Chapter 1: Introduction

Triacylglycerols (TAGs) are one of the main forms of energy storage in living organisms. Natural fats, which are basically multicomponent TAG systems, are the major components of food. They are also used as matrices of medicine and cosmetics. They are made up of more than 30 TAG species and their major constituent fatty acid is generally oleic acid. Industrial demands promote the studies on thermophysical properties of the multicomponent TAG systems for a long time; however, the whole picture of their phase behavior has not been completely understood yet because of the complexity.

In order to understand the phase behaviors of these complex systems, many studies have been carried out for simple TAG systems as the models. Powder X-ray diffraction has been the most used experimental approach and has revealed interesting phenomena such as crystal polymorphism and "molecular compound" formation. However, the technique suffers from serious limitations when studying coexistence of multiple phases.

To study these phenomena within multicomponent TAG systems, Raman spectroscopy has a distinct advantage. Raman spectrum as a whole is often called "molecular finger print" which is distinctive to a unique phase. By using this characteristic, one can extract the information of the phases coexisting in a complex system. Also, lipids are appropriate molecules for Raman spectroscopy because their large polarizability volumes give strong Raman scattering. Their structural changes are reflected in the spectra with high sensitivity.

In this study, multicomponent TAG systems are investigated by the use of Raman spectroscopy to characterize the phases occurring in these systems. For this purpose, the structure and phase behavior of TAGs are firstly summarized with emphasis on the recent developments (Chapter 2) and then their Raman spectral features are described (Chapter 3). Based on these, the phase behavior of some multicomponent systems are studied by Raman spectroscopy (Chapters 4 and 5).

Chapter 2: Structure and phase behavior of TAGs

TAGs possess the basic structure of lipids: a glycerol backbone and acyl chains attached to it (Fig. 1a). The chemical composition of the acyl chains of natural fats is genetically determined and affects their phase behavior.

One of the important phase behaviors of TAGs is polymorphism (Fig. 1b). Three polymorphic phases, α, β' and β, are generally observed. α and β' are the metastable phases and β is the most stable one. The polymorphic phases differ in their hydrocarbon subcell structure.

It is also known that TAGs form "molecular compound" in their binary systems (Fig. 1c). A molecular compound behaves like a new, pure TAG species with unique phase behavior that differs from those of its component TAGs. The formation of a molecular compound is thought to occur in terms of the specific interactions between oleic acyl moieties of the component TAG molecules.

Chapter 3: Raman spectra of TAGs

Raman spectral features of TAGs are described in relation to TAG structure of each phase. On the basis of the spectroscopic data of basic molecules such as polyethylene, paraffin, n-alkanes and fatty acids, the detailed TAG structures can be characterized from Raman spectra. Their spectra are largely determined by acyl moieties that dominate the compositions. The conformation and inter-chain interaction of acyl moieties differ among the phases and are reflected in their Raman spectra. There is little information on the glycerol conformation so far; however, some spectral regions do indicate the difference in this moiety for the different polymorphic phases.

Chapter 4: Investigation of molecular compound formation in a TAG binary system

1,3-dipalmitoyl-2-oleoyl-sn-glycerol (POP, Fig. 1a) and 1,3-dioleoyl-2-palmitoyl-sn-glycerol (OPO, Fig. 1a) binary system was investigated using Raman spectroscopy.

POP and OPO were completely melted and mixed to prepare the samples with different molar ratios of POP and OPO. The crystals of the samples were prepared by cooling down the melt to 4°C followed by incubation. Raman spectra of the polycrystal were measured by a 532-nm excitation Raman spectrometer which is developed in the laboratory. The Raman spectral data of the samples are assembled into a matrix and subjected to singular value decomposition to analyze the number of independent spectral components. The spectrum and the concentration profile of each component were reconstructed under the constraints in order to minimize ambiguities.

It is found that two spectral components are not enough to explain the data set. On the other hand, three components successfully explain the data. Their concentration profiles and spectra are shown in Fig. 2. From these results, the existence of the third component in the binary system is shown spectrometrically. The components 1 and 2 are POP and OPO, respectively. The third component, component 3, is thought to be the POP-OPO molecular compound. However, it seems that the compound is formed at the molecular ratio of POP:OPO=1:2 and not 1:1 as reported previously.1 This is may be due to the difference in thermal treatment on crystal preparation. Further studies are needed to identify this component and its structure

Chapter 5: TAG polymorphic phase behavior in biological systems

It is empirically known that the mixing of multicomponent systems, accompanied by a large TAG compositional change, would indicate a transition to a completely different fat with different phase behavior. However, because of the complexity, the underlying causes for such transition are not known so far.

Adopting bovine and porcine fats as the instance of TAG multicomponent systems, the influence of the difference in TAG composition on their phase behavior and the phase behavior of their mixture are investigated. Bovine fats have high concentration of TAGs which have oleoyl acyls primarily substituted in their sn-2 position (Fig. 3). On the other hand, porcine fats have TAG molecules with oleoyls in their sn-1 and-3 positions.

Crystals of these two fats and their mixture fats were prepared by cooling down the melt to 0°C and holding for 5 min. Raman spectra of the polycrystal were measured by a 785-nm Raman spectrometer developed in the laboratory. The samples were kept at 0°C during the measurements.

The porcine fats show a band at 1417cm(-1) (Fig. 4), while the bovine fats do not exhibit this band.2 This band is assigned to the CH2-scissors mode characteristic of the orthorhombic subcell structure of β'-polymorph. It is therefore shown that the porcine fats contain the β'-polymorph while bovine fats do not contain them. The difference arises due to the TAG compositional difference between the two fats. The major TAG species in porcine fats (OSatO) is likely to form β'-polymorphs in the present experimental conditions.

In bovine-porcine mixture systems, however, β'-polymorphs scarcely exist even in the presence of porcine fat upto 50% (Fig. 5). The SatOSat-OSatO type molecular compound formation is the most likely reason why the addition of the bovine fat disturbs the β'-polymorph formation. The empirically known drastic changes of phase behavior which are caused by mixing multicomponent systems probably arise due to molecular compound formation.

Chapter 6: Conclusion

By the use of Raman spectroscopy, the characterization of TAG phases in multicomponent systems has been conducted. In the POP-OPO binary system, the formation of the third component has been shown. In the biological bovine-porcine system, the molecular compound formation is suggested. It has been shown that Raman spectroscopy is the powerful tool to study about the phase behavior of multicomponent TAG systems. Future prospects of studies on TAG structural chemistry through the application of Raman spectroscopy have also been discussed.

Fig. 1 Structures and interesting phase behaviors of TAGs

Fig. 2 Results of reconstruction (a) Concentration related index profiles. ▲, component 1; ●, component 2; ■, component 3; +, residuals. (b) Raman spectra of the components of POP-OPO binary system.

Fig. 3 Major TAG molecules in bovine and porcine fats. Sat: Saturated acyl chain O: Oleoly chain

Fig. 4 Raman spectra of porcine and bovine fats.2

Fig. 5 Relation between A(1417)cm(-1) and porcine-fat concentration.2

本論文は、トリアシルグリセロール多成分系の相挙動およびその構造に関するラマン分光学的研究について記述しており、全六章から構成される。

第一章では導入として、トリアシルグリセロールの構造と相挙動に関するこれまでの研究についての抄録と、本研究の位置付けが述べられている。トリアシルグリセロールは脂質の基本構造を有する分子で自然界では多成分系として存在し、その構造と相挙動は一成分あるいは二成分モデル系を用いて主に粉末X線解析により調べられてきたが、多成分系自体についてはほとんど明らかにされていないことが記述されている。また、"分子の指紋"を得ることのできるラマン分光法は、多成分系の解析には有利であることが説明されている。

第二章では、モデル系を使ってこれまで明らかにされてきたトリアシルグリセロールの結晶構造や相挙動、およびそれに影響を及ぼす因子に関する知見が解説されている。結晶多形、結晶化過程、molecular compound形成など、相挙動を複雑にする要因について最近の知見を中心に述べられており、次章以降の実験データ解釈のための根拠となっている。

第三章では、数種のトリアシルグリセロールについて液相および各結晶相を調整し、得られたラマンスペクトルと構造との対応が示されている。脂質のラマンスペクトルについては、これまでにモデル化合物であるポリエチレンやアルカン、脂肪酸について詳細に調べられており、それらの知見に基づいてトリアシルグリセロールの構造も解釈できることが述べられている。

第四章では、トリアシルグリセロールニ成分系(POP-OPO系)の相挙動について、ラマン分光を用いて調べた結果とその考察について記述されている。スペクトルデータセットを特異値分解することで第三の成分が存在することを示し、それがmolecular compoundであると考察している。また、その構造がこれまで報告されているPOP:OPO=1:1ではなく、1:2とみなせることから、molecular compoundが結晶化方法の影響を強く受ける動的な構造持つと考察している。

第五章では、生体由来トリアシルグリセロール多成分系であるウシ由来油脂とブタ由来油脂およびそれらの混合油脂の相挙動を解析した結果とその考察について述べられている。混合によりブタ由来油脂に特徴的な結晶多形の形成が阻害されることを見出し、molecular compolmdはモデル系のみでなく天然油脂においても生成すると考察している。また、結晶多形の違いを示す一つのラマンバンドにより、油脂の由来判別が可能であることを示している。

第六章では、トリアシルグリセロールの構造と相挙動に関する研究の今後の展望が述べられている。本分野の発展に寄与すると考えられるいくつかの研究が提案され、ラマン分光装置の性能はこれらの研究に十分であり、ラマン分光法によりトリアシルグリセロール多成分系の相挙動が今後さらに明らかになることが期待できるとしている。

本研究により、トリアシルグリセロールの構造および相挙動解析におけるラマン分光法の有用性が示されたとともに、多成分系の相挙動に関して新たな知見が得られた。特に、トリアシルグリセロールの結晶過程のダイナミクスは本研究分野において残された最大の課題の一つとされており、molecular compolmdが結晶化方法の影響を受ける動的な構造である可能性を示した点は高く評価される。さらに、多成分系の相挙動の違いをラマン分光で検出できることに着目し、天然油脂の由来判別に応用した点も独創性が高く、本分光手法のさらなる応用可能性の拡大に貢献したものと考えられる。このように、ラマン分光法を用いたトリアシルグリセロールの構造および相挙動解析に関する本論文の業績は高く評価できる。

本論文第五章の主要部分はApphed Spectroscopy誌に公表済み(安藤正浩・佐々木啓介・濱口宏夫との共著)であるが、論文提出者が主体となって実験および解析を行なっており、その寄与が十分であるので、学位論文の一部とすることに何ら問題はないと判断する。

以上の理由から、論文提出者本山三知代に博士(理学)の学位を授与することが適当であると認める。