条斑紫菜多糖提取及物理降解研究
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摘要
紫菜是中国重要的经济藻类,其多糖具有多重药理活性,提取工艺及增值化技术是影响紫菜多糖利用的关键问题。本研究以条斑紫菜为原料,探讨了紫菜多糖(PSPY)的制备技术、理化性质、及其超声降解和微波降解技术。主要研究工作与结论如下:
(1)对条斑紫菜中蛋白质、脂肪、总糖等营养成分进行了测定。采用Plackett-Burman设计法,从9个相关因素中筛选出了影响条斑紫菜多糖提取率的主要影响因子,分别为浸提温度(P=0.0017),浸提时间(P=0.0043)和料液比(P=0.0180)。
(2)通过二次回归通用旋转组合设计对提取工艺进行研究,得到了多糖得率最高的萃取条件为料液比1.16:100(g/mL)、时间4.7h、温度100℃;多糖纯度最高的萃取条件为料液比1.16:100(g/mL)、时间1.3h、温度73.2℃。在试验范围内,影响紫菜多糖得率的顺序为温度＞料液比＞时间,对多糖纯度影响顺序为料液比＞时间＞温度。经试验验证,回归模型预测性良好。
(3)在得率优化条件下获得PSPY的得率19.5%,纯度94.5%,硫酸基含量7.2%,3,6-AG含量为12.3%,单糖组成为Gal、Xyl、Fuc、Ara,摩尔比为:46:2:1:1。水溶液中,25℃时特性粘度为3.38dL/g;PSPY等当点为7.15。抗肿瘤细胞研究发现,PSPY在低浓度时(＜200μg/mL),对293细胞有一定的促进生长作用,而在浓度相对较高时(＞200μg/mL),对293细胞有抑制作用;对SGC-7901和95D细胞有一定的抑制作用,在考察浓度范围内最大抑制达到20%;但对U937细胞有明显的促进生长作用,在浓度为1000μg/mL时,生长促进作用达到26.82%。抗氧化研究表明,PSPY清除超氧负离子EC_(50)=0.20mg/mL,清除羟基自由基EC_(50)=1.32mg/mL,同时都有较好的对数量效关系(r＞0.95)。对PSPY进行分级分离获得三个均一性组分PSPY3、PSPY4和PSPY5,其分子量分别为2.2×10~5 Da、3.9×10~5Da和6.1×10Da,硫酸基含量分别为7.4%、10.5%和7.9%,单糖组成及摩尔比分别为:25.8(Gal):1.1(Ara):1(Fuc),27(Gal):1(Ara),PSPY5为Gal。
(4)通过超声降解研究,建立了相应的Mark-Houwink方程,单因素考察发现超声功率的增大及体系温度的增加均有利于PSPY超声降解作用增强;浓度的影响不是单调变化的,在0.75g/dL时降解较快。建立了不同温度下描述反应速率常数随反应时间变化的PSPY超声降解动力学方程,发现反应速率常数与反应时间之间呈显著的指数关系。以动力学方程为基础,通过Arrhenius方程,计算出PSPY超声降解反应的活化能为52.13kJ/mol。PSPY特性粘度与超声处理时间之间有指数关系,且从理论推导进行了验证。在结构方面,降解产物——超声降解后紫菜多糖(UD-PSPY)的分子量分布发生明显变化,向低分子量迁移,但红外光谱特征未出现变化。在抗肿瘤活性方面,UD-PSPY抗SGC-7901和U937肿瘤细胞效果极显著提高,而抗95D和293细胞效果没有明显变化;在抗氧化方面,UD-PSPY清除超氧负离子(UD-PSPY:EC_(50)=0.14mg/mL,PSPY:EC_(50)=0.20mg/mL)和清除羟基自由基(UD-PSPY:EC_(50)=0.59mg/mL,PSPY:EC_(50)=1.32mg/mL)有显著提高。
(5)从UD-PSPY中获得了三个均一性组分F2,F3和F4,分子量分别为2.1×10~5Da,3.1×10~5Da和4.0×10~5Da,硫酸基含量分别为7.6%,10.3%和11.7%,单糖组成均为Gal、Ara、Fuc,其摩尔比分别为:48.5:1.2:1、39.0:1.1:1和36.4:1.1:1。经红外光谱和核磁共振分析,结果其分子骨架中的重复结构单元分别为:F2为[G-A]和[G-L6S];F3为[G-A]、[G-L6S]和[G-A2M];F4为[G-A]、[G-L6S]和[G-A2 or 6M]。抗氧化研究发现,清除超氧负离子能力大小顺序为:F4＞F3＞F2＞Vc;清除羟基自由基能力大小顺序为:F2＞F3＞F4＞Mannitol;螯合能力大小顺序为:EDTA＞F4＞F3＞F2;还原力大小顺序为:Vc＞F2＞F3＞F4。从构效角度推测,在清除超氧负离子或螯合能力方面,多糖中的硫酸基含量可能起主要作用;在清除羟基自由基或还原力方面,多糖的分子量大小起主导作用。
(6)进行了PSPY的微波降解试验研究。发现该微波系统的响应时间约为4s,工作体积在20～40mL范围内,经t检验温度变化率没有显著差异。采用恒温比较法以美拉德反应(MR)作为模型反应对微波加热的非热效应进行了探讨,通过褐变程度、紫外可见光光谱、同步荧光光谱及GC-MS分析发现,在本实验条件下,微波加热具有非热效应,且可推测微波只加速了MR的进程,并没有改变其反应途径。PSPY微波降解试验表明,在考察实验范围内,微波本身对PSPY没有降解作用,但微波辅助H_2O_2降解PSPY有明显提高。随着H_2O_2添加量的增加,降解作用增强,但添加量为2%和4%之间的降解作用差异较小;随着微波功率的增大,微波辅助降解作用更加明显。PSPY微波降解试验同时证实了微波加热中存在一定的非热效应。
Porphyra is an impotant and economical alga in China.Its polysaccharides possess multi-pharmaceutic activities,and extractive and value-added technologies of polysaccharides from porphyra are key problems in the deep processing.Preparation technology,physicochemical property,ultrasonic degradation(kinetics and modification),microwave degradation and its non-thermal effects of polysaccharide from Porphyra yezosensis(PSPY) were studied.The detail contents and conclusions are as follows.
The contents of protein,fat and total sugar in P.yezoensis were determined.The Plackett-Burman design method was used to the screening of the main impact factors on the extraction rate of P.yezoensis polysaccharide,and results are extraction temperature (P=0.0017),extraction time(P=0.0043),ratio of sample to solvent(P=0.0180).
Extracting technology of polysaccharide was designed and carried out in the method of quadratic regression rotation design.The optimal condition with rather higher rate(purity) for extraction of polysaccharide are determined as follows:ratio of sample to solvent 1.16:100(1.16:100)(g/mL),extraction time 4.7(1.3) h and extraction temperature 100(73.2)℃.In the present test,the order for affecting rate in the extraction is extraction temperature＞ratio of sample to solvent＞extraction time,and so the corresponding order for purity is ratio of sample to solvent＞extraction time＞extraction temperature.The regression model was effective and predictability, which was confirmed by experimental verification.
PSPY was obtained in the optimal condition(with rather higher rate),the rate is 19.5%,purity is 94.5%,sulfated group is 7.2%,3,6-AG is 12.3%,and monosaccharide composition is Gal、Xyl、Fuc、Ara,in the molar ratio of 46:2:1:1,respectively.The intrinsic viscosity of PSPY in distilled water at 25℃is 3.38dL/g.,Test with maillard reaction,browing is rather higher in acid condition than in the alkaline one,the least is in neutral condition,this may be affect by hydrolysis of PSPY.Equivalent point of PSPY is 7.15.PSPY has inhibition retrogradation of starch.Effects of PSPY on tumor cells growth were investigated.Results are as follows:in rather low concentration(＜200μg/mL),PSPY has a certainty promotion on cell 293,but in rather high concentration(＞200μg/mL) PSPY has a certainty inhibition on cell 293.PSPY also has a certainty inhibition on cell SGC-7901and 95D,the highest inhibition activity as much as 20%.As cell U937 is concerned,PSPY has markedly promotion effect,and in concentration 1000μg/mL,the promotion activity is up to 26.82%.Antioxidation activity of PSPY was also studied.EC_(50) of scavenging superoxide anion radical and scavenging hydroxyl radical is 0.18mg/mL and 1.32mg/mL,respectively,and good (r＞0.95) logarithmic activity-dose relationship was found here.Three homogeneity fractions were obtained,and molecular weight is 2.2×10~5 Da、3.9×10~5 Da and 6.1×10~5 Da,sulfated group content is 7.4%、10.5%and 7.9%,monosaccharide composition in the molar ratio of PSPY3(Gal:Ara:Fuc,25.8:1.1:1),PSPY4(Gal:Ara,27:1), PSPY5(Gal),respectively.
Ultrasound degradation of PSPY was systematic studied.The Mark-Houwink equation was also extablished firstly,then,augmentation of power and enhance temperature can promote the degradation,and effect of concentration on the degradation is not monotony,the rather higher degradation is at concentration of 0.75g/dL,these were concluded from single factor investigation.Remarkable exponential relation between reaction rate constant and reaction time was found from dynamics equation established in different temperature conditions.Activation energy of the degradation is 52.13kJ/mol,which was estimated according to Arrhenius and dynamics equations. There is exponential relation between intrinsic viscosity of PSPY and reaction time, which was validated from theoretical derivation.Further study,as structure of UD-PSPY(Ultrasound degradation of PSPY) is concerned,distribution of molecular weight was notability altered and moved to the rather lower.As for anti-tumor activity of cell SGC-7901 and U937,the activities were observably improved,but the effects on activities for 95D and 293 were not too obviously changed.As about antioxidation, scavenging superoxide anion radical(UD-PSPY:EC_(50)=0.14mg/mL,PSPY:EC_(50)= 0.20mg/mL) and scavenging hydroxyl radical(UD-PSPY:EC_(50)=0.59mg/mL,PSPY: EC_(50)=1.32mg/mL) were also notedly improved.
Three homogeneity fractions(F2,F3 and F4) were obtained from UD-PSPY, molecular weight is 2.1×10~5Da,3.1×10~5 Da and 4.0×10~5 Da,sulfated group content is 7.6%,10.3%and 11.7%,monosaccharide composition in the molar rato of(Gal、Ara、Fuc)F2(48.5:1.2:1)、F3(39.0:1.1:1) and F4(36.4:1.1:1),respectively.It has a linear backbone of alternating[G-A]and[G-L6S]in F2,[G-A]and[G-L6S]and[G-A2M]in F3,[G-A]and[G-L6S]and[G-A2or6M]in F4,which were analyzed by FT-IR and NMR spectroscopies.The order of activity scavenging superoxide anion radical is F4＞F3＞F2＞Vc,activity scavenging hydroxyl radical is F2＞F3＞F4＞Mannitol,chelating ability is EDTA＞F4＞F3＞F2,reducing power is Vc＞F2＞F3＞F4,respectively.Decrease of molecular weight was dominant reason for increases on activities of scavenging hydroxyl radical and reducing power of F1,F2 and F3,and increase of sulfate content in molecule was dominant reason for increases on activities of scavenging superoxide anion radical and chelating ferrous ions.
Parameters of the microwave reaction instrument were mensurated.The apparatus log time is about 4s,and there is no markedly difference in rate of temperature increasing was found between volumes between 20mL to 40mL,which was confirmed by t test check.The non-thermal effects in microwave reaction were investigated by constant temperature-compare methord adopting MR as model reaction.Non-thermal effect(accelerating the MR,without changing the reaction path) in microwave reaction was found in the present study,which was authenticated and confirmed by browning, spectrum of ultraviolet-visible scanning,spectrum of synchronous fluorescent scanning and GC-MS analyses.There is no degradation for microwave on PSPY,but it can accelerate degradation of H_2O_2 on PSPY.As content of H_2O_2 is concerned,increasing of H_2O_2 content can promote the degradation,but up to 2%and 4%,the increasing action is very weak.Increasing microwave anode current can promote the degradation of H_2O_2 on PSPY.Non-thermal effect in microwave reaction was also validated in the degradation of H_2O_2 on PSPY.
(1)对条斑紫菜中蛋白质、脂肪、总糖等营养成分进行了测定。采用Plackett-Burman设计法,从9个相关因素中筛选出了影响条斑紫菜多糖提取率的主要影响因子,分别为浸提温度(P=0.0017),浸提时间(P=0.0043)和料液比(P=0.0180)。
(2)通过二次回归通用旋转组合设计对提取工艺进行研究,得到了多糖得率最高的萃取条件为料液比1.16:100(g/mL)、时间4.7h、温度100℃;多糖纯度最高的萃取条件为料液比1.16:100(g/mL)、时间1.3h、温度73.2℃。在试验范围内,影响紫菜多糖得率的顺序为温度＞料液比＞时间,对多糖纯度影响顺序为料液比＞时间＞温度。经试验验证,回归模型预测性良好。
(3)在得率优化条件下获得PSPY的得率19.5%,纯度94.5%,硫酸基含量7.2%,3,6-AG含量为12.3%,单糖组成为Gal、Xyl、Fuc、Ara,摩尔比为:46:2:1:1。水溶液中,25℃时特性粘度为3.38dL/g;PSPY等当点为7.15。抗肿瘤细胞研究发现,PSPY在低浓度时(＜200μg/mL),对293细胞有一定的促进生长作用,而在浓度相对较高时(＞200μg/mL),对293细胞有抑制作用;对SGC-7901和95D细胞有一定的抑制作用,在考察浓度范围内最大抑制达到20%;但对U937细胞有明显的促进生长作用,在浓度为1000μg/mL时,生长促进作用达到26.82%。抗氧化研究表明,PSPY清除超氧负离子EC_(50)=0.20mg/mL,清除羟基自由基EC_(50)=1.32mg/mL,同时都有较好的对数量效关系(r＞0.95)。对PSPY进行分级分离获得三个均一性组分PSPY3、PSPY4和PSPY5,其分子量分别为2.2×10~5 Da、3.9×10~5Da和6.1×10Da,硫酸基含量分别为7.4%、10.5%和7.9%,单糖组成及摩尔比分别为:25.8(Gal):1.1(Ara):1(Fuc),27(Gal):1(Ara),PSPY5为Gal。
(4)通过超声降解研究,建立了相应的Mark-Houwink方程,单因素考察发现超声功率的增大及体系温度的增加均有利于PSPY超声降解作用增强;浓度的影响不是单调变化的,在0.75g/dL时降解较快。建立了不同温度下描述反应速率常数随反应时间变化的PSPY超声降解动力学方程,发现反应速率常数与反应时间之间呈显著的指数关系。以动力学方程为基础,通过Arrhenius方程,计算出PSPY超声降解反应的活化能为52.13kJ/mol。PSPY特性粘度与超声处理时间之间有指数关系,且从理论推导进行了验证。在结构方面,降解产物——超声降解后紫菜多糖(UD-PSPY)的分子量分布发生明显变化,向低分子量迁移,但红外光谱特征未出现变化。在抗肿瘤活性方面,UD-PSPY抗SGC-7901和U937肿瘤细胞效果极显著提高,而抗95D和293细胞效果没有明显变化;在抗氧化方面,UD-PSPY清除超氧负离子(UD-PSPY:EC_(50)=0.14mg/mL,PSPY:EC_(50)=0.20mg/mL)和清除羟基自由基(UD-PSPY:EC_(50)=0.59mg/mL,PSPY:EC_(50)=1.32mg/mL)有显著提高。
(5)从UD-PSPY中获得了三个均一性组分F2,F3和F4,分子量分别为2.1×10~5Da,3.1×10~5Da和4.0×10~5Da,硫酸基含量分别为7.6%,10.3%和11.7%,单糖组成均为Gal、Ara、Fuc,其摩尔比分别为:48.5:1.2:1、39.0:1.1:1和36.4:1.1:1。经红外光谱和核磁共振分析,结果其分子骨架中的重复结构单元分别为:F2为[G-A]和[G-L6S];F3为[G-A]、[G-L6S]和[G-A2M];F4为[G-A]、[G-L6S]和[G-A2 or 6M]。抗氧化研究发现,清除超氧负离子能力大小顺序为:F4＞F3＞F2＞Vc;清除羟基自由基能力大小顺序为:F2＞F3＞F4＞Mannitol;螯合能力大小顺序为:EDTA＞F4＞F3＞F2;还原力大小顺序为:Vc＞F2＞F3＞F4。从构效角度推测,在清除超氧负离子或螯合能力方面,多糖中的硫酸基含量可能起主要作用;在清除羟基自由基或还原力方面,多糖的分子量大小起主导作用。
(6)进行了PSPY的微波降解试验研究。发现该微波系统的响应时间约为4s,工作体积在20～40mL范围内,经t检验温度变化率没有显著差异。采用恒温比较法以美拉德反应(MR)作为模型反应对微波加热的非热效应进行了探讨,通过褐变程度、紫外可见光光谱、同步荧光光谱及GC-MS分析发现,在本实验条件下,微波加热具有非热效应,且可推测微波只加速了MR的进程,并没有改变其反应途径。PSPY微波降解试验表明,在考察实验范围内,微波本身对PSPY没有降解作用,但微波辅助H_2O_2降解PSPY有明显提高。随着H_2O_2添加量的增加,降解作用增强,但添加量为2%和4%之间的降解作用差异较小;随着微波功率的增大,微波辅助降解作用更加明显。PSPY微波降解试验同时证实了微波加热中存在一定的非热效应。
Porphyra is an impotant and economical alga in China.Its polysaccharides possess multi-pharmaceutic activities,and extractive and value-added technologies of polysaccharides from porphyra are key problems in the deep processing.Preparation technology,physicochemical property,ultrasonic degradation(kinetics and modification),microwave degradation and its non-thermal effects of polysaccharide from Porphyra yezosensis(PSPY) were studied.The detail contents and conclusions are as follows.
The contents of protein,fat and total sugar in P.yezoensis were determined.The Plackett-Burman design method was used to the screening of the main impact factors on the extraction rate of P.yezoensis polysaccharide,and results are extraction temperature (P=0.0017),extraction time(P=0.0043),ratio of sample to solvent(P=0.0180).
Extracting technology of polysaccharide was designed and carried out in the method of quadratic regression rotation design.The optimal condition with rather higher rate(purity) for extraction of polysaccharide are determined as follows:ratio of sample to solvent 1.16:100(1.16:100)(g/mL),extraction time 4.7(1.3) h and extraction temperature 100(73.2)℃.In the present test,the order for affecting rate in the extraction is extraction temperature＞ratio of sample to solvent＞extraction time,and so the corresponding order for purity is ratio of sample to solvent＞extraction time＞extraction temperature.The regression model was effective and predictability, which was confirmed by experimental verification.
PSPY was obtained in the optimal condition(with rather higher rate),the rate is 19.5%,purity is 94.5%,sulfated group is 7.2%,3,6-AG is 12.3%,and monosaccharide composition is Gal、Xyl、Fuc、Ara,in the molar ratio of 46:2:1:1,respectively.The intrinsic viscosity of PSPY in distilled water at 25℃is 3.38dL/g.,Test with maillard reaction,browing is rather higher in acid condition than in the alkaline one,the least is in neutral condition,this may be affect by hydrolysis of PSPY.Equivalent point of PSPY is 7.15.PSPY has inhibition retrogradation of starch.Effects of PSPY on tumor cells growth were investigated.Results are as follows:in rather low concentration(＜200μg/mL),PSPY has a certainty promotion on cell 293,but in rather high concentration(＞200μg/mL) PSPY has a certainty inhibition on cell 293.PSPY also has a certainty inhibition on cell SGC-7901and 95D,the highest inhibition activity as much as 20%.As cell U937 is concerned,PSPY has markedly promotion effect,and in concentration 1000μg/mL,the promotion activity is up to 26.82%.Antioxidation activity of PSPY was also studied.EC_(50) of scavenging superoxide anion radical and scavenging hydroxyl radical is 0.18mg/mL and 1.32mg/mL,respectively,and good (r＞0.95) logarithmic activity-dose relationship was found here.Three homogeneity fractions were obtained,and molecular weight is 2.2×10~5 Da、3.9×10~5 Da and 6.1×10~5 Da,sulfated group content is 7.4%、10.5%and 7.9%,monosaccharide composition in the molar ratio of PSPY3(Gal:Ara:Fuc,25.8:1.1:1),PSPY4(Gal:Ara,27:1), PSPY5(Gal),respectively.
Ultrasound degradation of PSPY was systematic studied.The Mark-Houwink equation was also extablished firstly,then,augmentation of power and enhance temperature can promote the degradation,and effect of concentration on the degradation is not monotony,the rather higher degradation is at concentration of 0.75g/dL,these were concluded from single factor investigation.Remarkable exponential relation between reaction rate constant and reaction time was found from dynamics equation established in different temperature conditions.Activation energy of the degradation is 52.13kJ/mol,which was estimated according to Arrhenius and dynamics equations. There is exponential relation between intrinsic viscosity of PSPY and reaction time, which was validated from theoretical derivation.Further study,as structure of UD-PSPY(Ultrasound degradation of PSPY) is concerned,distribution of molecular weight was notability altered and moved to the rather lower.As for anti-tumor activity of cell SGC-7901 and U937,the activities were observably improved,but the effects on activities for 95D and 293 were not too obviously changed.As about antioxidation, scavenging superoxide anion radical(UD-PSPY:EC_(50)=0.14mg/mL,PSPY:EC_(50)= 0.20mg/mL) and scavenging hydroxyl radical(UD-PSPY:EC_(50)=0.59mg/mL,PSPY: EC_(50)=1.32mg/mL) were also notedly improved.
Three homogeneity fractions(F2,F3 and F4) were obtained from UD-PSPY, molecular weight is 2.1×10~5Da,3.1×10~5 Da and 4.0×10~5 Da,sulfated group content is 7.6%,10.3%and 11.7%,monosaccharide composition in the molar rato of(Gal、Ara、Fuc)F2(48.5:1.2:1)、F3(39.0:1.1:1) and F4(36.4:1.1:1),respectively.It has a linear backbone of alternating[G-A]and[G-L6S]in F2,[G-A]and[G-L6S]and[G-A2M]in F3,[G-A]and[G-L6S]and[G-A2or6M]in F4,which were analyzed by FT-IR and NMR spectroscopies.The order of activity scavenging superoxide anion radical is F4＞F3＞F2＞Vc,activity scavenging hydroxyl radical is F2＞F3＞F4＞Mannitol,chelating ability is EDTA＞F4＞F3＞F2,reducing power is Vc＞F2＞F3＞F4,respectively.Decrease of molecular weight was dominant reason for increases on activities of scavenging hydroxyl radical and reducing power of F1,F2 and F3,and increase of sulfate content in molecule was dominant reason for increases on activities of scavenging superoxide anion radical and chelating ferrous ions.
Parameters of the microwave reaction instrument were mensurated.The apparatus log time is about 4s,and there is no markedly difference in rate of temperature increasing was found between volumes between 20mL to 40mL,which was confirmed by t test check.The non-thermal effects in microwave reaction were investigated by constant temperature-compare methord adopting MR as model reaction.Non-thermal effect(accelerating the MR,without changing the reaction path) in microwave reaction was found in the present study,which was authenticated and confirmed by browning, spectrum of ultraviolet-visible scanning,spectrum of synchronous fluorescent scanning and GC-MS analyses.There is no degradation for microwave on PSPY,but it can accelerate degradation of H_2O_2 on PSPY.As content of H_2O_2 is concerned,increasing of H_2O_2 content can promote the degradation,but up to 2%and 4%,the increasing action is very weak.Increasing microwave anode current can promote the degradation of H_2O_2 on PSPY.Non-thermal effect in microwave reaction was also validated in the degradation of H_2O_2 on PSPY.
引文
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