特色资源果胶的提取和物理化学性质表征及微射流降解机理
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摘要
果胶是一系列具有相同特性,但精细结构不同的结构杂多糖的总称,从植物中提取的果胶已经被广泛用于食品,医药和化妆品行业中。目前,低酯果胶正日益受到关注。然而商业低酯果胶常通过对高酯果胶进行酶法或酸碱去酯化得到,步骤复杂且容易引起降解,较好的方法是从自然资源中直接提取果胶。薜荔籽和豆腐柴叶在民间常被用于制作“凉粉”和“神仙豆腐”,然而关于其制作机理目前尚不清楚。初步研究表明,这两种资源中蕴含有丰富的果胶,可是关于其果胶的提取,物理化学和流变性能缺乏充分研究。本文首先采用各种溶剂从薜荔籽和豆腐柴叶中提取果胶；然后采用现代分析技术对果胶进行物理化学性质表征,考察所得果胶的流变性能和凝胶性质,解释传统食品“凉粉”和“神仙豆腐”的制作之谜。采用阴离子交换色谱分级薜荔籽果胶得到两组分,通过X-衍射等手段分析两组分溶解性差异的原因。此外本论文还特别关注透析溶液对果胶性质的影响,及果胶在动态高压微射流(DHPM)处理过程中的降解问题。研究发现,低酯果胶在透析过程中易于吸收透析溶液中的金属离子,特别是钙离子；DHPM处理会造成果胶的降解,经对DHPM引起的果胶降解的表征,探讨了DHPM降解果胶的机理。本文进一步采用DHPM制备果胶低聚糖,所得低聚糖具有明显的益生活性。主要研究结论如下：
1.采用水,草酸铵和盐酸分别提取薜荔籽中的凝胶物质,结果表明三种方法提取的主要成分均为低酯果胶,且该果胶主要分布于薜荔籽的表面,因此解释了传统“凉粉”制作工艺中采用挤压和揉搓方法的可行性。与草酸铵提和盐酸提果胶相比,水提果胶具有最高的半乳糖醛酸含量、粘均分子量和特征粘性,以及最低的甲酯化。这种水提低酯果胶为薜荔籽水提物的主要多糖组分,同时检测到水提物中存有大量的钙离子,暗示“凉粉”的自凝胶可能是基于低酯果胶的“蛋盒”模型。
2.薜荔籽水提果胶经阴离子交换柱分离,得到两组分WEP-0.3及WEP-0.4,这两个组分的溶解性表现出明显的差异性。WEP-0.3能较快溶解在水中,而约有89.5%的WEP-0.4不溶于水。该结果与红外光谱得出的WEP-0.4具有比WEP-0.3更低的酯化度,而酯化度越低,溶解性应该越好的常理相违背。扫描电镜表明WEP-0.4形成面条状的紧致结构,X-衍射分析表明WEP-0.4存在结晶区。而WEP-0.3结构比较松散且均为无定形区域,这些结果表明WEP-0.4不易溶解是因为其在透析的过程中形成了部分凝胶。凝胶形成可能是因为低酯果胶在透析过程中吸收了Ca2+,反映在金属元素分析中WEP-0.4含有更高的钙离子含量。
3.对豆腐柴叶进行组分分析得知,豆腐柴叶的细胞壁物质(AIS)占干叶质量的77.82%,AIS中总膳食纤维,不可溶性膳食纤维和半乳糖醛酸含量分别为69.45%,56.35%,23.94%。采用水,草酸铵,盐酸和氢氧化钠对AIS进行连续提取,发现草酸铵溶性果胶(OXSP)占总果胶的大部分(59.48%),其半乳糖醛酸含量为76.15%,酯化度为14.90%,平均分子量为980.67kDa.这些理化性质导致OXSP具有很好的凝胶性能。本研究能促进豆腐柴叶作为膳食纤维和低酯果胶资源而被开发利用。
4.本研究分别采用自来水和去离子水对豆腐柴叶提取的果胶进行透析,发现透析溶剂对果胶溶液外观,果胶得率,蛋白质含量及形貌产生重大的影响。水溶性果胶,草酸铵溶性果胶及碱溶性果胶经自来水透析后都形成或部分形成凝胶。这主要是由于低酯果胶在自来水透析过程中吸收钙离子形成了凝胶,这一推论与组分中Ca离子含量及灰分含量较高的结果一致。本章还考察了连续提取每个步骤后残渣的形貌,表明连续提取是个逐步释放果胶的过程,其中采用草酸铵提取后,细胞壁物质的形貌变化最大,印证草酸铵是提取豆腐柴叶果胶较为理想的提取剂。
5.采用动态高压微射流法对高酯果胶进行处理,发现高酯果胶的表观粘度,分子量和粒径随压力的升高而降低,还原糖含量则随压力的升高而升高。同时果胶的表观形貌也由块状向多孔碎片转变。傅里叶红外光谱表明动态高压微射流对果胶的基本结构没有影响,同时由于还原糖的升高和分子量的降低呈线性关系,表明果胶的降解可能源于分子内糖苷键的断裂。当H+离子浓度降低时降解得到抑制,表明这种糖苷键的断裂可能不仅仅来自于强烈的机械力作用也可能来自于酸水解作用。因此,本研究认为机械力可以打开果胶分子或者使果胶分子更加伸展,使得更多的糖苷键可以暴露给机械力和H+攻击。
6.采用动态高压微射流对苹果果胶进行降解制备果胶低聚糖,经响应曲面优化,在果胶浓度为1.84%,溶液温度63℃,微射流处理压力155MPa,循环次数6次时,果胶制备成果胶低聚糖的转化率为32.92%。所得果胶低聚糖含有29.56%的半乳糖醛酸以及58.53%的中性糖。粪便发酵实验表明,果胶低聚糖较母体果胶更能增加双歧杆菌和乳酸杆菌的数量,同时产生更多的乙酸,乳酸和丙酸。而且,果胶低聚糖能抑制拟杆菌和梭状芽胞杆菌但其母体果胶则促进这两种菌的生长。另外,果胶低聚糖对细菌生长的影响和短链脂肪酸的产生能力和最常用的益生元-低聚果糖相当。本研究结果表明动态高压微射流是制备果胶低聚糖比较适合的手段,所得果胶低聚糖可能是一种有效的新型益生元。
Pectin describes a family of structural hetropolysaccharides that have common features, but are extremely diverse in their fine structure. Pectins extracted from plants have been used widely as gelling agent, thicker, texturizer in food, pharmacy and cosmetic area. There is growing interest in low methoxyl pectin. However, LMP has to be prepared from HMP by reducing the DM through acid demethoxylation, alkali demethoxylation, or demethoxylating reaction of pectin methyl esterase. These methoxylations are complex and may cause potential depolymerization. An alternative approach for manufacturing LMP is to 'mine' plant cell walls that are enriched with LMP. Creeping fig (Ficus pumila Linn) seeds and Premna microphylla turcz leaves have been used to prepare a "Liangfen" and "Shenxian tofu", respectively by local Chinese people. However, their making secret have not been explored. Preliminary experiments indicated that both resources contain high amount of pectic polysaccharides. However, extraction, physicochemical and rheological properties of pectin was rarely reported. In this study, pectic polysaccharides were extracted from Creeping fig seeds and Premna microphylla turcz leaves using kinds of extracting agent, then the physicochemical properties and gelling capability of resulting pectin were investigated to explain why "Liangfen" and "Shenxian tofu" would be manufactured in the traditional methods. The pectin isolated from creeping fig seeds was fractionated into two components by ion exchange column, techniques such as X-ray diffraction were used to explain the reason why two fractions have significant different solublities. During investigation, we also pay attention to the effect of dialysis solution on properties of pectin, and degradation of pectin induced by dynamic high pressure microfluidization (DHPM). We found out that low methoxyl pectin tends to absorb metal ions from dialysis solution. After characterizing the extent of degradation of pectin induced by DHPM, the mechanism of degradation was discussed. This technique was also used to produce pectic oligosaccharides (POS), the resulting POS exhibit a notable prebiotic properties. Main conclusions are summarized as follows:
1. Gelling material was extracted from creeping fig seeds using water, ammonium oxalate and hydrochloric acid, respectively. Results showed the main component of three extracts is low methoxyl pectin. The pectin locates in a transparent layer on the surface of seeds, as revealed by an inverted microscope. Hence, explained the feasibility of the squeezing and rubbing method in traditional handcraft. Comparing with the other methods, water-extracted pectin has high galacturonic acid content, viscosity-average molecular weight and intrinsic viscosity but low degree of methoxylation. This pectin forms the major component of WE, together with the high amount of calcium ions present in WE, it suggests the spontaneous gelation may be based on the'egg-box'formation.
2. The water-extracted pectin (WEP) from creeping fig seeds can be fractionated into two components (WEP-0.3and WEP-0.4) by ion exchange column. These two fractions showed a notable difference in dissolution behavior, WEP-0.3can be easily dissolved into water while89.5%of WEP-0.4become insoluble after dialysis against running water and distilled water later and freeze-dried. This phenomenon was contradicted with the results of FTIR spectrum where WEP-0.4have lower degree of methoxylation should have better solubility. For WEP-0.4, partial crystal was observed by X-ray diffraction and noodle like-shape was found by environmental scanning electronic microscopy. In the case of WEP-0.3, it's surface topography was relatively loosely and it has amorphous structure. These results indicated a microgel was formed for WEP-0.4in the process of dialysis. The formation of microgel may due to the adsorption of Ca2+by WEP-0.4, as reflected in a high calcium content in WEP-0.4.
3. Premna microphylla turcz leaves (PMTL) have been used for preparing a "Shenxian tofu" by Chinese for a long history. Cell-wall material of PMTL extracted as alcohol insoluble solids (AIS) was analyzed. The AIS recovery accounted for77.82g/100g dried leaves, while the total, insoluble dietary fiber and galacturonic acid content was69.45,56.35and23.94g/100g of the AIS, respectively. A sequential extraction, using water, ammonium oxalate, hydrochloric acid and sodium hydroxide, was used to extract different pectic fractions. Oxalate-soluble pectin (OXSP) was found to be the major pectic fraction (59.48%), which contains high amounts of galacturonic acids (76.15%) with mainly homogalacturonan, and has low degree of methoxylation (14.90%) and high average molecular weight (980.67kDa). All of these characteristics have contributed an excellent gelling capability of OXSP. The results might allow an improved use of PMTL as a resource of dietary fiber and low-methoxyl pectin.
4. Pectin sequentially isolated from Premna microphylla turcz leaves were dialyzed against running tap water or deionized water. It was found that dialysis solution has significant impact on appearance, final yield, ash and protein content, and surface topography of pectin. Water soluble pectin, oxalate-soluble pectin and alkali-soluble pectin formed or partial formed gels. The formation of gel would be due to low methoxyl pectin associated with calcium, which absorbed into dialysis tube when dialysis against running taping water. This deduction agrees with that a high amount of calcium and ash content were detected in these samples. The morphology of plant cell wall after each isolation step was observed. The results showed that sequential isolation steps are processes, which gradually release pectin from plant cell wall materials. The most remarkable change of morphology of cell wall material was found when ammonium oxalate were used as extracting agent, indicating ammonium oxalate is suitable for isolation of pectin in Premna microphylla turcz leaves.
5. High-methoxyl pectin was degraded by dynamic high pressure microfluidization (DHPM). It was found that apparent viscosity, average molecular weight and particle size of pectin decreased, whereas the amount of reducing sugars increased with increasing DHPM pressure. At the same time, the surface topography of pectin was changed from large flake-like structure to smaller porous chips. The mechanism of DHPM-induced degradation of pectin was also investigated. Fourier transform infrared spectra showed DHPM had no effect on the primary structure of pectin. On the other hand, reducing sugars content increased linearly with decreasing average molecular weight, suggesting the degradation may derive from the rupture of glycosidic bond. The breakdown of glycosidic bond may not only result from intensive mechanical forces but also from acid hydrolysis, which was evidenced in the reduction of degradation when the concentration of H+was lowered. In addition, neither P-elimination nor demethoxylation occurred with DHPM. Based on these results, a model was proposed to illustrate the degradation of pectin induced by DHPM.
6. Pectic-oligosaccharides (POS) were prepared from apple pectin by dynamic high-pressure microfluidization. Operating under selected conditions (pectin concentration1.84%, solution temperature63℃, DHPM pressure155MPa and number of cycles6passes),32.92%of the pectin was converted into POS. The resulting POS contains29.56%galacturonic acid and58.53%neutral sugars. The prebiotic properties of POS were then evaluated using a fecal batch culture fermentation. The POS increased the number of Bifidobacteria and Lactobacilli, and produced a higher concentration of acetic, lactic, and propionic acid than their parent pectin. Furthermore, POS decreased the number of Bacteroides and Clostridia while their parent pectin increased them. Moreover, the effects of POS on the growth of these bacteria and production of short-chain fatty acids are comparable to those of the most studied prebiotic, fructooligosaccharide. These results indicated that the POS prepared by DHPM has a potential to be an effective prebiotic.
1.采用水,草酸铵和盐酸分别提取薜荔籽中的凝胶物质,结果表明三种方法提取的主要成分均为低酯果胶,且该果胶主要分布于薜荔籽的表面,因此解释了传统“凉粉”制作工艺中采用挤压和揉搓方法的可行性。与草酸铵提和盐酸提果胶相比,水提果胶具有最高的半乳糖醛酸含量、粘均分子量和特征粘性,以及最低的甲酯化。这种水提低酯果胶为薜荔籽水提物的主要多糖组分,同时检测到水提物中存有大量的钙离子,暗示“凉粉”的自凝胶可能是基于低酯果胶的“蛋盒”模型。
2.薜荔籽水提果胶经阴离子交换柱分离,得到两组分WEP-0.3及WEP-0.4,这两个组分的溶解性表现出明显的差异性。WEP-0.3能较快溶解在水中,而约有89.5%的WEP-0.4不溶于水。该结果与红外光谱得出的WEP-0.4具有比WEP-0.3更低的酯化度,而酯化度越低,溶解性应该越好的常理相违背。扫描电镜表明WEP-0.4形成面条状的紧致结构,X-衍射分析表明WEP-0.4存在结晶区。而WEP-0.3结构比较松散且均为无定形区域,这些结果表明WEP-0.4不易溶解是因为其在透析的过程中形成了部分凝胶。凝胶形成可能是因为低酯果胶在透析过程中吸收了Ca2+,反映在金属元素分析中WEP-0.4含有更高的钙离子含量。
3.对豆腐柴叶进行组分分析得知,豆腐柴叶的细胞壁物质(AIS)占干叶质量的77.82%,AIS中总膳食纤维,不可溶性膳食纤维和半乳糖醛酸含量分别为69.45%,56.35%,23.94%。采用水,草酸铵,盐酸和氢氧化钠对AIS进行连续提取,发现草酸铵溶性果胶(OXSP)占总果胶的大部分(59.48%),其半乳糖醛酸含量为76.15%,酯化度为14.90%,平均分子量为980.67kDa.这些理化性质导致OXSP具有很好的凝胶性能。本研究能促进豆腐柴叶作为膳食纤维和低酯果胶资源而被开发利用。
4.本研究分别采用自来水和去离子水对豆腐柴叶提取的果胶进行透析,发现透析溶剂对果胶溶液外观,果胶得率,蛋白质含量及形貌产生重大的影响。水溶性果胶,草酸铵溶性果胶及碱溶性果胶经自来水透析后都形成或部分形成凝胶。这主要是由于低酯果胶在自来水透析过程中吸收钙离子形成了凝胶,这一推论与组分中Ca离子含量及灰分含量较高的结果一致。本章还考察了连续提取每个步骤后残渣的形貌,表明连续提取是个逐步释放果胶的过程,其中采用草酸铵提取后,细胞壁物质的形貌变化最大,印证草酸铵是提取豆腐柴叶果胶较为理想的提取剂。
5.采用动态高压微射流法对高酯果胶进行处理,发现高酯果胶的表观粘度,分子量和粒径随压力的升高而降低,还原糖含量则随压力的升高而升高。同时果胶的表观形貌也由块状向多孔碎片转变。傅里叶红外光谱表明动态高压微射流对果胶的基本结构没有影响,同时由于还原糖的升高和分子量的降低呈线性关系,表明果胶的降解可能源于分子内糖苷键的断裂。当H+离子浓度降低时降解得到抑制,表明这种糖苷键的断裂可能不仅仅来自于强烈的机械力作用也可能来自于酸水解作用。因此,本研究认为机械力可以打开果胶分子或者使果胶分子更加伸展,使得更多的糖苷键可以暴露给机械力和H+攻击。
6.采用动态高压微射流对苹果果胶进行降解制备果胶低聚糖,经响应曲面优化,在果胶浓度为1.84%,溶液温度63℃,微射流处理压力155MPa,循环次数6次时,果胶制备成果胶低聚糖的转化率为32.92%。所得果胶低聚糖含有29.56%的半乳糖醛酸以及58.53%的中性糖。粪便发酵实验表明,果胶低聚糖较母体果胶更能增加双歧杆菌和乳酸杆菌的数量,同时产生更多的乙酸,乳酸和丙酸。而且,果胶低聚糖能抑制拟杆菌和梭状芽胞杆菌但其母体果胶则促进这两种菌的生长。另外,果胶低聚糖对细菌生长的影响和短链脂肪酸的产生能力和最常用的益生元-低聚果糖相当。本研究结果表明动态高压微射流是制备果胶低聚糖比较适合的手段,所得果胶低聚糖可能是一种有效的新型益生元。
Pectin describes a family of structural hetropolysaccharides that have common features, but are extremely diverse in their fine structure. Pectins extracted from plants have been used widely as gelling agent, thicker, texturizer in food, pharmacy and cosmetic area. There is growing interest in low methoxyl pectin. However, LMP has to be prepared from HMP by reducing the DM through acid demethoxylation, alkali demethoxylation, or demethoxylating reaction of pectin methyl esterase. These methoxylations are complex and may cause potential depolymerization. An alternative approach for manufacturing LMP is to 'mine' plant cell walls that are enriched with LMP. Creeping fig (Ficus pumila Linn) seeds and Premna microphylla turcz leaves have been used to prepare a "Liangfen" and "Shenxian tofu", respectively by local Chinese people. However, their making secret have not been explored. Preliminary experiments indicated that both resources contain high amount of pectic polysaccharides. However, extraction, physicochemical and rheological properties of pectin was rarely reported. In this study, pectic polysaccharides were extracted from Creeping fig seeds and Premna microphylla turcz leaves using kinds of extracting agent, then the physicochemical properties and gelling capability of resulting pectin were investigated to explain why "Liangfen" and "Shenxian tofu" would be manufactured in the traditional methods. The pectin isolated from creeping fig seeds was fractionated into two components by ion exchange column, techniques such as X-ray diffraction were used to explain the reason why two fractions have significant different solublities. During investigation, we also pay attention to the effect of dialysis solution on properties of pectin, and degradation of pectin induced by dynamic high pressure microfluidization (DHPM). We found out that low methoxyl pectin tends to absorb metal ions from dialysis solution. After characterizing the extent of degradation of pectin induced by DHPM, the mechanism of degradation was discussed. This technique was also used to produce pectic oligosaccharides (POS), the resulting POS exhibit a notable prebiotic properties. Main conclusions are summarized as follows:
1. Gelling material was extracted from creeping fig seeds using water, ammonium oxalate and hydrochloric acid, respectively. Results showed the main component of three extracts is low methoxyl pectin. The pectin locates in a transparent layer on the surface of seeds, as revealed by an inverted microscope. Hence, explained the feasibility of the squeezing and rubbing method in traditional handcraft. Comparing with the other methods, water-extracted pectin has high galacturonic acid content, viscosity-average molecular weight and intrinsic viscosity but low degree of methoxylation. This pectin forms the major component of WE, together with the high amount of calcium ions present in WE, it suggests the spontaneous gelation may be based on the'egg-box'formation.
2. The water-extracted pectin (WEP) from creeping fig seeds can be fractionated into two components (WEP-0.3and WEP-0.4) by ion exchange column. These two fractions showed a notable difference in dissolution behavior, WEP-0.3can be easily dissolved into water while89.5%of WEP-0.4become insoluble after dialysis against running water and distilled water later and freeze-dried. This phenomenon was contradicted with the results of FTIR spectrum where WEP-0.4have lower degree of methoxylation should have better solubility. For WEP-0.4, partial crystal was observed by X-ray diffraction and noodle like-shape was found by environmental scanning electronic microscopy. In the case of WEP-0.3, it's surface topography was relatively loosely and it has amorphous structure. These results indicated a microgel was formed for WEP-0.4in the process of dialysis. The formation of microgel may due to the adsorption of Ca2+by WEP-0.4, as reflected in a high calcium content in WEP-0.4.
3. Premna microphylla turcz leaves (PMTL) have been used for preparing a "Shenxian tofu" by Chinese for a long history. Cell-wall material of PMTL extracted as alcohol insoluble solids (AIS) was analyzed. The AIS recovery accounted for77.82g/100g dried leaves, while the total, insoluble dietary fiber and galacturonic acid content was69.45,56.35and23.94g/100g of the AIS, respectively. A sequential extraction, using water, ammonium oxalate, hydrochloric acid and sodium hydroxide, was used to extract different pectic fractions. Oxalate-soluble pectin (OXSP) was found to be the major pectic fraction (59.48%), which contains high amounts of galacturonic acids (76.15%) with mainly homogalacturonan, and has low degree of methoxylation (14.90%) and high average molecular weight (980.67kDa). All of these characteristics have contributed an excellent gelling capability of OXSP. The results might allow an improved use of PMTL as a resource of dietary fiber and low-methoxyl pectin.
4. Pectin sequentially isolated from Premna microphylla turcz leaves were dialyzed against running tap water or deionized water. It was found that dialysis solution has significant impact on appearance, final yield, ash and protein content, and surface topography of pectin. Water soluble pectin, oxalate-soluble pectin and alkali-soluble pectin formed or partial formed gels. The formation of gel would be due to low methoxyl pectin associated with calcium, which absorbed into dialysis tube when dialysis against running taping water. This deduction agrees with that a high amount of calcium and ash content were detected in these samples. The morphology of plant cell wall after each isolation step was observed. The results showed that sequential isolation steps are processes, which gradually release pectin from plant cell wall materials. The most remarkable change of morphology of cell wall material was found when ammonium oxalate were used as extracting agent, indicating ammonium oxalate is suitable for isolation of pectin in Premna microphylla turcz leaves.
5. High-methoxyl pectin was degraded by dynamic high pressure microfluidization (DHPM). It was found that apparent viscosity, average molecular weight and particle size of pectin decreased, whereas the amount of reducing sugars increased with increasing DHPM pressure. At the same time, the surface topography of pectin was changed from large flake-like structure to smaller porous chips. The mechanism of DHPM-induced degradation of pectin was also investigated. Fourier transform infrared spectra showed DHPM had no effect on the primary structure of pectin. On the other hand, reducing sugars content increased linearly with decreasing average molecular weight, suggesting the degradation may derive from the rupture of glycosidic bond. The breakdown of glycosidic bond may not only result from intensive mechanical forces but also from acid hydrolysis, which was evidenced in the reduction of degradation when the concentration of H+was lowered. In addition, neither P-elimination nor demethoxylation occurred with DHPM. Based on these results, a model was proposed to illustrate the degradation of pectin induced by DHPM.
6. Pectic-oligosaccharides (POS) were prepared from apple pectin by dynamic high-pressure microfluidization. Operating under selected conditions (pectin concentration1.84%, solution temperature63℃, DHPM pressure155MPa and number of cycles6passes),32.92%of the pectin was converted into POS. The resulting POS contains29.56%galacturonic acid and58.53%neutral sugars. The prebiotic properties of POS were then evaluated using a fecal batch culture fermentation. The POS increased the number of Bifidobacteria and Lactobacilli, and produced a higher concentration of acetic, lactic, and propionic acid than their parent pectin. Furthermore, POS decreased the number of Bacteroides and Clostridia while their parent pectin increased them. Moreover, the effects of POS on the growth of these bacteria and production of short-chain fatty acids are comparable to those of the most studied prebiotic, fructooligosaccharide. These results indicated that the POS prepared by DHPM has a potential to be an effective prebiotic.
引文
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