Chitins are present in the outer shells of crustaceans such as crab and shrimp, the cuticles of insects and the cell walls of some fungi, coexisting with proteins and certain minerals. More than 100 million tons of chitins are biosynthesized every year. Since a large amount of crab and shrimp shells are produced as food waste, further utilization of chitin as functionalized materials or commodities is highly required. The importance of chitin as a sustainable resource is increasing owing to not only its abundance but also unique structure and properties.

Isolated chitins are linear and crystalline hetero-polysaccharides consisting of N-acetylanhydroglucosamine and anhydroglucosamine units with various ratios linked by (1--04)-13-glycoside bonds. Followed by cellulose, chitin is one of abundant structural polysaccharides, which physically support living bodies, forming hierarchical structures, that increase in size from simple molecules and highly crystalline fibrils at the nanometer level to composites at the micron level upward in sea-animals, insects and fungi and so on. The lateral dimensions of the crystalline fibrils of chitins range from 2.5 to 25 nm, depending on their biological origins. Thus, chitins intrinsically have potential to be converted to crystalline nano-fibers or nano-whiskers by so-called downsizing processing. If chitins can be individualized at nano level without impairing the original high crystallinities or high molecular weights, they would be used as new functional and bio-based nano-materials with biodegradability, reproducibility, biocompatibility, and other advantages.

Preparation of nano-dispersed chitins by TEMPO-mediated oxidation

TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy1 radical)-mediated oxidation was applied to crab shell a-chitins and tubeworm B-chitins. When the TEMPO-oxidized a- and B-chitins were subjected to ultrasonication in water, the slurries were converted to highly viscous and translucent gels, which consisted of mostly individualized chitin nano-whiskers and nano-fibers, respectively. In these cases, the position-selective formation of dissociated C6 carboxylate groups on the chitin nano-crystallite surfaces by TEMPO-mediated oxidation is the necessary point to convert original chitins to individualized nano-whiskers or nano-fibers. Electrostatic repulsions and/or osmotic effects between anionically-charged chitin microfibrils are likely to be the key driving force for the nano-conversion. Therefore, the mechanism to prepare chitin nano-fibers/whiskers is intrinsically the same as that for preparation of cellulose nanofibers by TEMPO-mediated oxidation of native celluloses reported by Saito et al. (Biomacromolecules, 8, 2485 (2007)).

Preparation of nano-dispersed chitins by mechanical treatment under acid conditions

Based on the mechanism of the TEMPO-mediated oxidation method to prepare cellulose and chitin nano-fibers, cationization of chitin microfibrils were studied. When squid pen B-chitin was disintegrated in water at pH 3-4 for several minutes by ultrasonication, highly viscous and transparent gels were obtained. The TEM (Transmission electron microscope) observations revealed that the gels consisted of nanofibers 3-4 nm in cross sectional width and at least a few microns in length. Cationization or protonation of the C2 amino groups present on the crystalline fibril surfaces under acid conditions is likely to be one of the most significant and necessary conditions for the nanofiber conversion. Thus, nanofibers can be directly obtained from squid pen B-chitin at pH 3-4 without any chemical modification.

However, the cationization/individualization was applicable only to squid pen B-chitin, i.e. the nano-fiber conversion by the simple ultrasonication is characteristic for squid pen B-chitin. Hence, the simple disintegration method could not convert a-chitin to nano-fibers or nano-whiskers, although a-chitin is present more abundantly than B-chitin in nature and easy to be collected as food wastes.

The relatively low degree of N-acetylation (DNAc) and low crystallinity of squid pen B-chitin, which are different from a-chitins, are probably necessary conditions for the nano-fiber conversion. Thus, partial deacetylation was applied to a-chitins under heterogeneous solid/liquid conditions.

When partially de-acetylated a-chitin was disintegrated in water at pH 3-4 by magnetic stirring for several days, highly viscous and transparent gels consisting of mostly individual rod-like nano-whiskers 6-7 nm and 100-500 nm in width and length respectively, were obtained. This is the first finding to obtain a-chitin nano-whiskers having widths quite similar to crystal sizes determined by X-ray diffraction method. Thus, complete individualization of chitin fibrils can be achieved by the de-acetylation and the following disintegration in water at pH 3-4. Some long and individual nano-fibers with length up to microns were observed in the TEM images. Conversion of C2 acetylamide groups present on the crystallite surfaces of a-chitin to corresponding amino groups by heterogeneous de-acetylation and the following cationization/protonation under acid conditions is necessary for nano-fibrillation of a-chitin, providing strong inter-fibrillar electrostatic repulsions.

Characterization of chitins nano-fibers and nano-whiskers

a-Chitin nano-whisker. Two methods were used to prepare a-chitin nano-whiskers; one is TEMPO-mediated oxidation followed by mechanical disintegration in water (TEMPO-oxidized a-chitins), the other is heterogeneous de-acetylation followed by mechanical agitation in water under acid conditions (partially de-acetylated x-chitins). Properties of thus prepared a-chitin nano-whiskers were compared with those of nano-whiskers prepared by conventional acid hydrolysis (hydrolyzed a-chitins). The water dispersion of de-acetylated a-chitins showed somewhat different rheological properties, and had the highest light transmittance and the highest viscositys, indicating that de-acetylated a-chitins had the highest degree of individualization.

Squid pen B-chitin nanofiber films and aerogels. Due to high aspect ratios of individualized squid pen B-chitin nanofibers (3-4 nm in width and at least a few microns in length with aspect ratios more than 250), highly functional films or aerogels were expected to be obtained from B-chitin nanofibers. When squid pen B-chitin nanofiber/acidic water dispersions were filtrated on membranes, films with high light-transmittance and tensile strength were obtained Moreover, sponge-like aerogels with specific porous structures could be acquired from squid pen B-chitin nanofibers.

Figure 1: Nano-fibrillation of crab shell a-chitin and tubeworm B-chitin by TEMPO-mediated oxidation followed by mechanical disintegration in water.

Figure 2: Nano-fibrillation of partially de-acetylated crab shell a-chitin and squid pen B-chitin by simple mechanical treatment under acid conditions.

本研究では、バイオマス多糖としてセルロースに次ぐ量が毎年生産・蓄積されているキチンが高結晶性でナノサイズ幅のフィブリルを構成単位としていることに注目し、近年その基礎および応用研究が進んでいるナノ分散化と、そのナノ構造およびナノ分散機構解析の研究を行った。ナノ分散化方法としては、反応条件を制御したTEMPO(2,2,6,6-tetramethylpiperiniyl-1-oxyl radical)触媒酸化、およびその結果と原理に基づいたフィブリル表面荷電制御条件での解繊処理を選択し、多くの新しい知見を得ることができた。

市販精製α-キチン、単離精製した高結晶性のハオリムシ由来のβ-キチン、単離精製した低結晶性のβ-キチンのTEMPO触媒酸化を、共酸化剤である次亜塩素酸ナトリウムの添加量を制御して行い、水不溶分の構造解析と水中解繊処理を行った。その結果、いずれも十分な次亜塩素酸ナトリウムを添加した場合には、水不溶性のキチンが反応の進行と共に水可溶性となり、そのC6位の1級水酸基が位置選択的に全て酸化されてカルボキシル基のナトリウム塩に変換したキトウロン酸が得られた。

一方、α-キチンのTEMPO触媒酸化では、次亜塩素酸ナトリウムの添加量を制御することにより、カルボキシル基量が最大で約0.85mmol/g含まれる水不溶成分を40~95%収率で得ることができた。この水不溶成分は元のキチンの結晶構造および結晶化度を維持している。従って、TEMPO触媒酸化によって、結晶性のα-キチンフィブリルの表面のみに位置選択的にカルボキシル基が導入されたことが明らかになった。

得られたα-キチン由来のTEMPO酸化キチンの水不溶分を水中で解繊処理したところ、透明で高粘度のゲルが得られた。このゲルを希釈乾燥させて透過型電子顕微鏡で観察したところ、個々に分散した紡錘形短繊維(ウィスカー)からなることが明らかになった。その平均幅は約8nmで、長さは340nmであった。TEMPO酸化により、α-キチンフィブリル表面に高密度でマイナス荷電を有するカルボキシル基のナトリウム塩が導入されたため、フィブリル間の荷電反発と機械的解繊処理によりナノ分散が可能になった。

一方、ハオリムシ由来のβ-キチンのTEMPO触媒酸化では、カルボキシル基が最大で約0.25mmol/g導入され、元の高結晶化度を維持した水不溶成分が得られた。水中解繊処理することで、幅が20~50nmで長さ数ミクロンのナノファイバーが得られた。しかし、イカの腱由来の低結晶性β-キチンに関しては、TEMPO酸化によってカルボキシル基は導入されたが、水不溶分の水中解繊処理でもナノ分散化は達成できなかった。その理由をフィブリル間ヘミアセタール結合の形成と、ポリイオン錯体形成で説明することができた。

上記のTEMPO酸化によるナノ分散の原理に基づき、精製キチンに元々存在しているC2位のアミノ基に荷電を付与させて、キチンの化学構造を変化させること無くナノ分散させる検討を行った。その結果、低結晶化度でC2位のアミノ基が単位当たり10%ほど存在するイカの腱由来のβ-キチンのみ、pH3~4の水中での超音波あるいは高速回転ミキサーによる解繊処理で、透明で高粘度のゲルを得ることができた。このゲルを希釈乾燥させて透過型電子顕微鏡で観察したところ、幅3~4nmで、長さ数ミクロンの孤立したナノファイバーに分散できることが明らかになった。しかし、C2位のアミノ基量の少ないハオリムシ由来のβ-キチン、およびカニやエビ由来のα-キチンからは同様の方法でナノ分散することはできなかった。

そこで、食品廃棄物として多量に排出され、一部精製されて市販されているα-キチンの弱酸性下での水中解繊処理のみでのナノ分散化を達成するため、フィブリル表面のみのC2位のN-アセチル基の一部を脱アセチル化し、1級アミノ基を露出させる検討を行った。その結果、33%水酸化ナトリウム水溶液で処理することにより、元のα-キチンの高結晶化度を維持し、水不溶性のまま収率85~90%で、C2位のアミの基による荷電量を1.8mmol/gまで増加させることができた。この水不溶性の一部脱アセチル化キチンを水中で解繊処理したところ透明高粘度ゲルが得られ、その希釈物の透過型電子顕微鏡観察から、幅6~7nmで長さ100~500nmの孤立した棒状ウィスカーにナノ分散できることが明らかになった。また、一部に長さが1ミクロン以上のナノファイバーも初めて確認された。得られたα-キチンナノウィスカーは安全性の課題もクリアでき、機能性食品としての利用も可能となる。

以上のように、一連の各種キチン試料のナノ分散化の研究によって、キチン系バイオマスの新しい高機能ナノ素材としての応用の可能性が拡がり、ナノ分散化のメカニズムに関連する貴重な基礎的知見が多く得られた。これらの成果は、キチン系バイオマスの利活用を進める観点からも高く評価されている。従って、審査委員一同は、本論文が博士(農学)の学位論文として価値あるものと認めた。