Reuterin的發酵生成與化學合成及其在生物組織材料上的應用; Evaluation of a natural compound (reuterin) fermented from glycerol using Lactobacillus reuteri in sterilizing and crosslinking biological tissues

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题名:	Reuterin的發酵生成與化學合成及其在生物組織材料上的應用;Evaluation of a natural compound (reuterin) fermented from glycerol using Lactobacillus reuteri in sterilizing and crosslinking biological tissues
作者:	陳俊男;Chung-Nun Chen
贡献者:	化學工程與材料工程研究所
关键词:	生物組織材料;滅菌處理;滅菌劑;reuterin;乳酸菌;生物相容性;biological tissues;sterilization
日期:	2001-06-28
上传时间:	2009-09-21 12:16:56 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	生物組織材料在植入人體前皆須經過適當的滅菌處理，以大量減少或完全除去材料上所帶有的微生物，以避免病人遭受致病微生物的感染。目前臨床上常使用的生醫材料滅菌法，可以分為物理方法與化學方法兩大類。物理方法包括了高壓蒸汽、γ射線與紫外線照射等；而化學方法則有ethylene oxide、glutaraldehyde或環氧化物等等。但是對於生物組織材料，上述的物理方法往往會因高溫或照射的射線能量，使得生物組織材料產生變性（denature）的現象。而化學方法目前所使用的液體或氣體，其細胞毒性皆相當的高，滅菌後在組織材料上的殘餘物，往往會影響到其生物相容性。為了克服上述的問題，本實驗室目前正著手研究一種新的天然滅菌劑-reuterin 。Reuterin是由人或動物的腸胃道所分離出的乳酸菌Lactobacillus reuteri在嫌氣的狀態下，代謝glycerol後所產生的廣效性滅菌物質。因為reuterin為三個碳的醇醛類，其醛基也可能會與生物組織的自由胺基反應形成交聯，所以本研究評估了reuterin對生物組織的交聯性質，以及以reuterin交聯處理的生物組織在動物體內生物相容性的探討。由於Lactobacillus reuteri代謝glycerol產生 reuterin的產率及產量比較低，所需的生產時間比較長，無法符合實際應用上的需求。為了提高reuterin的產量，我們也將嘗試以化學合成的方法來合成reuterin。因此本論文主要是評估reuterin的發酵生成與化學合成及其在生物組織材料上的應用的可行性。在第一部份的實驗裡，我們以Lactobacillus reuteri乳酸菌發酵glycerol生成reuterin及乳酸等其他產物，並以製備級的高效能液相層析儀分離純化出reuterin，接著應用分析級的高效能液相層析儀來定量分析reuterin和分析reuterin在酸性及鹼性的環境下，可能存在的形式。並以體外微生物培養的方式，評估了天然滅菌劑reuterin的最低抑菌濃度(Minimum Inhibitory Concentration, MIC)和最低滅菌濃度(Minimum Bactericidal Concentration, MBC)，以及在不同的溫度和pH值下，reuterin的滅菌效果。實驗中我們使用了臨床上最常感染生物組織的五種細菌：分別為大腸桿菌（Escherichia coli, E）、綠膿桿菌（Pseudomonas aeruguinosa, P）、金黃色葡萄球菌（Staphylococcus aureus, S）與枯草桿菌（Bacillus subtilis. B）和念珠菌（Candida albicans, C）。實驗中使用glutaraldehyde溶液做為我們的對照組。綜合實驗的結果顯示，reuterin的抑菌效果要比glutaraldehyde好約1.2倍∼3.9倍，而reuterin的滅菌效果要比glutaraldehyde來的好約1.6∼3.6倍。另外，reuterin對於革蘭氏陽性菌、革蘭氏陰性菌、霉菌等不同類別的菌株皆有滅菌效果，也初步證明了reuterin為廣效性的滅菌劑。reuterin的滅菌效果會隨著pH值和溫度的增加而提高，而在pH 8.5和37℃的條件下，reuterin的滅菌效果較佳。Reuterin的細胞毒性要比glutaraldehyde低約4倍，且glutaraldehyde會影響細胞的細胞週期進行，和引發細胞凋亡所需的濃度要比reuterin低約3∼4倍。而在第二部份的實驗裡，則探討了以reuterin做為生物組織交聯劑的可行性。實驗裡，我們分別測試了reuterin交聯處理的生物組織的體外酵素分解、交聯指數、變性溫度以及機械性質等。並探討在不同交聯反應條件下，reuterin交聯處理的生物組織的交聯指數、變性溫度、機械性質與抗酵素分解能力，以找出reuterin與生物組織交聯反應的最適化條件。另外，我們也初步探討了reuterin與生物組織可能的交聯反應機制和其細胞毒性。實驗結果顯示， reuterin能有效的交聯生物組織材料。Reuterin的交聯機制可能是在酸性的條件下，3個reuterin分子會形成acetal，並且帶有2個醛基，acetal能夠和生物組織形成交聯。鹼性和高溫的環境比較有利於reuterin的交聯反應，而reuterin交聯反應的最適化條件為： 0.068M的reuterin溶液，在37℃、pH 8.5下交聯處理生物組織3天，其變性溫度、交聯指數、抗酵素分解和機械性質等皆與glutaraldehyde交聯的生物組織相當，因此，reuterin為一個有效的交聯劑。在組織細胞毒性部分，以reuterin交聯處理的生物組織其細胞毒性要比glutaraldehyde交聯處理的生物組織來的低。在第三部份的實驗裡，我們評估了以天然交聯劑reuterin交聯處理的生物組織，在生物體內的生物相容性質。實驗裡，我們分別以glutaraldehyde及未經交聯處理的生物組織當做對照組。將試片植入老鼠背部的皮層底下，然後分別於3天、1週、4週、12週、24週與48週後，將這些植入的測試組織取出來，分別比較它們的生物相容性質，包括了：測試組織的免疫反應、機械強度的變化、變性溫度的變化、鈣化程度、組織形態的變化情形等。以上的實驗結果顯示了，以reuterin交聯處理的生物組織，因其較不具細胞毒性，這使得經reuterin交聯處理的組織，在植入生物體內後免疫反應較glutaraldehyde交聯處理的組織為輕微。因此，reuterin交聯處理的組織生物相容性，要比glutaraldehyde交聯處理的組織要來得好。另外，reuterin交聯處理的組織在生物體內的抗酵素分解能力，與glutaraldehyde交聯處理的組織相當。而且經reuterin交聯處理的組織可能可以抑制組織鈣化的形成。在第四部份的實驗裡，我們探討了以化學合成的方法來合成reuterin。實驗裡，首先我們利用acrolein在酸性、高溫的環境下水合生成reuterin。為了確定化學合成產物的分子結構式是reuterin，我們以分離純化後的reuterin 分別進行FT-IR 、GC-MS 和NMR分析。在reuterin合成反應最適化探討的部分，我們藉由改變不同的溫度、不同的H2SO4濃度和不同的反應時間，進一步探討了不同反應條件對合成reuterin的影響。實驗結果顯示，我們可以由acrolein化學合成的方法來製備reuterin，並經過分離純化後得到純的reuterin。Acrolein化學合成reuterin的最適化條件為：以0.5M的acrolein於0.6N H2SO4溶液中，在50℃下反應3小時。且微生物合成reuterin和化學合成reuterin具有相同的滅菌性質，細胞毒性相當，而以化學合成reuterin或微生物合成reuterin交聯處理的生物組織具有相當的交聯程度。綜合以上體外及體內的實驗結果，reuterin為一個有效的滅菌劑及交聯劑，reuterin的滅菌效果要比gluaraldehyde來的好，交聯性質與gluaraldehyde相當，且細胞毒性比gluaraldehyde低。另外，透過化學合成的方法來提高reuterin的產量，除了可以解決其微生物合成方式產量的不足，亦可增加reuterin在生物組織材料上應用的可行性 Bioprostheses derived from biological tissues must be fixed and subsequently sterilized before they can be implanted in humans. Sterilization is to destruct all forms of life on biomaterials and may be achieved via either physical or chemical means, including dry heat, steam under pressure, irradiation, filtration, and chemical solutions. Clinically, sterilization of biological tissues is generally accomplished by exposing the tissues to a chemical solution such as aqueous glutaraldehyde, formaldehyde, or epoxy compound. However, these chemical solutions are all highly cytotoxic, which may impair the biocompatibility of the sterilized tissues. It is therefore desirable to provide an antimicrobial agent that is of low cytotoxicity and may from germless and biocompatible products. To achieve this goal, a natural compound (reuterin) fermented from glycerol using Lactobacillus reuteri was used by our group to sterilize biological tissues. It is known that reuterin has an antimicrobial activity against a wide spectrum of microorganisms such as bacteria, fungi, and protozoan. Additionally, reuterin (β-hydroxypropioaldehyde, CH2OHCH2CHO) has an aldehyde and an alcohol functional groups in its chemical structure. Therefore, similar to formaldehyde, reuterin may be used as a crosslinking agent to fix biological tissues employing its aldehyde group. In the study, a preparative high-performance liquid chromatographer (HPLC) was employed successfully to isolate reuterin fermented from glycerol using Lactobacillus reuteri. The conversion rate of reuterin fermented from glycerol was approximately 11%. The antimicrobial activities of reuterin against the microorganisms commonly observed clinically, the cytotoxicity of reuterin, and the crosslinking characteristics and the biocompatibility (subcutaneous implantation in a growing rat model) of the reuterin-fixed tissues were studied. Additionally, a chemical method in synthesizing reuterin was developed and its optimal synthetic conditions were investigated. The results obtained in the studies of the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) showed that the antimicrobial activity of reuterin is significantly superior to glutaraldehyde. The cytotoxicity of reuterin was assessed in vitro using 3T3 fibroblasts (BALB/3T3 C1A31-1-1). The results demonstrated that the cytotoxicity of reuterin was significantly lower than glutaraldehyde. The crosslinking characteristics of the reuterin-fixed tissues were investigated using porcine pericardia as raw materials. The results demonstrated that reuterin is an effective crosslinking agent for biological tissue fixation, and its optimal fixation condition was: 0.068M reuterin in PBS solution at pH 8.5, 37ºC for 3 days. It was found that the reuterin-fixed tissues had comparable fixation index, denaturation temperature, mechanical strength and resistance against enzymatic degradation as the glutaraldehyde-fixed tissues. It is suggested that reuterin may form acetal products in the acidic condition. The acetal form of reuterin may be able to crosslink biological tissues with its di-aldehyde functional groups. In the biocompatibility study, a reuterin-fixed porcine pericardium was implanted subcutaneously in a growing rat model. Fresh, and the glutaraldehyde-fixed counterparts were used as controls. It was noted that the inflammatory reactions of the reuterin-fixed tissues were significantly less than its glutaraldehyde-fixed counterparts. Also, the reuterin-fixed tissues had comparable tensile strength and resistance against in vivo degradation as the glutaraldehyde-fixed tissues throughout the entire course of the study. In addition, the calcification rate of the reuterin-fixed tissues was significantly less than their glutaraldehyde-fixed counterparts. A chemical method in synthesizing reuterin was developed using acrolein as the raw material. The chemical structure of the synthesized reuterin was studied by the FT-IR, GC-Mass and NMR analyses. The results showed that the chemical-synthesized reuterin and the microbial-fermented reuterin were identical. Additionally, it was found that the optimal condition in synthesizing reuterin was 0.6N sulfuric acid in distilled water at 50ºC for 3 h. The results obtained in the aforementioned studies indicated that reuterin is an effective crosslinking agent and sterilant. Additionally, as far as the cytotoxicity and biocompatibility are concerned, reuterin is a promising crosslinking agent for biological tissue fixation.
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