介質研磨對幾丁質之物性與酵素水解之影響

Abstract

幾丁質為多功能性的天然高分子多醣聚合物，主要存在於海洋無脊椎動物、昆蟲的外殼與真菌菌體細胞壁。由於幾丁質及其衍生物具生物活性、生物相容性、生物可降解性等特質，使其應用範圍廣泛。然而幾丁質不水溶的特性使其應用受限，水解後之N-乙醯幾丁寡醣及幾丁寡醣，分子量小且水溶性佳，有較佳之利用性。 本研究主旨在探討經介質研磨之幾丁質的物性改變，及其對後續酵素水解效率之影響。實驗所使用之蟹殼幾丁質原料的體經平均粒徑為224.50 ± 13.44 μm。藉由0.8 mm釔鋯珠作為介質，取5克及12.5克幾丁質原料與500克去離子水混合形成濃度為1及2.5%的幾丁質懸浮液，於研磨0、30、60及120分鐘後，濃度1%幾丁質懸浮液之體積平均粒徑分別為224.5、76.08、58.45及7.13 μm；2.5%幾丁質懸浮液之體積平均粒徑則為224.5、82.40、52.40及23.80 μm。經由掃描式電子顯微影像觀察幾丁質懸浮液顆粒表面樣貌，確定次微米顆粒的存在。以動態流變儀進行流動測試，懸浮液黏度隨幾丁質濃度提高而增加，隨剪切速率增加而下降，屬於剪切致稀性流體。利用傅立葉紅外線光譜儀測量幾丁質之去乙醯度，發現介質研磨可提高幾丁質之去乙醯度；從原來的30.5%提高至43%。 在酵素反應的部分，採用Streptomyces griseus產生之幾丁質酶水解經介質研磨後之幾丁質。結果發現隨著研磨時間增加，水解後N-乙醯幾丁二醣之產率明顯提高。未經研磨的幾丁質懸浮液，經酵素水解8小時，N-乙醯幾丁二醣之產率為16.04%；使用濃度為1%經研磨30、60及120分鐘之幾丁質懸浮液進行酵素水解時，N-乙醯幾丁二醣之產率分別為33.73、34.09及41.35%；使用濃度為2.5%經研磨30、60及120分鐘之幾丁質懸浮液作酵素水解時，產率則為31.51、34.15及44.28%。就最終水解產物N-乙醯幾丁二醣之產率而言，發現經研磨120分鐘之幾丁質懸浮液其產率較未研磨者提高約2.5倍以上。

Being abundant polysaccharides in nature and multi-applications such as biological, biocompatible and biodegradable functions, chitin and its derivatives are useful biopolymers and taken a lot of attentions by scientists and researchers in decades. They are present mainly in exoskeletons of invertebrates and the cell walls of fungi. The water insolubility constrains the application of chitin, however, the hydrolytic products of chitin such as N-acetyl chitooligosaccharides and chitooligosaccharides are water soluble and get lots of employments. The objective of this study was to investigate the influence of media milling on the particle sizes and physical properties of chitin as well as the enhancement of media milled chitin on the enzymatic hydrolysis. The volume mean diameter of purchased chitin was 224.50 ± 13.44 μm. It was processed by media mill to reduce the particle size to micron and submicron scales. Physical properties such as particle size distribution, pH, deacetylation, rheology and morphology were compared between the unmilled and milled chitin. Finally the yield of N-acetyl diglucosamine ((GlcNAc)2) was applied to explore the effect of milling on chitin hydrolysis. Chitin (5 or 12.5 g) was blended with 500 g deionized water to prepare a 1% or 2.5% slurry for media milling, where 0.8 mm YTZ (yttria-stabilized tetragonal zirconia) beads was selected as the milling media. After 30-, 60- and 120-min milling, the volume mean diameters were reduced to 76.08, 58.45 and 7.13 μm, respectively, for 1% solution and 82.40, 52.40 and 23.80 μm for 2.5% one. The particle size distribution and SEM morphology showed the presence of submicron chitin particles for 120-min milled products. The rheological studies of the milled chitin-suspension showed the viscosity was increased with the increase of concentration, and, however it decreased with increase of shear rate; i.e., the milled chitin-suspension appeared to be a shear-thinning fluid. The degree of deacetylation of chitin evaluated by FTIR was increased from 30.5 to 43% by media milling. The chitinase obtained from Streptomyces griseus was applied to proceed the hydrolysis of milled chitin. It showed that a longer milling product resulted in a higher yield of (GlcNAc)2 as the same hydrolytic duration was considered (8 hr). The yield of (GlcNAc)2 from unmilled chitin-suspension was 16.04%. For the hydrolysis of 30-, 60-, 90-min milled 1% chitin suspension, the yield of (GlcNAc)2 was 33.73, 34.09 and 41.35%, respectively; while for the hydrolysis of 30-, 60-, 120-min milled 2.5% chitin suspension, the yield of (GlcNAc)2 was 31.51, 34.15 and 44.28%.