14917 had the ability to adhere to the milk fat globule surface and aggregated in the MFGM material. There was also shown that MFGM material could increase acid and bile tolerance of L. plantarum in a dose-dependent manner at pH 3 and 0.3% bile conditions. Above all, the hydroxyapatite pretreatment combining with microfiltration might be an efficiency way to isolate MGFM comparing to other methods. Besides, the MFGM material obtained from this process shows the potential to protect lactic acid bacteria species against acid and bile challenge.致謝 i 中文摘要 ii Abstract iii 表次 vii 圖次 viii 縮寫對照表 x 第一章 前言 1 1. 研究動機 1 2. 研究目的 2 第二章 文獻探討 3 1. 乳脂肪球膜之組成份 3 1.1 極性脂肪 3 1.2 膜蛋白 5 1.2.1 嗜乳脂蛋白 5 1.2.2 黃嘌呤脫氫/氧化酶 5 1.2.3 乳黏著蛋白 6 1.2.4 黏蛋白1 6 1.2.5 親脂蛋白 6 1.2.6 脂肪酸結合蛋白 7 1.2.7 Cluster of differentiation 36 7 2. 乳脂肪球膜的結構與形成機制 9 3. 乳脂肪球膜分離策略 14 3.1 三酸甘油酯核心去除策略 16 3.1.1 物理法 16 3.1.2 化學萃取法 16 3.2 水溶性蛋白質去除策略 17 3.2.1 乳脂清洗法 17 3.2.2 凝乳法 18 3.2.3 酪蛋白膠粒解離法 20 3.3 乳脂肪球膜收集策略 22 3.3.1 超高速離心 22 3.3.2 膜過濾 22 4. 乳脂肪球膜應用潛力 24 4.1 健康機能潛力 24 4.1.1 抑制癌症 24 4.1.2 降低血膽固醇 25 4.1.3 促進神經系統發育 27 4.1.4 促進腸道成熟與修復 27 4.1.5 其他機能性 28 4.2 乳化性 29 4.3 物質運輸潛力 30 5. 微生物與乳脂肪球膜之交互作用 32 5.1 吸附現象 32 5.1.1 病原菌結合位競爭效應 32 5.1.2 乳酸菌與乳製品之乳脂肪球膜吸附現象 34 5.2 生長代謝影響 35 5.2.1 抑制病原菌生長與調控致病性 35 5.2.2 供給乳酸菌能量來源以及代謝路徑改變 36 第三章 材料與方法 37 1. 試驗設計 37 2. 乳脂肪球膜分離 39 2.1 酪乳製備 39 2.2 酪蛋白膠粒解離處理 39 2.3 酪乳明度分析 40 2.4 微過濾 40 3. 化學成分分析 42 3.1 碳水化合物 42 3.2 膽鹼相關磷脂 43 3.2 粗蛋白 44 3.4 蛋白質電泳 45 4. 乳酸菌與乳脂肪球膜之交互作用 49 4.1 乳酸菌菌種來源 49 4.2 菌體表面疏水性試驗 49 4.3 共軛焦雷射掃描顯微影像 50 4.4 耐酸與耐膽鹽試驗 53 5. 統計方法 53 第四章 結果與討論 54 1. 氫氧磷灰石與檸檬酸鈉對酪乳中酪蛋白膠粒解離之影響 54 2. 微過濾及其前處理對分離產物的影響 58 2.1 總碳水化合物回收率 58 2.2 膽鹼相關磷脂回收率 60 2.3 蛋白質組成份變化 63 2.4 乳脂肪球膜結構變化 70 3. 乳脂肪球膜與乳酸菌之交互作用 74 3.1 乳酸菌分離株菌體疏水性分布 74 3.2 乳脂肪球膜與乳酸菌之吸附現象 76 3.3 乳脂肪球膜對乳酸菌耐酸特性之影響 82 3.4 乳脂肪球膜對乳酸菌耐膽鹽特性之影響 86 第五章 結論 89 第六章 參考文獻 9