臺灣博碩士論文加值系統

細菌性纖維(bacterial cellulose, BC)係一種由微生物所生產之纖維素，其具有如高保水性、良好機械性質以及生物相容性等優點，可被用以作為食品加工、香妝工業及醫療應用等用途。
本研究以提高BC的商業價值為主，可分為四個方向：(1) BC之半連續式生產系統建立; (2)半連續式系統之產量提升;(3) BC生產菌株之鑑定以及 (4) BC於癒創敷材之應用。
首先，我們將轉動式生物反應器(rotating disk bioreactor, RDB)結合塑化基材(plastic composite support, PCS)以開發為塑化基材轉動式生物反應器(PCS-RDB)。塑化基材為一種於高溫高壓下擠壓而成之支架，其成分為黃豆殼、黃豆粉、豬血球粉、酵母粗萃物以及聚丙烯等材料。過去研究發現PCS具有粗糙表面可幫助微生物附著，並可緩慢釋出養分以利微生物生長。本研究結果發現PCS-RDB可用以生產BC產量達0.24 g/L/day，並可於無再次接種之情況下持續生產BC達到五個循環以上。由掃描式電子顯微鏡(Scanning electron microscope, SEM)結果可知，PCS具有粗糙之表面，並於使用後可被發現仍有許多BC生產菌種Gluconacetobacter xylinus (G. xylinus)附著於PCS表面，這些附著之細菌可於下一次生產時做為種菌之來源。於X光繞射(X-ray diffraction patterns, XRD)之結果可知，與靜置培養(static culture, SC)之BC (SC/BC)相比，由PCS-RDB生產之BC (PCS-RDB/BC)具有較低之結晶度。於機械性質分析(mechanical property analysis)可發現PCS-RDB/BC具有較低的應力以及較高的應變，顯示其具有較脆弱但有彈性之材料特性。
而於第二部分之研究，我們嘗試提升PCS-RDB生產BC之能力。我們選擇了四種不同聚合物如微晶纖維(microcrystalline cellulose, Avicel was used herein)、甲基化纖維(carboxymethyl cellulose, CMC)、瓊脂(agar)以及藻醣酸鈉(sodium alginate)作為培養基添加物。並對添加不同添加物之PCS-RDB/BC進行材料特性分析。結果顯示於CMC及Avicel添加下可顯著增加BC於PCS-RDB系統之產量，並在0.8% Avcel添加之組別中可達最高產量約3.84 g/L。由傅立葉轉換紅外光譜分析(transform infrared spectroscopy, FTIR)、熱重分析儀(thermogravimetric analysis, TGA)、SEM以及XRD等分析可知CMC及Avicel於BC生產過程中會被再吸收進入BC內部進而影響其材料特性。此外，這些添加物會增加培養基之黏性，進而使得BC生產菌株得以抵禦PCS-RDB轉動過程所產生之剪切力而導致的傷害。而機械性質及保水性分析顯示於Avicel添加組別之PCS-RDB/BC仍具有高保水性、高彈性以及較低之應力。
除了生產系統之建立及其優化之外，挑選具潛力之BC生產菌株亦能提升其於產業之實際應用性。於此部分之研究，我們嘗試由坊間之水果發酵產物取得未知之BC生產菌種，透過分子生物學以及生化分析等方式鑑定未知菌種，並對其生產特性進行測試以了解該未知菌種是否與常用BC生產菌株G. xylinus 23769相比是否有所不同。藉由16s rDNA 序列比對以及生化特性分析結果可知，該菌種為Komagataeibacter intermedius (K. intermedius)，因此我們將其命名為K. intermedius FST213-1。藉由將其培養於不同起始pH值之環境中，我們發現K. intermedius FST213-1 具有耐鹼性之特性，其可於pH值達到8之環境中存活並生產BC。而在產量之比較上，K. intermedius FST213-1具有比G. xylinus 23769更高之BC產量(1.2 g/L v.s. 0.6 g/L)。於材料特性分析之結果可知，與G. xylinus 23769相比，K. intermedius FST213-1所生產之BC具有高保水性、較低之熱穩定性以及結晶性等。而在機械性質分析上，兩者具有相似的應變以及應力。
BC因為其特殊材料特性而具有廣泛的應用，而在其所有應用中，醫療應用具有最高之商業價值，因此我們將BC與右旋糖酐(dextran)透過化學交聯以形成水凝膠，並希望其能作為癒創敷材以促進傷口癒合。於本研究中，我們使用10%, 20%以及30% dextran與BC交聯形成dextran/BC水膠。由機械性質分析可知交聯後之dextran/BC，其應力會隨著交聯之dextran濃度增加而下降，而其應變則是於10-20%之交聯組別保持彈性，但會於30%之交聯組別急速下降，此結果顯示於高濃度之dextran交聯組別具有易碎及不穩定之特性。而於細胞實驗中，我們使用NIH-3T3纖維母細胞來測試dextran是否具有細胞毒性(cytotoxicity)以及其細胞增生能力(cell proliferation)之測試。結果顯示於所有組別之dextran/BC均不具有細胞毒性。而在細胞增生實驗上，10% dextran/BC具有最佳之細胞增生能力以及穩定性。而於動物實驗中，我們將小鼠背部打孔形成切割傷口，經10% dextran/BC處理後可發現其具有加速小鼠傷口癒合之能力，顯示10% dextran/BC具有研發成為癒創敷材之潛力。
總結來說，本研究透過半連續生產系統之建立及優化、新式菌種之發現及生醫敷材之應用等方向來提昇BC之應用價值。透過PCS-RDB之研發，我們僅需接種一次，便可半連續式生產BC達五個循環以上，並可透過不同添加物的添加來提昇該系統生產BC之能力。此外，我們也鑒定出一新式BC生產菌株-K. intermedius FST213-1，並發現其具有鹼耐受性以及高產量等特性。最後我們將BC與dextran結合以研發出新式水凝膠，並於細胞以及動物實驗發現其不具有細胞毒性、可促進纖維母細胞增生以及幫助小鼠傷口癒合等能力，適合作為癒創敷材之應用。這些結果將可用以降低BC於工業生產之成本並提升其應用性。

Bacterial cellulose (BC), a kind of cellulose produced from microorganism, provides a lot of advantages such as high water content, great strength and good biocompatibility. Because of these advantages, BC has been applied in manufacturing industry, food processing, cosmetics industry and medical application.
The purpose of this research aimed to improve the application value of BC. It can be classified to four areas: (1) establishment of semi-continuous BC producing system, (2) improving the BC yield in the semi-continuous system, (3) selection of novel BC producing strain and (4) application of modified BC in wound healing.
First of all, A rotating disk bioreactor (RDB) with plastic composite support (PCS) as the solid support was developed for BC production. The functions of PCS are to provide the rough surface for microorganism adhesion and enhance microbe growth by mean of slowly nutrition release. The material properties of BC produced from this system were estimated. The results indicated that BC can be produced in a semi-continuous manner. The BC productivity reached around 0.24 g/L/day, and can be sustrained for at least five consecutive runs. Scanning electron microscopy (SEM) results showed that the BC producing strain Gluconacetobacter xylinus (G. xylinus) attached on the PCS surface leading to BC production in next round fermentation no need for inoculation. In the X-ray diffraction patterns (XRD), the BC produced from PCS-RDB presented lower crystallinity when compared to the BC obtained from static culture. In mechanical analysis, PCS-RDB/BC revealed the lower stress and higher strain suggesting that it exhibits a fragile but flexible material property.
For improving BC production in PCS-RDB system, we selected four kinds of polysaccharide including microcrystalline cellulose (MCC; Avicel was used herein), carboxymethyl cellulose (CMC), agar and sodium alginate as the medium additives. The material properties of BC produced from PCS-RDB were also analyzed using fourier transform infrared spectroscopy (FTIR), SEM, TGA, XRD and dynamic mechanical analysis (DMA). The results demonstrated that the addition of CMC and Avicel can help increase BC production, and the highest yield of BC from Avicel addition group reached 3.84 g/L, which is much higher than non-addition group (1.8 g/L). The FTIR, SEM and XRD results indicated that CMC and Avicel was incorporating into BC inside, and influencing BC structure during fermentation. Besides, the additives may provide high viscosity environment to help BC producing strain avoiding the damage from cut force (or shear force) which is produced from rotation of PCS. Therefore, the addition of CMC or Avicel can be used to improve BC production in PCS-RDB. The produced BC can still have the similar water content and strain but lower stress compared to BCs produced from static culture.
Moreover, the selection of microorganism is crucial for BC production in different cultivation system. In our previous study, G. xylinus was used to produce BC due to its high BC production and easy-to-culture. Consequence, exploring a new potential BC producing strain is the other direction for improving BC production. An unknown strain isolated from fermented fruit juice was identified as Komagataeibacter intermedius (K. intermedius) FST213-1 by 16s rDNA sequencing analysis and biochemical characteristics test. K. intermedius FST213-1 processed high alkaline tolerance which can produce BC at pH 8 in short-term (4-day) cultivation. It also provided higher BC production compared to G. xylinus 23769 at pH value of 8 (1.2 g/L vs. 0.6 g/L). Results of material property analysis indicated that BC produced from K. intermedius FST213-1 exhibits higher water content ability (99.5%), lower thermostability (315◦C), lower crystallinity (79.3%) and similar mechanical properties in comparison with the specimen of model BC producer- G. xylinus 23769.
Among of all applications of BC, medical application provides highly valuable commercial value. We, therefore, tried to combine BC with dextran as the hydrogel for medical application. Mechanical property result showed that the stress of modified hydrogel was decreased dependent on the added dextran concentration. And 10-20% dextran/BC groups exhibit the highest strain. The cell based experiment results presented that no cytotoxicity in all dextran/BC groups, and 10% dextran/BC shows the highest cell proliferation ability. In mice based animal model experiment, 10% dextran/BC revealed good wound healing ability that can reduce wound closure quickly and accumulate skin mature suggesting that dextran/BC can be developed as potential wound dressing in clinical application.
After all, our study focused on enhancing the application value of BC. At first, we successfully established a semi-continuous BC production system. And the effects of various additions added in this system were also estimated. Through the system with additives adjunction, BC production can be semi-continuously proceeded with high yield. In the other hand, a new BC producing substrain- K. intermedius FST213-1 was found that can produce BC in alkaline environment with high yield. Last but not least, a novel dextran modified BC hydrogel was used as wound dressing for medical healing. The hydrogel dressing can improve fibroblast cell proliferation. In addition, it can also accelerate wound healing in vivo. In the end, these results clearly disclosed that the application value of BC is increased utilizing improving production system, increasing the yield of BC, selecting novel BC producing strain and developing new application.

口試委員審定書 ii
致謝 iii
中文摘要 v
ABSTRACT viii
LIST of FIGURES xv
LIST of TABLES xvii
CHAPTER 1. INTRODUCTION 1
CHAPTER 2. LITERATURE REVIEW 4
2.1. General outline 4
2.2. Bacterial cellulose 4
2.3. Bacterial cellulose biosynthesis 5
2.3.1. Cellulose producing strain 5
2.3.2. Gluconacetobacter xylinus 6
2.3.3. Other BC producing strains 9
2.3.4. Genetics and enzyme regulation 10
2.4. Production of BC 12
2.4.1. Static cultures for BC production 13
2.4.2. Submerged fermentation 14
2.4.3. Culture media 15
2.4.4. Long-term fermentation 16
2.4.5. Effects of other additives on BC production 17
2.4.6. Bioreactor design and usage 19
2.5. Application of BC 28
2.5.1. Acoustic transducer diaphragm 28
2.5.2. Paper manufacturing 28
2.5.3. Filtration 29
2.5.4. Pharmaceutical applications 30
2.5.5. Food applications 38
2.5.6. Other applications 39
CHAPTER 3. SEMI-CONTINUOUS BACTERIAL CELLULOSE PRODUCTION in a ROTATING DISK BIOREACTOR and ITS MATERIALS PROPERTIES ANALYSIS. 44
3.1. Abstract 44
3.2. Introduction 44
3.3. Materials and methods 46
3.3.1 Microorganisms 46
3.3.2 Medium 47
3.3.3 Plastic composite support 47
3.3.4 Selection of BC production strains 48
3.3.5 Semi-continuous BC production 48
3.3.6 Materials properties analysis of BC 49
3.3.7 Statistical analysis 51
3.4. Results and discussion 52
3.4.1 Selection of BC production strain 52
3.4.2 Semi-continuous cultivation of BC in PCS-RDB 53
3.4.3 Materials properties analysis of BC 55
3.5. Conclusion 61
CHAPTER 4. PRODUCTION of BACTERIAL CELLULOSE with VARIOUS ADDITIVES in PCS ROTATING DISK BIOREACTOR and ITS MATERIAL PROPRTIES ANALYSIS. 62
4.1. Abstract 62
4.2. Introduction 62
4.3. Materials and methods 64
4.3.1. Microorganisms 64
4.3.2. Media 64
4.3.3. Plastic composite support 65
4.3.4. BC production with various additives 66
4.3.5. Materials properties analysis of BC 66
4.4. Results and discussion 68
4.4.1 Effects of various additives on BC production 68
4.4.2 Fourier transform infrared spectroscopy (FTIR) 72
4.4.3 X-ray diffraction 73
4.4.4 Morphology of BCs 74
4.4.5 Thermogravimetric analysis and water content analysis 76
4.4.6 Strength measurement 77
4.5. Conclusion 78
CHAPTER 5. ISOLATION and IDENTIFICATION of CELLULOSE-PRODUCING STRAIN Komagataeibacter intermedius FST213-1 from FERMENTED FRUIT JUICE. 80
5.1. Abstract 80
5.2. Introduction 80
5.3. Materials and methods 82
5.3.1. Fermented fruit juice preparation 82
5.3.2. Isolation of BC-producing strain 82
5.3.3. Identification of BC-producing strain 82
5.3.4. Cultivation and harvest of BCs 83
5.3.5. Material property analysis 84
5.3.6. Statistical analysis 86
5.4. Results and discussion 86
5.4.1. Isolation and identification of cellulose-producing strain 86
5.4.2. Materials properties of bacterial cellulose 92
5.5. Conclusion 96
CHAPTER 6. EVALUATION of DEXTRAN MODIFIED BACTERIAL CELLULOSE HYDEOGEL ACCELELERATING CUTANEOUS WOUND HEALING 97
6.1. Abstract 97
6.2. Introduction 97
6.3. Materials and methods 99
6.3.1. Production and preparation of dextran modified BC hydrogel 99
6.3.2. Material property analysis 100
6.3.3. Cell based experiments 101
6.3.4. Wound healing modeling 102
6.3.5. Statistical analysis 103
6.4. Results and discussion 104
6.4.1. Morphology of dextran/BC hydrogel 104
6.4.2. Thermogravimetric analysis 105
6.4.3. Mechanical property and water content ability 107
6.4.4. Cell cytotoxicity and cell proliferation 109
6.4.5. Wound healing in vivo 111
6.5. Conclusion 114
CHAPTER 7. CONCLUSIONS and SCOPE for FUTURE RESEARCH 115
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