天然高性能乳化剂—印度树胶的精细分子结构和构象特性研究
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摘要
印度树胶(Gum ghatti),又名达瓦树胶,主产于印度和斯里兰卡,是由宽叶榆绿木(Anogeissus latifolia)树干自然渗出的半透明胶状物质纯化而来。印度树胶具有比阿拉伯胶更加优良的乳化稳定性,因此已作为增稠剂、稳定剂、被膜剂和乳化剂被广泛的应用于国际食品工业中。印度树胶在传统乳化剂难以起作用的食品体系中具有尤其重要的应用价值。此外,在造纸业、制蜡业和制药业等领域也发挥着及其重要的作用。印度树胶的主要组成成分为天然多聚糖,其功能特性受分子量大小及分布、分子结构(单糖组成、糖苷键连接方式和分支度)、溶解度和黏度等理化特性的影响。
目前国际上关于印度树胶的研究仅集中于其功能特性,而对其多糖的分子结构、糖链在溶液中的空间构象几乎为空白,更未见任何结构和功能特性关系的报道;国内更无任何关于印度树胶的研究。本课题首先研究了印度树胶多糖的理化特性,应用甲基化结合气质联用分析,一维和二维核磁共振(COSY、TOCSY、HMQC和HMBC)技术解析出多糖的精细分子结构,并应用激光光散射和计算机分子模拟技术得出糖链的空间构象。同时应用基质辅助激光解吸电离飞行时间质谱(Maldi-TOF-MS)等技术对糖蛋白的结构进行了初步探讨,并由此进一步讨论了印度树胶的构效关系,这对从分子水平阐明印度树胶的作
用机制奠定了基础,为印度树胶乳化稳定剂新产品的开发提供了理论依据和指导。印度树胶化学组成为:还原糖89.67%,蛋白4.34%,灰分2.25%,水分8.04%。单糖组成(摩尔比):Ara:Gal:Xyl:Man:Glc=1.7:38.7:22.9:1.8:1.9:0.7,糖醛酸含量为13.2% (GlcA:GlaA=5:13)。本研究用乙醇分级沉淀法将印度树胶分为四个组分:F50 (50%乙醇沉淀), F65(65%乙醇沉淀物), F80(80%乙醇沉淀物)和FS (80%乙醇沉淀后的上清液)。流变学研究表明,当印度树胶水溶液的浓度小于20%(w/v)时,溶液呈现牛顿流体特性;当树胶水溶液浓度大于20%(w/v)时,溶液呈现非牛顿流体特征,具有剪切变稀现象。在相同浓度、剪切速率及温度的情况下,四个组分的表观粘度为F50>F65>F80>FS.在相同的测定条件下,FS的表面活性高于原始样品和其他组分,甚至高于阿拉伯胶。单糖组分和初步结构分析表明四个组分的链分支程度按F50, F65和F80的次序依次增大。糖链的分支程度、蛋白和糖醛酸含量可能是造成各个组分在乙醇中具有不同溶解度的原因。FS组分的分子结构和其他组分有较大差异。傅立叶变换红外光谱结果表明印度树胶结构中不存在甲酯基。
F80在印度树胶中占较大比例,是印度树胶的主要成分之一,且和树胶的另一主要成分F65的结构很相近; FS虽含量较低,却具有极高的表面活性。因此本课题选择F80和FS进行进一步的分子结构研究。甲基化分析结果表明:F80具有较多的分支,其非还原末端占所有链接方式的40.8%,其中t-α-L-Araf为主要的末端糖基,另外含有少量的t-GlcpA,t-Arap, t-Rhap和t-Galp基团。3,4位连有支链的“→6)-β-D-Galp-(1→”糖单元大约占总糖的14.2%。甲基化和2D NMR的结果表明F80的整体结构为:1,6-β-键连接的Galp组成主链;且在主链Galp的3-,4-位分别连有支链,构成了此结构的“毛发区”。“光滑区”由α-1,2键连接的Araf,β-1,4糖苷键连接的GlcpA和β-1,6连接的Galp组成,即→2)-Araf-(1→4)-GlcpA-(1→6)-Galp-(1→6)-Galp-(1→。
FS是含有乙酰基的多支链糖,具有比F80更多、更长的支链,其结构上只有“毛发区”,没有“光滑区”,且支链还可以再连接次级支链。其具体结构特征如下:主链由β-1,6连接而成,且在3,4位分别连有支链;支链片段主要有:Araf-(1→3)-Araf-(1→2)-Araf-(1→4)-GlcpA-(1→6)-Galp-(1→,Araf-(1→3)-Araf-(1→2)-Araf-(1→,Araf-(1→5)-Araf-(1→和Araf-(1→3)-Araf-(1→5)-Araf-(1→6)-Galp-(1→5)-Araf-(1→5)-Araf-(1→,其中→5)-Araf-(1→在3,4端还可以连接支链。FS的特殊理化性质(如高表面活性和高酒精溶解性)可能是由这种高度分支的结构特征所决定。
因含蛋白的组分可能在乳化中起到关键性的作用,所以本研究首先通过部分酸水解、阿拉伯糖酶和半乳糖酶水解方法,证明了印度树胶中的多糖和蛋白是通过共价键方式连接,且蛋白并不与末端阿拉伯糖基和半乳糖基相连,而是直接连到糖的主链上。氨基酸组分分析也表明印度树胶的糖蛋白链接方式不同于阿拉伯胶。
利用MALDI-TOF-MS和NMR对印度树胶的糖蛋白连接方式做了进一步研究,发现糖蛋白的结构为:由以β-1,6连接的半乳糖为主并含有少量木糖和甘露糖的主链上间隔连有糖支链和蛋白或多肽片段,糖和蛋白的连接片段为:(Hex)n-GlcNAc-Asn。
采用静态和动态光散射技术对印度树胶及其四个组分(F50, F65, F80和FS)在溶液中的构象进行了研究。经研究发现,印度树胶分子在纯水溶液中和0.2 M NaCl溶液中均存在聚集现象,具有较大的水合半径(180.3和108.7 nm),但在0.5M NaOH溶液中不存在分子聚集现象,水合半径仅为(11.5 nm)。多糖分子在0.5M NaOH溶液中48h内不降解。
采用配备多重检测器的高效凝胶过滤色谱(HPSEC)对印度树胶及其四个组分(F50, F65, F80和FS)的空间构象进行研究,结果表明:印度树胶及其组分的特征黏度为0.4728-0.7571 dL/g,绝对重均分子量(Mw)为643.8-1011.0 kDa。Mark-houwink方程参数α结果表明: F50分子具有较刚硬的链构象,F65的构象相对比较松散伸展,具有接近球状的构象特征,这种构象缘于其多支链的结构特征。
利用静态和动态光散射研究了印度树胶及其四个组分(F50, F65, F80和FS)在无聚集状态下的构象特征,结果表明:在0.5M NaOH溶液中印度树胶具有较高的第二维里系数(A2),说明0.5M NaOH是印度树胶极好的溶剂。均方旋转半径(Rg)值按F80, F65, F50和FS的顺序依次降低,水合半径(Rh)和其绝对重均分子量(Mw)呈同样的趋势。由Rg/Rh计算得到构象参数ρ,F50较小的ρ值表示其在0.5M NaOH溶液中呈无规蜷曲构象,这是由其结构上支链较少的特征决定的。而F65和F80呈一定的星型构象,这也由其多支链结构所决定。
根据已经得出的F80和FS的一级结构,依据主体选择规则和能量最低原理,用计算机分子模拟技术,建立了F80和FS多糖的分子结构的3D构象模型,结果表明,F80和FS都呈现星型的链构象,同时,通过RMMC模拟算法计算所得的构象参数和实验测得值相吻合。
本研究以印度树胶作为研究材料,首次系统地分析了该树胶的结构特性和构象特征,探讨了印度树胶中糖蛋白的连接方式,成功建立了印度树胶的构效关系;从结构和构象角度(高度分支状的多糖结构、糖和蛋白的共价连接形式,以及相对紧密的无规蜷曲构象),对其优良乳化稳定性的机理进行了探讨;为印度树胶在工业生产中的应用提供了理论支持和保障,并为其它复杂多糖尤其是树分泌胶的结构研究提供了方法参考。
Gum Ghatti is versatile translucent exudate (water-soluble gum) from Anogeissus latifolia, a tree that is native to India and Sri Lanka. The excellent emulsifying properties of gum ghatti offer great potential for its use in the food industry. However, information about the characterisation of its molecular structure, conformational properties in solution and the linkage between protein and polysaccharide is very limited. Since the structure information is very important to understand the structure-property relationship of gum ghatti, this research focused on fractionation, physical and chemical investigation of gum ghatti, and elucidation the detailed molecular structure of two fractions of gum ghatti, followed by study the structure of a glycoprotein and conformational analysis of gum ghatti fractions. Numerous techniques were involved, such as, methylation analysis-GC-MS, Maldi-TOF MS and 2D NMR spectroscopy including homonuclear 1H/1H correlations spectroscopy (COSY, TOCSY), heteronuclear 13C/1H multiple-quantum coherence spectroscopy (HMQC) and heteronuclear multiple bond correlation (HMBC) etc.
The fractionations, chemical and physical characterization of processed gum Ghatti (Gatifolia SD) were first investigated, and the source of its surface activity was identified. Four fractions were separated using the gradual ethanol precipitation method. With the increase of alcohol concentration, chemical composition of the fractions exhibited a pattern: arabinose content increased, but the galactose, protein and uronic acid contents decreased in the order of: F50 (50% ethanol precipitate), F65 (65% ethanol precipitate), F80 (80% ethanol precipitate) and FS (the supernatant after 80% ethanol precipitation). Rheologically gum ghatti and its fractions exhibited Newtonian flow behaviour until gum concentrations reached to 20% (w/v), at which point gum ghatti showed some shear-thinning flow behavior. At the same shear rate and concentration, the apparent viscosities of these fractions decreased in the order of F50>F65>F80>FS. When compared at same concentration, the FS fraction had the highest surface activity relative to Gatifolia SD, the other fractions and even gum Arabic. Monosaccharide composition and preliminary structural analysis showed that the branching of the polymer increased in the order of F50, F65 and F80. The degree of branching levels, protein and uronic acid content could be responsible for the different solubility of the fractions in alcohol. However, the molecular structure of FS is significantly different from the other fractions. FT-IR spectroscopy revealed no esterified carboxyl group in gum ghatti.
The major structure of gum ghatti as represented by F80 was characterized using methylation analysis, 1D and 2D NMR spectroscopy. The detailed structure information about the main polysaccharide of gum ghatti, including linkage patterns, configuration of and the sequences of each sugar unit was presented. Methylation and GC-MS analysis indicated that F80 was a highly branched polysaccharide; the terminal sugar residues were about 40.8% of the total sugars. The majority of the terminal units wereα-L-Araf, with small amounts of t-GlcpA, t-Arap, t-Rhap and t-Galp. About 14.2% of the total sugar residues were→6)-β-D-Galp-(1→branched at 3 and 4 positions. The linear portion of the arabinogalactan was composed of→4)-GlcpA(1→,→6)-Galp(1→and→2)-L-Araf-(1→linkages. Based on the results from methylation analysis, 1D and 2D spectroscopy, a schemed structure was proposed and summarized as follows: The backbone is composed of 1,6-linked galactopyransyl (Galp) residues substituted at O-3 and O-4 position, which can be called“hairy region”, while the“smooth region”consists of→2)-Araf-(1→4)-GlcpA-(1→6)-Galp-(1→6)-Galp-(1→. Side chains are terminated by arabinofuranosyl (Araf) and occasionally by rhamnopyranosyl (Rhap), arabinopyranosyl (Arap), galactopyranosyl (Galp) and glucuronopyranosyl (GlcpA) residues.
The structure of a globular gum ghatti fraction (FS) with high surface activity was investigated using the same methods as F80. FS is proposed to be a highly branched polysaccharide with small amount of acetyl substitution. It consists of 1,6-linked galactopyransyl (galp) backbone, branched at O-3 and O-4 position by various of sugar residues, including→6)-β-D-Galp-(1→,→2)-α-L-Araf-(1→,→5)-α-L-Araf-(1→, t-α-L-Araf and→2,3,5)-α-L-Araf-(1→. Moreover, the→2,3,5)-α-L-Araf-(1→residue on the side chain can have two side chains at O-2 and O-3 position, which gives the multi-branched structure of FS. Most of the side chains have terminal arabinofuranosyl (Araf), and are occasionally terminated by rhamnopyranosyl (Rhap), arabinopyranosyl (Arap), Galp and glucuronopyranosyl (GlcpA) residues. Some of the side chains are as long as five sugar units. Comparing with the main polysaccharide (F80) of gum ghatti, fraction FS is much more branched and has longer side chains. These structural features are consistent with the physical properties such as solubility for the FS fraction (FS is more soluble than F80). This multi-branched structure most likely accounts for the special physical properties including the excellent surface activities exhibited by FS.
Since the protein content of gum ghatti was 4.34% (w/w), proteinaceous part was reported to play an important role to the emulsification properties. In this study, enzymatic degradation (α-L-arabinofuranosidase andβ-D-galactosidase) and chemical hydrolysis results indicated that most of the protein was covalently linked to the backbone of the polysaccharide. The amino acid composition and protease hydrolysis results indicated that the linkage between polysaccharide and protein were different from that of gum Arabic, which had a wattle blossom structure.
The structure of gum ghatti glycoprotein was investigated by Maldi-TOF MS and 1D&2D NMR spectroscopy. Combined with the polysaccharide structure results, a structure model was proposed: it was a 1,6-linked galactose backbone with numerous of side chains, occasionally, xylose and mannose were also appeared on the backbone. Proteins or polypeptides attached directly to the core part of the polysaccharide. The linkage site of amino acids and polysaccharides was determined as N-linked (Hex)n-GlcNAc-Asn.
Conformational properties of original gum ghatti and four fractions (F50, F65, F80 and FS) were investigated by dynamic and static light scattering techniques. For the known structure fractions (F80 and FS), two models were built to simulate structure properties, and the conformational parameters calculated after RMMC simulation were compared with experimental results.
Gum ghatti molecules formed aggregates in pure water and 0.2M NaCl solution, with the apparent mean diameter of 360.52 and 217.43 nm, respectively. The aggregates were successfully eliminated by dissolving in 0.5M NaOH solution and the solution was stable in two days at room temperature without visible degradation
HPSEC coupled with multiple detectors gave numerous properties including intrinsic viscosity [η], weight average molecular weight (Mw), number average molecular weight (Mn), Mark-houwink equation parameters (α) and logκ., Theαvalues indicated the random coil conformation of four fractions: F50 was much rigid than other fractions, while F65 exhibited loosely extended chain close to the spherical conformation, suggested a highly branched structure.
The results from DLS and SLS in aggregate free (0.5M NaOH) solution further confirmed the conformation properties of gum ghatti obtained from the HPSEC. More parameters were derived including Rg, A2 andρ=Rg/Rh. The high positive value of A2 for each fraction suggested that 0.5 M NaOH solution was a good solvent for gum ghatti. The values of Rg decreased in the order of F80, F65, F50 and FS, which is consistent with molecular weight. The hydrodynamic radius Rh had the same trend which was obtained from CONTIN method. The less branched fraction F50 (ρ=1.694) demonstrated a random coil conformation in 0.5M NaOH solution, whereas F65 and
F80 exhibited regular stars conformation due to the highly branched structure Combining with the computational method, the structure-functionality relationship for each fraction of gum ghatti was partially established. The 3D molecular model of gum ghatti fractions F80 and FS was created and the regular star chain conformation was first visualized. The conformational properties of F80 and FS calculated by RMMC simulation were in good agreement with experimental results.
The structure-function relationship of gum ghatti was established in the present study. The highly branched molecular structure of gum ghatti, to some extend, led to the regular star and globular conformation of molecules. The structural characteristics and the relative compact conformation, as well as the conjugation between polysaccharide and protein contributed to the excellent emulsification and stabilization properties.
The elucidation of the fine structure and conformation of gum ghatti can provide guidances for the study of other polysaccharide especially exudates gums. Also, the establishment of the structure-function relationship of gum ghatti will not only expand its applications of gum ghatti, but also provide the theoretical base for physicochemical modification of natural gums for the production of high quality natural emulsifiers and stabilizers.
目前国际上关于印度树胶的研究仅集中于其功能特性,而对其多糖的分子结构、糖链在溶液中的空间构象几乎为空白,更未见任何结构和功能特性关系的报道;国内更无任何关于印度树胶的研究。本课题首先研究了印度树胶多糖的理化特性,应用甲基化结合气质联用分析,一维和二维核磁共振(COSY、TOCSY、HMQC和HMBC)技术解析出多糖的精细分子结构,并应用激光光散射和计算机分子模拟技术得出糖链的空间构象。同时应用基质辅助激光解吸电离飞行时间质谱(Maldi-TOF-MS)等技术对糖蛋白的结构进行了初步探讨,并由此进一步讨论了印度树胶的构效关系,这对从分子水平阐明印度树胶的作
用机制奠定了基础,为印度树胶乳化稳定剂新产品的开发提供了理论依据和指导。印度树胶化学组成为:还原糖89.67%,蛋白4.34%,灰分2.25%,水分8.04%。单糖组成(摩尔比):Ara:Gal:Xyl:Man:Glc=1.7:38.7:22.9:1.8:1.9:0.7,糖醛酸含量为13.2% (GlcA:GlaA=5:13)。本研究用乙醇分级沉淀法将印度树胶分为四个组分:F50 (50%乙醇沉淀), F65(65%乙醇沉淀物), F80(80%乙醇沉淀物)和FS (80%乙醇沉淀后的上清液)。流变学研究表明,当印度树胶水溶液的浓度小于20%(w/v)时,溶液呈现牛顿流体特性;当树胶水溶液浓度大于20%(w/v)时,溶液呈现非牛顿流体特征,具有剪切变稀现象。在相同浓度、剪切速率及温度的情况下,四个组分的表观粘度为F50>F65>F80>FS.在相同的测定条件下,FS的表面活性高于原始样品和其他组分,甚至高于阿拉伯胶。单糖组分和初步结构分析表明四个组分的链分支程度按F50, F65和F80的次序依次增大。糖链的分支程度、蛋白和糖醛酸含量可能是造成各个组分在乙醇中具有不同溶解度的原因。FS组分的分子结构和其他组分有较大差异。傅立叶变换红外光谱结果表明印度树胶结构中不存在甲酯基。
F80在印度树胶中占较大比例,是印度树胶的主要成分之一,且和树胶的另一主要成分F65的结构很相近; FS虽含量较低,却具有极高的表面活性。因此本课题选择F80和FS进行进一步的分子结构研究。甲基化分析结果表明:F80具有较多的分支,其非还原末端占所有链接方式的40.8%,其中t-α-L-Araf为主要的末端糖基,另外含有少量的t-GlcpA,t-Arap, t-Rhap和t-Galp基团。3,4位连有支链的“→6)-β-D-Galp-(1→”糖单元大约占总糖的14.2%。甲基化和2D NMR的结果表明F80的整体结构为:1,6-β-键连接的Galp组成主链;且在主链Galp的3-,4-位分别连有支链,构成了此结构的“毛发区”。“光滑区”由α-1,2键连接的Araf,β-1,4糖苷键连接的GlcpA和β-1,6连接的Galp组成,即→2)-Araf-(1→4)-GlcpA-(1→6)-Galp-(1→6)-Galp-(1→。
FS是含有乙酰基的多支链糖,具有比F80更多、更长的支链,其结构上只有“毛发区”,没有“光滑区”,且支链还可以再连接次级支链。其具体结构特征如下:主链由β-1,6连接而成,且在3,4位分别连有支链;支链片段主要有:Araf-(1→3)-Araf-(1→2)-Araf-(1→4)-GlcpA-(1→6)-Galp-(1→,Araf-(1→3)-Araf-(1→2)-Araf-(1→,Araf-(1→5)-Araf-(1→和Araf-(1→3)-Araf-(1→5)-Araf-(1→6)-Galp-(1→5)-Araf-(1→5)-Araf-(1→,其中→5)-Araf-(1→在3,4端还可以连接支链。FS的特殊理化性质(如高表面活性和高酒精溶解性)可能是由这种高度分支的结构特征所决定。
因含蛋白的组分可能在乳化中起到关键性的作用,所以本研究首先通过部分酸水解、阿拉伯糖酶和半乳糖酶水解方法,证明了印度树胶中的多糖和蛋白是通过共价键方式连接,且蛋白并不与末端阿拉伯糖基和半乳糖基相连,而是直接连到糖的主链上。氨基酸组分分析也表明印度树胶的糖蛋白链接方式不同于阿拉伯胶。
利用MALDI-TOF-MS和NMR对印度树胶的糖蛋白连接方式做了进一步研究,发现糖蛋白的结构为:由以β-1,6连接的半乳糖为主并含有少量木糖和甘露糖的主链上间隔连有糖支链和蛋白或多肽片段,糖和蛋白的连接片段为:(Hex)n-GlcNAc-Asn。
采用静态和动态光散射技术对印度树胶及其四个组分(F50, F65, F80和FS)在溶液中的构象进行了研究。经研究发现,印度树胶分子在纯水溶液中和0.2 M NaCl溶液中均存在聚集现象,具有较大的水合半径(180.3和108.7 nm),但在0.5M NaOH溶液中不存在分子聚集现象,水合半径仅为(11.5 nm)。多糖分子在0.5M NaOH溶液中48h内不降解。
采用配备多重检测器的高效凝胶过滤色谱(HPSEC)对印度树胶及其四个组分(F50, F65, F80和FS)的空间构象进行研究,结果表明:印度树胶及其组分的特征黏度为0.4728-0.7571 dL/g,绝对重均分子量(Mw)为643.8-1011.0 kDa。Mark-houwink方程参数α结果表明: F50分子具有较刚硬的链构象,F65的构象相对比较松散伸展,具有接近球状的构象特征,这种构象缘于其多支链的结构特征。
利用静态和动态光散射研究了印度树胶及其四个组分(F50, F65, F80和FS)在无聚集状态下的构象特征,结果表明:在0.5M NaOH溶液中印度树胶具有较高的第二维里系数(A2),说明0.5M NaOH是印度树胶极好的溶剂。均方旋转半径(Rg)值按F80, F65, F50和FS的顺序依次降低,水合半径(Rh)和其绝对重均分子量(Mw)呈同样的趋势。由Rg/Rh计算得到构象参数ρ,F50较小的ρ值表示其在0.5M NaOH溶液中呈无规蜷曲构象,这是由其结构上支链较少的特征决定的。而F65和F80呈一定的星型构象,这也由其多支链结构所决定。
根据已经得出的F80和FS的一级结构,依据主体选择规则和能量最低原理,用计算机分子模拟技术,建立了F80和FS多糖的分子结构的3D构象模型,结果表明,F80和FS都呈现星型的链构象,同时,通过RMMC模拟算法计算所得的构象参数和实验测得值相吻合。
本研究以印度树胶作为研究材料,首次系统地分析了该树胶的结构特性和构象特征,探讨了印度树胶中糖蛋白的连接方式,成功建立了印度树胶的构效关系;从结构和构象角度(高度分支状的多糖结构、糖和蛋白的共价连接形式,以及相对紧密的无规蜷曲构象),对其优良乳化稳定性的机理进行了探讨;为印度树胶在工业生产中的应用提供了理论支持和保障,并为其它复杂多糖尤其是树分泌胶的结构研究提供了方法参考。
Gum Ghatti is versatile translucent exudate (water-soluble gum) from Anogeissus latifolia, a tree that is native to India and Sri Lanka. The excellent emulsifying properties of gum ghatti offer great potential for its use in the food industry. However, information about the characterisation of its molecular structure, conformational properties in solution and the linkage between protein and polysaccharide is very limited. Since the structure information is very important to understand the structure-property relationship of gum ghatti, this research focused on fractionation, physical and chemical investigation of gum ghatti, and elucidation the detailed molecular structure of two fractions of gum ghatti, followed by study the structure of a glycoprotein and conformational analysis of gum ghatti fractions. Numerous techniques were involved, such as, methylation analysis-GC-MS, Maldi-TOF MS and 2D NMR spectroscopy including homonuclear 1H/1H correlations spectroscopy (COSY, TOCSY), heteronuclear 13C/1H multiple-quantum coherence spectroscopy (HMQC) and heteronuclear multiple bond correlation (HMBC) etc.
The fractionations, chemical and physical characterization of processed gum Ghatti (Gatifolia SD) were first investigated, and the source of its surface activity was identified. Four fractions were separated using the gradual ethanol precipitation method. With the increase of alcohol concentration, chemical composition of the fractions exhibited a pattern: arabinose content increased, but the galactose, protein and uronic acid contents decreased in the order of: F50 (50% ethanol precipitate), F65 (65% ethanol precipitate), F80 (80% ethanol precipitate) and FS (the supernatant after 80% ethanol precipitation). Rheologically gum ghatti and its fractions exhibited Newtonian flow behaviour until gum concentrations reached to 20% (w/v), at which point gum ghatti showed some shear-thinning flow behavior. At the same shear rate and concentration, the apparent viscosities of these fractions decreased in the order of F50>F65>F80>FS. When compared at same concentration, the FS fraction had the highest surface activity relative to Gatifolia SD, the other fractions and even gum Arabic. Monosaccharide composition and preliminary structural analysis showed that the branching of the polymer increased in the order of F50, F65 and F80. The degree of branching levels, protein and uronic acid content could be responsible for the different solubility of the fractions in alcohol. However, the molecular structure of FS is significantly different from the other fractions. FT-IR spectroscopy revealed no esterified carboxyl group in gum ghatti.
The major structure of gum ghatti as represented by F80 was characterized using methylation analysis, 1D and 2D NMR spectroscopy. The detailed structure information about the main polysaccharide of gum ghatti, including linkage patterns, configuration of and the sequences of each sugar unit was presented. Methylation and GC-MS analysis indicated that F80 was a highly branched polysaccharide; the terminal sugar residues were about 40.8% of the total sugars. The majority of the terminal units wereα-L-Araf, with small amounts of t-GlcpA, t-Arap, t-Rhap and t-Galp. About 14.2% of the total sugar residues were→6)-β-D-Galp-(1→branched at 3 and 4 positions. The linear portion of the arabinogalactan was composed of→4)-GlcpA(1→,→6)-Galp(1→and→2)-L-Araf-(1→linkages. Based on the results from methylation analysis, 1D and 2D spectroscopy, a schemed structure was proposed and summarized as follows: The backbone is composed of 1,6-linked galactopyransyl (Galp) residues substituted at O-3 and O-4 position, which can be called“hairy region”, while the“smooth region”consists of→2)-Araf-(1→4)-GlcpA-(1→6)-Galp-(1→6)-Galp-(1→. Side chains are terminated by arabinofuranosyl (Araf) and occasionally by rhamnopyranosyl (Rhap), arabinopyranosyl (Arap), galactopyranosyl (Galp) and glucuronopyranosyl (GlcpA) residues.
The structure of a globular gum ghatti fraction (FS) with high surface activity was investigated using the same methods as F80. FS is proposed to be a highly branched polysaccharide with small amount of acetyl substitution. It consists of 1,6-linked galactopyransyl (galp) backbone, branched at O-3 and O-4 position by various of sugar residues, including→6)-β-D-Galp-(1→,→2)-α-L-Araf-(1→,→5)-α-L-Araf-(1→, t-α-L-Araf and→2,3,5)-α-L-Araf-(1→. Moreover, the→2,3,5)-α-L-Araf-(1→residue on the side chain can have two side chains at O-2 and O-3 position, which gives the multi-branched structure of FS. Most of the side chains have terminal arabinofuranosyl (Araf), and are occasionally terminated by rhamnopyranosyl (Rhap), arabinopyranosyl (Arap), Galp and glucuronopyranosyl (GlcpA) residues. Some of the side chains are as long as five sugar units. Comparing with the main polysaccharide (F80) of gum ghatti, fraction FS is much more branched and has longer side chains. These structural features are consistent with the physical properties such as solubility for the FS fraction (FS is more soluble than F80). This multi-branched structure most likely accounts for the special physical properties including the excellent surface activities exhibited by FS.
Since the protein content of gum ghatti was 4.34% (w/w), proteinaceous part was reported to play an important role to the emulsification properties. In this study, enzymatic degradation (α-L-arabinofuranosidase andβ-D-galactosidase) and chemical hydrolysis results indicated that most of the protein was covalently linked to the backbone of the polysaccharide. The amino acid composition and protease hydrolysis results indicated that the linkage between polysaccharide and protein were different from that of gum Arabic, which had a wattle blossom structure.
The structure of gum ghatti glycoprotein was investigated by Maldi-TOF MS and 1D&2D NMR spectroscopy. Combined with the polysaccharide structure results, a structure model was proposed: it was a 1,6-linked galactose backbone with numerous of side chains, occasionally, xylose and mannose were also appeared on the backbone. Proteins or polypeptides attached directly to the core part of the polysaccharide. The linkage site of amino acids and polysaccharides was determined as N-linked (Hex)n-GlcNAc-Asn.
Conformational properties of original gum ghatti and four fractions (F50, F65, F80 and FS) were investigated by dynamic and static light scattering techniques. For the known structure fractions (F80 and FS), two models were built to simulate structure properties, and the conformational parameters calculated after RMMC simulation were compared with experimental results.
Gum ghatti molecules formed aggregates in pure water and 0.2M NaCl solution, with the apparent mean diameter of 360.52 and 217.43 nm, respectively. The aggregates were successfully eliminated by dissolving in 0.5M NaOH solution and the solution was stable in two days at room temperature without visible degradation
HPSEC coupled with multiple detectors gave numerous properties including intrinsic viscosity [η], weight average molecular weight (Mw), number average molecular weight (Mn), Mark-houwink equation parameters (α) and logκ., Theαvalues indicated the random coil conformation of four fractions: F50 was much rigid than other fractions, while F65 exhibited loosely extended chain close to the spherical conformation, suggested a highly branched structure.
The results from DLS and SLS in aggregate free (0.5M NaOH) solution further confirmed the conformation properties of gum ghatti obtained from the HPSEC. More parameters were derived including Rg, A2 andρ=Rg/Rh. The high positive value of A2 for each fraction suggested that 0.5 M NaOH solution was a good solvent for gum ghatti. The values of Rg decreased in the order of F80, F65, F50 and FS, which is consistent with molecular weight. The hydrodynamic radius Rh had the same trend which was obtained from CONTIN method. The less branched fraction F50 (ρ=1.694) demonstrated a random coil conformation in 0.5M NaOH solution, whereas F65 and
F80 exhibited regular stars conformation due to the highly branched structure Combining with the computational method, the structure-functionality relationship for each fraction of gum ghatti was partially established. The 3D molecular model of gum ghatti fractions F80 and FS was created and the regular star chain conformation was first visualized. The conformational properties of F80 and FS calculated by RMMC simulation were in good agreement with experimental results.
The structure-function relationship of gum ghatti was established in the present study. The highly branched molecular structure of gum ghatti, to some extend, led to the regular star and globular conformation of molecules. The structural characteristics and the relative compact conformation, as well as the conjugation between polysaccharide and protein contributed to the excellent emulsification and stabilization properties.
The elucidation of the fine structure and conformation of gum ghatti can provide guidances for the study of other polysaccharide especially exudates gums. Also, the establishment of the structure-function relationship of gum ghatti will not only expand its applications of gum ghatti, but also provide the theoretical base for physicochemical modification of natural gums for the production of high quality natural emulsifiers and stabilizers.
引文
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[1] Castellani, O., Gaillard, C., Vié, V., et al., Hydrocolloids with emulsifying capacity. Part 3 - Adsorption and structural properties at the air-water surface. Food Hydrocolloids, 2010, 24, 131-141.
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[3] Castellani, O., Al-Assaf, S., Axelos, M., et al., Hydrocolloids with emulsifying capacity. Part 2 - Adsorption properties at the n-hexadecane-Water interface. Food Hydrocolloids, 2010, 24, 121-130.
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[1] Castellani, O., Al-Assaf, S., Axelos, M., et al., Hydrocolloids with emulsifying capacity. Part 2 - Adsorption properties at the n-hexadecane-Water interface. Food Hydrocolloids, 2010, 24, 121-130.
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[17] Rantanen, H., Virkki, L., Tuomainen, P., et al., Preparation of arabinoxylobiose from rye xylan using family 10 Aspergillus aculeatus endo-1,4-[beta]-d-xylanase. Carbohydrate Polymers, 2007, 68, 350-359.
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[1] Al-Assaf, S., Phillips, G.O., & Amar, V., 'Gum ghatti'. in: Phillips, G.O., &Williams, P.A., (Eds.), Handbook of hydrocolloids, Woodhead Publishing, 2009, pp. 477-497.
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