Chapter 1:Introduction

　A major cacao industry has emerged with the development of cocoa, cocoa butter, syrups, pastes and all kind of chocolates. Therefore, processing of cocoa leads to increasing disposal of wastes such as hulls, shells, and so on. Among the cacao by-products, cacao hull is regarded as potential and valuable sources of food industry, live stock feed, and is gaining considerable interest in developed countries. In order to improve its effective utilization, structural characterization of predominant polyphenolic compound, which is most important factor for its potential use, is prerequisite. However, there is a paucity of information on their structural features of predominant polyphenolic compounds. This investigation reported herein provides elucidative and valuable information for the above aims. In addition, the cacao hull has unusual developing pattern of secondary wall thickening such as tracheary elements(TEs). The morphological structure of TEs and their physico-chemical properties were also examined.

Chapter 2:Compositional Characterization of Cacao(Theobroma cacao L.)Hulls

　Cacao(Theobroma cacao L.)hull is regarded as potential and valuable resources for the food industry and livestock feed, and is gaining considerable interest in developed countries. The chemical composition and structural characteristics of cacao(Theobroma cacao L.)hull were investigated in this Chapter. Holocellulose, Klason residue, total amino acid, lipid and ash were quantified as 334 g kg-1, 320 g kg-1, 96 g kg-1, 99 g kg-1 and 93 g kg-1 respectively. Neutral sugar composition of cell wall polysaccharides was also analysed. It was suggested by an alkaline nitrobenzene oxidation of cacao hull that the Klason residue was composed of structurally unknown polyphenols together with small amounts of lignin and tannin. The results for total amino acid(96 g kg-1), and nitrogen content of the Klason residue(25 g kg-1)and samples treated with various surfactants(21-27 g kg-1), suggested the presence of polyphenol-protein complexes in cacao hull. In addition, relatively high amount of ash(potassium)and(essential)amino acid would be interesting in their potential applications. The effective utilization such as nutrition for ruminants was discussed.

Chapter 3:Structural Features of Predominant Polyphenolic Compounds in Cacao(Theobroma cacao L.)Hulls

　Extracts with 70% acetone before(E-1)and after(E-2)ball-milling of cacao(Theobroma cacao L.)hulls were investigated for their chemical compositions as well as comprehensive structural characterization of predominant polyphenolic compounds. The extract after ball-milling E-2)contained much higher amounts of rhamnose, arabinose, and galactose than E-1. Total yields of those sugars were 20.9% and 47.2% of the total determined neutral sugar in E-1 and E-2, respectively. In addition, small amounts of uronic acid(0.2%)and calcium(8 μg g-1)in E-1 were determined and relatively high amounts of uronic acid(4.3%)and calcium(46 μg g-1)in E-2. These results suggest that E-2 is composed higher amounts of pectic substances than E-1. Klason residue(KR)and acid soluble phenolics(ASP)in E-1 were 22.8% and 23.7% respectively, while E-2 contained 50.4% KR and 9.3% ASP. These results suggest that E-1 is highly composed of low molecular weights and acid soluble phenolics, which have usually contained carboxyl groups. The suggestion was confirmed by FTIR. The pyrogram of E-2 showed high intensities of catechol and 4-methylcatechol, while E-1 pyrogram showed high intensities of alkaloid compounds such as theobromine, theophylline and caffeine, phenol, and p-cresol. The existence of catechol units in E-2 was confirmed by 13C-NMR spectrometry. According to all results, lignin is not dominant constituent in cacao hull and unknown polyphenolic compounds which gave catechol by analytical pyrolysis would be predominant polyphenolic compounds.

Chapter 4:Assumption of Lignin Content Under Co-Existence with Non-Lignin PolyphenolicCcompounds in Cacao(Theobroma cacao L.)Hulls and Shells

　Structural characteristics of polyphenolic components of cacao(Theobroma cacao L.)bean hulls and shells were investigated by the use of four indications, Klason residue, methoxyl group, alkaline nitrobenzene oxidation, and ozonation products. Trials were made to assume the lignin content on the basis of these analyses and to compare the assumed lignin contents with the amount of Klason residue. Although Klason residues were obtained in rang of 272-320 g kg-1 of those samples, the methoxyl content and the yields of nitrobenzene oxidation and ozonation products were much smaller than the value expected from the level of Klason lignin content. Among those three indications, the methoxyl group content was relatively high for every sample but, even in this case, the lignin contents assumed from the methoxyl group content were in the range of 17-30, 67-115 and 73-126 g kg-1 of oven dry samples of cacao bean hulls as a residue from food industry, intact cacao bean hulls and shells of cacao fruits, respectively. Assumed lignin contents obtained from other indications were much smaller. All results of those approaches are suggesting that the lignin is not predominant polyphenolic component among cacao sub-products, especially cacao bean hulls.

Chapter 5:Morphological Characteristics of the Tracheary Elements in Cacao(Theobroma cacao L.)Hulls

　The tracheary elements(TEs), which are the distinctive function of the xylem, are characterized during the formation of a secondary wall with annular, spiral, reticulate, or pitted wall thickenings. The spiral TEs are likely of functional significance due to their ability to expand and transport water to plant organs. The morphological structure of TEs of cacao hull was characterized by scanning electron microscopy as well as physical properties. The spiral structures of TEs covered with web-like thin layer of primary wall on the outside surfaces were observed. The TEs had various sizes of diameter of spiral circularity ranging from about 5-10 μm and the thickness of secondary wall about 1.3-2.7 μm. Polarized microscopy and X-ray diffraction analysis revealed that the orientation of cellulose microfibrils was aligned in parallel, following the spiral thickening and the crystallite size, similar to cellulose-I of the cotton. The data for this study provide three-dimensional understanding of such extensive TE structures including some valuable information for plant physiology.

Chapter 6:Differences in Chemical Constituents between Vascular Bundles and Non-Vascular Bundles of Cacao(Theobroma cacao L.)Hulls

　Cacao hulls were physically separated into vascular bundles(VB)and non-vascular bundles(NVB)to investigate their chemical compositions as well as abundant and structural features of polyphenolic compounds. Xylose content of VB was significantly higher(13.1%)than that of NVB(2.8%). Non-vascular bundles were composed of relatively high proportions of rhamnose, arabinose, and galactose and significantly rich in uronic acid(12.9%), suggesting less secondary walls in NVB.

These data suggest that the high amount of glucose(21.4%)together with remarkable amount of xylose(13.1%)in VB is correlated with development of secondary wall formation. Total(poly)phenolic compounds(35.9-39.1%)quantified as Klason residues(KR)and acid soluble phenolic compounds(ASP)were similar in both cell types, however there were great differences in structural characteristics of polyphenolic compounds. The pyrogram of VB clearly showed high intensities of guaiacol and 4-vinylguaiacol, together with low intensities of catechol and 4-methylcatechol. On the other hand, that of NVB showed opposite trends. These results were also confirmed by total yields of vanillin and syringaldehyde of the products of alkaline nitrobenzene oxidation Therefore, the accumulation of different polyphenolic compounds in cacao hull is strongly relying on cell types, which have correlated with development of secondary wall.

　本論文の骨子は、次のとおりである。

　近年チョコレート等カカオを原料とする製品の消費が拡大し、それとともに種子殻等廃棄物の料も増加している。これら廃棄物のうち、種子殻は家畜飼料としてだけでなく、先進国において食品工業の原料として注目を集めてきている。しかしながら、これまで種子殻の成分、とりわけ主成分の一つであるPPhの化学構造に関する知見はほとんどなく、これを明らかにすることは、種子殻のさらなる有効利用開発にとって、重要である。本研究は、種々の分析手法を用いてカカオ種子殻ポリフェノール(PPh)の化学構造に関する知見を得ることを目的として進められた。加えてカカオ種子殻の維管束と維管束以外の組織の化学構造について検討した。

　まず、食品工場から廃棄物として出てくる種子殻(以下、廃棄種子殻という)の化学組成を分析した。種子殻の全アミノ酸量は96 g kg-1であり必須アミノ酸を含み、カリウムに富んでいることから家畜の飼料として有効であることを示した。リグニン定量法であるKlason法残さが320 g kg-1であったが、Klason残さはリグニンだけでなく、タンニンおよび未知のPPhで構成されていると考えられた。

　そこでKlason残さ中のPPhの構造について検討した。種子殻を70%アセトン抽出(抽出分をE1、残さをR1)した後、R1を微粉砕し再度70%アセトン抽出(抽出分をE2、残さをR2)して、各区分の化学組成および化学構造の分析を行った。E2はE1に比べてラムノース、ガラクトースおよびウロン酸が多く、E2はペクチンに富んでいた。E1およびE2のKlason残さと酸可溶性PPh量からE1のPPhはE2に比べて低分子であり、カルボキシル基に富んでいることが判明した。熱分解ガスクロマトによりE2からは高い収量でカテコール類が、E1からはカフェインのようなアルカロイドとともにフェノールおよびp-クレゾールが得られた。E2中にカテコール構造が存在することは13C-NMRでも確認された。これらの結果から、カカオ種子殻のPPhの主成分はリグニンでもタンニンでもなく、これまで知られていないPPhであることを見出した。

　それでは種子殻PPhのうちどの程度がリグニンであろうか?Klason残さ、メトキシル基量、ニトロベンゼン酸化生成物およびオゾン酸化分解生成物量から廃棄種子殻、インドネシアのプランテーションから採取した新鮮な果実の種子殻および種子ポッドのリグニン量を推定することを試みた。メトキシル基量もとに推定されたそれらのリグニン量は17-30,67-115および73-126 g kg-1であり、リグニンはKlason残さ量の1/10から1/3に過ぎないことが明らかになった。なお、ニトロベンゼン酸化生成物およびオゾン酸化分解生成物量から求めた値はさらに低かった。構造未知のPPh中のリグニン量を推定する新たな方法として、今後の応用が可能であろう。

　ついで、種子殻組織中でのPPhの分布を検討することを目的として、種子殻繊維束の光学および走査電子顕微鏡観察により、維管束は極めて特徴的な微細な螺旋組織で満たされていることが判明し、二次壁分化・肥厚の初期段階であるtracheary element(TE)であると同定された。このTEには右および左螺旋の両方があり、螺旋の口径は5-10μmであり、一般の植物で認められているTEのそれ(12-20μm)に比べて小さかった。カカオ種子殻のTE螺旋組織はその構成糖分析等の結果から一次壁と同定された伸縮性に富む薄膜で覆われており、種子の成長に対応できるようになっていることが判明した。種子殻繊維束が二次壁肥厚を完結しないで留まっているのは種子への水供給の圧力が極めて低いためであろう。螺旋組織を構成している繊維束の直径は1.3-2.7μm程度であるが、X線回折等からセルロース繊維は螺旋繊維束方向に配列しており、その結晶化度は木材細胞壁中のセルロース繊維のそれに比べ高い値を与えだ。

　顕微鏡下で種子殻をTEで構成される維管束(VB)および非維管束組織(NVB)に分画し、その化学成分について分析した。多糖を構成する中性糖の組成から、VBは成熟した二次壁であり、NVBは一次壁を含む極めて未成熟な二次壁によって構成されていることを明らかにした。NVBには大量のウロン酸を含んでいた。Klason残さ量はVBおよびNVBともに36-39%で、大きな差は認められなかったが、熱分解ガスクロ分析、ニトロベンゼン酸化およびオゾン酸化などにより、PPhの組成は大きく異なっていること、VBではリグニンに富むがNVBのPPhはタンニンおよび構造未知のPPhによって構成されていることを明らかにした。

　以上のように、食品工業から大量に廃棄されているカカオ種子殻のポリフェノールについての知見が得られたことは、種子殻のポリフェノール原料とした接着剤等有効利用開発への道を開くとともに、種子殻の成長プロセスを解明するなど数多くの新知見を与えており、応用上、学術上貢献することが少なくない。よって、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のあるものと認めた。