改性玉米皮膳食纤维的酶法制备及其降血脂机理研究
摘要
玉米属禾本科禾亚科玉黍属植物。玉米皮由玉米种子胚乳的糊粉层、残留的胚乳厚壁组织、种皮和果皮构成。现代营养学研究证明玉米皮作为膳食纤维具有吸附诱变剂、降血脂、通便、提高免疫力等生理功能。本论文以玉米皮为原料,去除淀粉和蛋白质后制得粗玉米皮膳食纤维CDF,通过非淀粉多糖酶酶种的筛选和酶解条件的优化,对CDF进行改性,进一步提高了其降血脂活性并得到改性玉米皮膳食纤维XMF,对XMF的降血脂机理进行了深入的研究。
原料玉米皮的化学组成为:TDF 65.38%、蛋白质10.32%、淀粉17.50%、脂肪2.65%以及灰份0.75%;其膳食纤维主要由纤维素14.18%、半纤维素48.31%和木质素5.28%组成;IDF占65.38%,SDF含量0.58%。CDF的主要组成是:TDF 82.76%、蛋白质5.55%、淀粉1.26%、脂肪2.04%、以及灰份2.12%;CDF膳食纤维主要由纤维素18.82%、半纤维素50.57%和木质素8.15%组成;IDF占82.90%,SDF含量0.62%。CDF的口感优于玉米皮。
单因素实验发现纤维素酶不能提高CDF对胆酸盐的体外结合能力;木聚糖酶使CDF结合胆酸钠、鹅脱氧胆酸钠、脱氧胆酸钠和牛磺胆酸钠的能力均得到了提高;纤维素酶和木聚糖酶复合酶解也能提高CDF的BSCDC、BSDC和BSTC值,但效果不如木聚糖酶单独酶解好;玉米皮膳食纤维的胆酸盐吸附能力同持水和持油能力之间有一定关系。通过响应面分析,优化了木聚糖酶酶解提高其产物改性玉米皮膳食纤维XMF吸附胆酸盐能力的最佳条件,具体为pH 5.3、1.75 h和酶用量56 Iu/g CDF,相应的BSC、BSCDC、BSDC和BSTC值分别为64.60、48.34、75.79和60.68μmol/ g XMF,它们分别是CDF的1.88、2.34、1.67和2.08倍。XMF对胆酸钠、鹅脱氧胆酸钠、脱氧胆酸钠和牛磺胆酸钠的吸附不存在两两相关性,表明XMF对不同胆酸盐的吸附机制存在差异,不是以竟争性吸附为主。不能用XMF对某一种胆酸盐的吸附量来代表它对所有胆酸盐的吸附能力。
XMF的SDF、IDF、TDF、纤维素及木质素含量得到了显著提高(p<0.05)。XMF中膳食纤维的主要组成是半纤维素,其次是纤维素,果胶含量低。高效凝胶色谱法测得,S-XMF相对分子质量在11,588左右的部分所占比例为S-CDF的1.64倍,且其相对分子质量在900以下的含量仅达到S-CDF的62.6%。气相色谱分析发现,XMF的主要单糖组成与CDF相同;I-XMF的鼠李糖含量降低到I-CDF的44%;S-XMF中聚阿拉伯糖木糖的摩尔含量提高了约10%。可溶性半纤维素和阿拉伯木聚糖的提高能改善XMF的降血脂效果。XMF的溶胀性、持水力和持油力也都得到了显著提高(p<0.05)。
X-射线衍射和SEM观察到XMF的无定形区产生了一定程度的降解,在超微结构上表现得比CDF更无序、具有较多大的空腔和孔隙,这是为什么XMF能吸附更多的胆酸盐及油和水的结构解释。
动物实验表明,CDF和XMF均对高脂膳食引起的TC、TG上升有明显的抑制作用,使LDL-C和AI显著下降(p<0.05),XMF还能使HDL– C显著升高(p<0.05),并有效地改善肝脏脂肪状况,减轻肝细胞的脂肪化变性。因此二种膳食纤维制品都有显著的降血脂作用,但XMF的效果更突出。XMF还表现出时间效应的特点,随着饲喂时间的延长,在有效降低TG增幅的同时更能有效控制TC和LDL-C水平,使之不再升高。
RT-PCR及体内抗氧化研究发现,XMF通过有效地积极调节肝脏中与胆固醇分解代谢有关的CYP7A1和FXR,与脂肪分解代谢有关的PPARα、PPARγ、Lpl和Lpic的基因表达,并提高机体抗氧化能力,而实现对脂代谢的积极调控;但CDF只能对CYP7A1、PPARα和Lpl的基因表达起到一定的积极调节作用,其提高机体抗氧化能力的效果也不如XMF。而且XMF比CDF能更有效地调节小肠中I-BABP和FXR的基因表达,有效地减少胆汁酸的重吸收,促进肝脏中胆固醇的分解代谢。XMF在肠道中束缚鹅脱氧胆酸钠和脱氧胆酸钠的能力强于CDF。与CDF相比,XMF还能更显著地促进机体对脂肪、胆固醇和胆汁酸的排出(p<0.05)。
HPLC测定结果表明,CDF和XMF吸附胆酸盐时都存在最大吸附量。CDF中的IDF和SDF对胆酸钠、鹅脱氧胆酸钠和脱氧胆酸钠的吸附不存在增强的协同作用,对牛磺胆酸钠的吸附表现轻微的增强协同作用。但XMF中的IDF和SDF在吸附胆酸钠、鹅脱氧胆酸钠、脱氧胆酸钠和牛磺胆酸钠时均表现出强烈的增强协同作用。CDF和XMF同胆酸盐间以疏水相互作用为主。CDF对胆酸钠和牛磺胆酸钠的吸附表现为完全竞争关系,牛磺胆酸钠的竞争力强于胆酸钠;对鹅脱氧胆酸钠和脱氧胆酸钠是部分竞争性吸附,鹅脱氧胆酸钠的竞争力强于脱氧胆酸钠。XMF对胆酸钠和牛磺胆酸钠的吸附是非竞争性的;对鹅脱氧胆酸钠和脱氧胆酸钠的吸附具有部分竞争特性,脱氧胆酸钠的竞争力大于鹅脱氧胆酸钠。
胆酸钠、鹅脱氧胆酸钠、脱氧胆酸钠和牛磺胆酸钠的吸附动力学曲线表明,XMF具有更大的吸附量,更强的吸附能力。AFM力谱表明,XMF同脱氧胆酸钠和牛磺胆酸钠间的相互作用均强于CDF,进一步证明了XMF具有更强的吸附胆酸盐的能力,因而能更显著地促进机体对胆酸盐的排出,有效地起到预防和抑制高脂血症发生和发展的生物活性功能。
Maize (Zea. mays L.) is of Zea strain, a member of Graminaceae, Poaccae family. Corn bran is composed of aleurone, endosperm thick-walled tissue and seed capsule of Z. mays L.
It is reported that corn bran has many physiological benefits such as mutagens adsorption, hypolipidemic effects, relaxation etc. In this dissertation, crude dietary fiber (CDF) was prepared from corn bran after the removal of most of starch and protein through enzyme hydrolysis. The influence of non-starch enzymes on the hypolipidemic function of CDF was investigated, and xylanase modified fiber (XMF) was abtained. The hypolipidemic mechanisms of XMF were then studied in a model animal system.
The chemical composition of corn bran was analyzed and found to be as follows: TDF 65.38%, protein 10.32%, starch 17.50%, lipid 2.65% and ash 0.75%. The dietary fiber of corn bran was composed of cellulose (14.18%), hemicellulose (48.31%) and lignin (5.28%). In the corn bran, IDF and SDF content were 65.38% and 0.58%, respectively. The main composition of CDF was TDF (82.76%), protein (5.55%), starch (1.26%), lipid (2.04%) and ash (2.12%). The dietary fiber of CDF was composed of cellulose (18.82%), hemicellulose (50.57%) and lignin (8.15%). IDF and SDF content in CDF were 82.9% and 0.62%, respectively. CDF presented better acceptability of color, taste, texture and aroma than corn bran.
Factorial experiments showed that there was no improvement for the binding capacity of CDF to bile salts in vitro through cellulase hydrolysis; the binding capacity of CDF to sodium cholate, sodium chenodeoxycholate, sodium deoxycholate and sodium taurocholate in vitro was increased to the highest level following xylanase hydrolysis. The effect of the combined treatment of cellulase and xylanase was better than cellulase alone but weaker than xylanase alone. Parameters of xylanase hydrolysis were optimized by response surface methodology, that is to say, hydrolysis pH of 5.3, hydrolysis time of 1.75 h and enzyme amount of 56 Iu/g CDF. The binding capacities of XMF to sodium cholate, sodium chenodeoxycholate, sodium deoxycholate and sodium taurocholate in vitro was 64.60, 48.34, 75.79 and 60.68μmol/ g XMF under the optimized conditions. There was no correlation between the bindings of any two bile salts by XMF, which indicates that the binding mechanisms of different bile salts by XMF studied here are different.
The swelling capacity, water holding capacity and oil binding capacity of XMF were significantly higher than CDF (p<0.05). Additionally, SDF, IDF, TDF, cellulose and lignin content of XMF were also increased significantly (p<0.05). High performance gel filtration chromatography (HPGFC) analysis showed that the relative molecular weight distribution of S-XMF was mainly around 11588, which content was 1.64 fold of S-CDF, the part in S-XMF with relative molecular weight lower than 900 was only 62.6% of that in S-CDF. The neutral single sugar composition of XMF and CDF were studied through gas chromatography method. The main neutral sugar in I-CDF and I-XMF were xylose and arabinose, rhamnose in I-XMF was decreased to 44% of that in I-CDF. S-CDF and S-XMF were mainly soluble hemicellulose with high content of arabinose, xylose, mannose and glucose. In S-XMF, the mole content of every neutral single sugar was defferrent from S-CDF, and the content of arabinxylan was almost 10% higher than that in S-CDF. Soluble hemicellulose and arabinxylan can improve the hypolipidemic effects of XMF.
X-ray diffraction and SEM results suggested that the amorphous region in XMF was decomposed partially, there were more big pores in XMF, which made the structure of XMF more out-of-order with larger capacity to bind water, lipids and bile acids.
In rats with atherogenic diet induced hyperlipidemia, ingestion of XMF suppressed the increase of serum TG, TC, LDL-C and AI more significantly than CDF (p<0.05), and increased serum HDL-C level significantly at the same time (p<0.05). Furthermore, XMF significantly decreased epididymal fat, liver fat and liver cholesterol concentration more than CDF in rats (p<0.05), and then decreased the fat denaturation of liver cells.
RT-PCR studies of several genes indicated that XMF could bind more chenodeoxycholate and deoxycholate than CDF in intestine, XMF enhanced the catabolism of lipids by up-regulating the expression of hepatic CYP7A1, FXR, PPARα, PPARγ, Lpl and Lpic, down-regulating the expression of ileal I-BABP and FXR. While CDF only down-regulated I-BABP expression, up-regulated hepatic PPARα, CYP7A1 and Lpl expression. Furthermore, XMF enhanced the excretion of lipid, cholesterol and bile acids more efficiently than CDF with significance (p<0.05). The antioxydation properties of CDF and XMF were also investingated in vivo. XMF ingestion could improve rats’antioxydation capability.
For CDF and XMF, there were maximum adsorbing capacities against bile salts. There was no positive cooperation between the IDF and SDF parts in CDF during the adsorbing process of sodium cholate, sodium chenodeoxycholate or deoxycholate. There was somewhat cooperation between the two parts when adsorbing sodium taurocholate. However, the IDF and SDF parts in XMF presented strong cooperation when adsorbing the four bile salts. The main interaction between cholate and CDF/XMF was hydrophobic interaction. For CDF, the adsorption of sodium cholate and sodium taurocholate was completely competitive, sodium taurocholate was more competitive than sodium cholate; and the adsorption of sodium deoxycholate and chenodeoxycholate was partially competitive, the latter was more competitive than the former. For XMF, the adsorption of sodium cholate and taurocholate was non-competitive; while the adsorption of sodium chenodeoxycholate and sodium deoxycholate was partially competitive, sodium deoxycholate was more efficient than sodium chenodeoxycholate.
The adsorption kinetics of the four cholates indicated that there were a larger adsorbing capacity and stronger adsorbing ability for XMF. Force measurements by AFM showed that both the interactions between XMF and sodium deoxycholate, XMF and sodium taurocholate were stronger than CDF.
原料玉米皮的化学组成为:TDF 65.38%、蛋白质10.32%、淀粉17.50%、脂肪2.65%以及灰份0.75%;其膳食纤维主要由纤维素14.18%、半纤维素48.31%和木质素5.28%组成;IDF占65.38%,SDF含量0.58%。CDF的主要组成是:TDF 82.76%、蛋白质5.55%、淀粉1.26%、脂肪2.04%、以及灰份2.12%;CDF膳食纤维主要由纤维素18.82%、半纤维素50.57%和木质素8.15%组成;IDF占82.90%,SDF含量0.62%。CDF的口感优于玉米皮。
单因素实验发现纤维素酶不能提高CDF对胆酸盐的体外结合能力;木聚糖酶使CDF结合胆酸钠、鹅脱氧胆酸钠、脱氧胆酸钠和牛磺胆酸钠的能力均得到了提高;纤维素酶和木聚糖酶复合酶解也能提高CDF的BSCDC、BSDC和BSTC值,但效果不如木聚糖酶单独酶解好;玉米皮膳食纤维的胆酸盐吸附能力同持水和持油能力之间有一定关系。通过响应面分析,优化了木聚糖酶酶解提高其产物改性玉米皮膳食纤维XMF吸附胆酸盐能力的最佳条件,具体为pH 5.3、1.75 h和酶用量56 Iu/g CDF,相应的BSC、BSCDC、BSDC和BSTC值分别为64.60、48.34、75.79和60.68μmol/ g XMF,它们分别是CDF的1.88、2.34、1.67和2.08倍。XMF对胆酸钠、鹅脱氧胆酸钠、脱氧胆酸钠和牛磺胆酸钠的吸附不存在两两相关性,表明XMF对不同胆酸盐的吸附机制存在差异,不是以竟争性吸附为主。不能用XMF对某一种胆酸盐的吸附量来代表它对所有胆酸盐的吸附能力。
XMF的SDF、IDF、TDF、纤维素及木质素含量得到了显著提高(p<0.05)。XMF中膳食纤维的主要组成是半纤维素,其次是纤维素,果胶含量低。高效凝胶色谱法测得,S-XMF相对分子质量在11,588左右的部分所占比例为S-CDF的1.64倍,且其相对分子质量在900以下的含量仅达到S-CDF的62.6%。气相色谱分析发现,XMF的主要单糖组成与CDF相同;I-XMF的鼠李糖含量降低到I-CDF的44%;S-XMF中聚阿拉伯糖木糖的摩尔含量提高了约10%。可溶性半纤维素和阿拉伯木聚糖的提高能改善XMF的降血脂效果。XMF的溶胀性、持水力和持油力也都得到了显著提高(p<0.05)。
X-射线衍射和SEM观察到XMF的无定形区产生了一定程度的降解,在超微结构上表现得比CDF更无序、具有较多大的空腔和孔隙,这是为什么XMF能吸附更多的胆酸盐及油和水的结构解释。
动物实验表明,CDF和XMF均对高脂膳食引起的TC、TG上升有明显的抑制作用,使LDL-C和AI显著下降(p<0.05),XMF还能使HDL– C显著升高(p<0.05),并有效地改善肝脏脂肪状况,减轻肝细胞的脂肪化变性。因此二种膳食纤维制品都有显著的降血脂作用,但XMF的效果更突出。XMF还表现出时间效应的特点,随着饲喂时间的延长,在有效降低TG增幅的同时更能有效控制TC和LDL-C水平,使之不再升高。
RT-PCR及体内抗氧化研究发现,XMF通过有效地积极调节肝脏中与胆固醇分解代谢有关的CYP7A1和FXR,与脂肪分解代谢有关的PPARα、PPARγ、Lpl和Lpic的基因表达,并提高机体抗氧化能力,而实现对脂代谢的积极调控;但CDF只能对CYP7A1、PPARα和Lpl的基因表达起到一定的积极调节作用,其提高机体抗氧化能力的效果也不如XMF。而且XMF比CDF能更有效地调节小肠中I-BABP和FXR的基因表达,有效地减少胆汁酸的重吸收,促进肝脏中胆固醇的分解代谢。XMF在肠道中束缚鹅脱氧胆酸钠和脱氧胆酸钠的能力强于CDF。与CDF相比,XMF还能更显著地促进机体对脂肪、胆固醇和胆汁酸的排出(p<0.05)。
HPLC测定结果表明,CDF和XMF吸附胆酸盐时都存在最大吸附量。CDF中的IDF和SDF对胆酸钠、鹅脱氧胆酸钠和脱氧胆酸钠的吸附不存在增强的协同作用,对牛磺胆酸钠的吸附表现轻微的增强协同作用。但XMF中的IDF和SDF在吸附胆酸钠、鹅脱氧胆酸钠、脱氧胆酸钠和牛磺胆酸钠时均表现出强烈的增强协同作用。CDF和XMF同胆酸盐间以疏水相互作用为主。CDF对胆酸钠和牛磺胆酸钠的吸附表现为完全竞争关系,牛磺胆酸钠的竞争力强于胆酸钠;对鹅脱氧胆酸钠和脱氧胆酸钠是部分竞争性吸附,鹅脱氧胆酸钠的竞争力强于脱氧胆酸钠。XMF对胆酸钠和牛磺胆酸钠的吸附是非竞争性的;对鹅脱氧胆酸钠和脱氧胆酸钠的吸附具有部分竞争特性,脱氧胆酸钠的竞争力大于鹅脱氧胆酸钠。
胆酸钠、鹅脱氧胆酸钠、脱氧胆酸钠和牛磺胆酸钠的吸附动力学曲线表明,XMF具有更大的吸附量,更强的吸附能力。AFM力谱表明,XMF同脱氧胆酸钠和牛磺胆酸钠间的相互作用均强于CDF,进一步证明了XMF具有更强的吸附胆酸盐的能力,因而能更显著地促进机体对胆酸盐的排出,有效地起到预防和抑制高脂血症发生和发展的生物活性功能。
Maize (Zea. mays L.) is of Zea strain, a member of Graminaceae, Poaccae family. Corn bran is composed of aleurone, endosperm thick-walled tissue and seed capsule of Z. mays L.
It is reported that corn bran has many physiological benefits such as mutagens adsorption, hypolipidemic effects, relaxation etc. In this dissertation, crude dietary fiber (CDF) was prepared from corn bran after the removal of most of starch and protein through enzyme hydrolysis. The influence of non-starch enzymes on the hypolipidemic function of CDF was investigated, and xylanase modified fiber (XMF) was abtained. The hypolipidemic mechanisms of XMF were then studied in a model animal system.
The chemical composition of corn bran was analyzed and found to be as follows: TDF 65.38%, protein 10.32%, starch 17.50%, lipid 2.65% and ash 0.75%. The dietary fiber of corn bran was composed of cellulose (14.18%), hemicellulose (48.31%) and lignin (5.28%). In the corn bran, IDF and SDF content were 65.38% and 0.58%, respectively. The main composition of CDF was TDF (82.76%), protein (5.55%), starch (1.26%), lipid (2.04%) and ash (2.12%). The dietary fiber of CDF was composed of cellulose (18.82%), hemicellulose (50.57%) and lignin (8.15%). IDF and SDF content in CDF were 82.9% and 0.62%, respectively. CDF presented better acceptability of color, taste, texture and aroma than corn bran.
Factorial experiments showed that there was no improvement for the binding capacity of CDF to bile salts in vitro through cellulase hydrolysis; the binding capacity of CDF to sodium cholate, sodium chenodeoxycholate, sodium deoxycholate and sodium taurocholate in vitro was increased to the highest level following xylanase hydrolysis. The effect of the combined treatment of cellulase and xylanase was better than cellulase alone but weaker than xylanase alone. Parameters of xylanase hydrolysis were optimized by response surface methodology, that is to say, hydrolysis pH of 5.3, hydrolysis time of 1.75 h and enzyme amount of 56 Iu/g CDF. The binding capacities of XMF to sodium cholate, sodium chenodeoxycholate, sodium deoxycholate and sodium taurocholate in vitro was 64.60, 48.34, 75.79 and 60.68μmol/ g XMF under the optimized conditions. There was no correlation between the bindings of any two bile salts by XMF, which indicates that the binding mechanisms of different bile salts by XMF studied here are different.
The swelling capacity, water holding capacity and oil binding capacity of XMF were significantly higher than CDF (p<0.05). Additionally, SDF, IDF, TDF, cellulose and lignin content of XMF were also increased significantly (p<0.05). High performance gel filtration chromatography (HPGFC) analysis showed that the relative molecular weight distribution of S-XMF was mainly around 11588, which content was 1.64 fold of S-CDF, the part in S-XMF with relative molecular weight lower than 900 was only 62.6% of that in S-CDF. The neutral single sugar composition of XMF and CDF were studied through gas chromatography method. The main neutral sugar in I-CDF and I-XMF were xylose and arabinose, rhamnose in I-XMF was decreased to 44% of that in I-CDF. S-CDF and S-XMF were mainly soluble hemicellulose with high content of arabinose, xylose, mannose and glucose. In S-XMF, the mole content of every neutral single sugar was defferrent from S-CDF, and the content of arabinxylan was almost 10% higher than that in S-CDF. Soluble hemicellulose and arabinxylan can improve the hypolipidemic effects of XMF.
X-ray diffraction and SEM results suggested that the amorphous region in XMF was decomposed partially, there were more big pores in XMF, which made the structure of XMF more out-of-order with larger capacity to bind water, lipids and bile acids.
In rats with atherogenic diet induced hyperlipidemia, ingestion of XMF suppressed the increase of serum TG, TC, LDL-C and AI more significantly than CDF (p<0.05), and increased serum HDL-C level significantly at the same time (p<0.05). Furthermore, XMF significantly decreased epididymal fat, liver fat and liver cholesterol concentration more than CDF in rats (p<0.05), and then decreased the fat denaturation of liver cells.
RT-PCR studies of several genes indicated that XMF could bind more chenodeoxycholate and deoxycholate than CDF in intestine, XMF enhanced the catabolism of lipids by up-regulating the expression of hepatic CYP7A1, FXR, PPARα, PPARγ, Lpl and Lpic, down-regulating the expression of ileal I-BABP and FXR. While CDF only down-regulated I-BABP expression, up-regulated hepatic PPARα, CYP7A1 and Lpl expression. Furthermore, XMF enhanced the excretion of lipid, cholesterol and bile acids more efficiently than CDF with significance (p<0.05). The antioxydation properties of CDF and XMF were also investingated in vivo. XMF ingestion could improve rats’antioxydation capability.
For CDF and XMF, there were maximum adsorbing capacities against bile salts. There was no positive cooperation between the IDF and SDF parts in CDF during the adsorbing process of sodium cholate, sodium chenodeoxycholate or deoxycholate. There was somewhat cooperation between the two parts when adsorbing sodium taurocholate. However, the IDF and SDF parts in XMF presented strong cooperation when adsorbing the four bile salts. The main interaction between cholate and CDF/XMF was hydrophobic interaction. For CDF, the adsorption of sodium cholate and sodium taurocholate was completely competitive, sodium taurocholate was more competitive than sodium cholate; and the adsorption of sodium deoxycholate and chenodeoxycholate was partially competitive, the latter was more competitive than the former. For XMF, the adsorption of sodium cholate and taurocholate was non-competitive; while the adsorption of sodium chenodeoxycholate and sodium deoxycholate was partially competitive, sodium deoxycholate was more efficient than sodium chenodeoxycholate.
The adsorption kinetics of the four cholates indicated that there were a larger adsorbing capacity and stronger adsorbing ability for XMF. Force measurements by AFM showed that both the interactions between XMF and sodium deoxycholate, XMF and sodium taurocholate were stronger than CDF.
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