阿拉伯木聚糖的功能性质及其应用研究
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摘要
全麦食品富含膳食纤维、维生素、矿物质及多种植物营养素。经常食用全麦食品会降低多种慢性病的发生率。全麦食品中全麦面包是西方国家首选的主食,是补充膳食纤维及多种营养素的最佳途径。在我国,由于全麦面包结构粗糙,消费者接受程度差,因而消费量受到极大的限制。面筋蛋白、阿拉伯木聚糖(Arabinoxylan,AX)及它们之间的相互作用对全麦面团的流变性质和全麦面包品质有很大影响。因此研究AX的功能性质,能改善全麦面包质构,提高民众健康水平,具有重要的理论意义和实用价值。本论文以全麦面粉为原料,优化水不溶性阿拉伯木聚糖(Water unextractable arabinoxylan,WUAX)的提取工艺,制得高纯度的水溶性阿拉伯木聚糖(Water extractable arabinoxylan,WEAX)和WUAX;研究了WEAX及WUAX的氧化胶凝机理以及AX与面筋蛋白之间的相互作用;研究了氧化酶在实际体系中的应用。
从全麦粉中提取纯化得到纯度较高的WEAX,相对重均分子质量为414,459。其单糖组成为阿拉伯糖42.60%,木糖51.55%,葡萄糖3.89%,甘露糖1.96%。FA含量较低为0.25%。
采用Ba(OH)2提取WUAX,优化了提取工艺。碱提WUAX预处理结果发现高压蒸煮法可提高WUAX的提取率及提取物中WUAX的含量。Ba(OH)2提取WUAX的最佳工艺条件为:料液比1:188,提取温度63℃,提取时间3h,提取率为31.60%;方差分析结果显示各因素对提取率影响的模型显著(P<0.05),提取温度的平方项对提取率的影响高度显著(P<0.001);最佳工艺条件下得到的提取物中WUAX含量为92.09%。
采用毛细管黏度法测定漆酶(laccase,LAC)与氧化酶体系POX/H2O2体系、LOX/LA体系及化学氧化剂KBrO3、ADA、BPO及过硫酸铵对AX粘度的影响,结果表明LAC较其他两种氧化酶体系和四种化学氧化剂更有效的作用于AX,使AX黏度明显增加。添加氧化酶前后,WEAX和WUAX的重均相对分子质量分布测定结果表明,加入氧化酶后WEAX和WUAX的重均相对分子质量均增加,且WUAX增加更明显。加入LAC时,重均相对分子质量增加最为明显。这说明LAC催化AX氧化交联能力较强。加入LAC后,AX中阿魏酸(Ferulic acid,FA)含量降低较明显,证明在LAC的作用下,更多的FA参与了氧化交联。在AX和LAC混合体系中加入游离的FA,反应1h后经反相高效液相色谱法(RP-HPLC)测定混合物中游离FA的含量,结果表明,游离的FA,在LAC的作用下,参与形成聚合物,因此WEAX在LAC的作用下可通过FA产生氧化交联。在AX和LAC混合体系中加入富马酸时,WEAX及WUAX溶液中FA浓度与未加入富马酸时FA浓度下降速度差别不大。说明富马酸不参与LAC催化的AX的氧化交联反应。而加入香草酸时,溶液中FA较未加入时降低速度明显加快。且随香草酸浓度的增大,FA降低速度加快。说明加入香草酸对AX的氧化交联反应有影响。进而证明了AX的氧化交联中心为酚羟基而非双键。
采用蠕变试验与小振幅振荡动态流变性质的测定相结合,研究WEAX和WUAX对面筋蛋白黏弹性的影响,结果表明面筋蛋白(gluten)、面筋蛋白与WEAX混合体系(G-WEAX)及面筋蛋白与WUAX的混合体系(G-WUAX)当NaCl浓度为4.0%(w/v)时,面筋蛋白、G-WEAX黏弹性最大;G-WUAX在NaCl浓度为3.0%(w/v)时弹性最大,在NaCl浓度为4.0%(w/v)时黏度最大。当加水量为50%时,面筋蛋白及G-WEAX和G-WUAX混合体黏弹性最佳。WEAX和WUAX可显著提高面筋蛋白的黏弹性。加入WEAX和WUAX后,面筋蛋白的LMWG中自由巯基的数量明显减少,这是面筋蛋白黏弹性增大的原因之一。
采用荧光各向异性法研究了AX对醇溶蛋白和麦谷蛋白荧光各向异性的影响,结果表明,麦谷蛋白中加入AX,其各向异性值显著提高,说明AX与麦谷蛋白分之间存在相互作用。
采用13C NMR法研究AX和面筋蛋白的相互作用,结果表明麦谷蛋白中加入WEAX时化学位移δ为136.5和128.8ppm的吸收峰(归属于芳香烃类化合物),分别向低场强方向移动至137.6ppm和129.9ppm;当加入WUAX时,化学位移δ为136.5和128.8ppm的吸收峰也分别向低场强方向移动至137.8ppm和130.1ppm。对于醇溶蛋白,当加入WEAX或WUAX时,未发现化学位移的变化。结果表明AX与面筋蛋白的相互作用主要是由AX分子侧链上的阿魏酸与麦谷蛋白分子上的酪氨酸基团相互作用引起的。
采用小振幅动态流变试验测定面团的黏弹性,面包体积测定、感官评定法与质构测定法相结合对面包品质进行综合评价,并对面包进行了贮存试验,结果表明LAC可提高普通面团和全麦面团的黏弹性,对全麦面团的作用更强。全麦面包的比容普遍小于普通面包,全麦面包中加入LOX和LAC能显著增加全麦面包的比容,并以LAC提高比容最为明显。当加入LAC时,评分员对全麦面包切面的组织结构、口感的评分的平均值较对照面包提高较大,说明LAC对全麦面包质构的改善作用最明显,且能改善全麦面包的风味。质构测定结果表明当加入LOX和LAC的普通面包硬度较对照面包减小。加入LAC的面包弹性增大和硬度降低较明显。说明LAC对全麦面包质构的改善作用最强。面包贮存过程中面包心硬度的变化和面包中水分含量的变化测定表明,加入LAC的全麦面包经贮存7天后硬度变化较小,水分下降速度较全麦面包对照面包明显减慢,说明LAC可明显延长面包的老化时间。
Whole meal foods are rich in dietary fiber, vitamin, minerals and various phytonutrients. Regular consumption of whole meal food will reduce the incidence of multiple chronic diseases. Whole meal bread as the staple food in western countries, is the best way to supplement the dietary fiber and a variety of nutrients. In our country, the coarse structure of whole meal bread leads to poor acceptance of the consumer and tremendous restrictions of the consumption. Gluten protein, arabinoxylan (AX) and their interactions have great influence on rheological properties of the whole meal dough and quality of the whole meal bread. The texture of whole meal bread can be improved and national health can be siginificantly enhanced by studying the functional properties of AX, which has important theoretical and practical value.
In this paper, whole wheat flour was used as material, the extraction process of water unextractable arabinoxylan (WUAX) was optimized, and the high purity water extractable arabinoxylan (WEAX) and WUAX was obtained. The oxidative gelation mechanism of WEAX and WUAX as well as the interaction between gluten protein and AX was studied.
The relative weight-average molecular weight of extracted WEAX is 414, 459 and WEAX is composed of 42.60% of arabinose, 51.55% of xylose, 3.89% of glucose and 1.96% of mannose. WEAX contains 0.25% of ferulic acid (FA), which play an important role in the functional properties of WEAX. The pretreatment results of extraction of WUAX by Ba(OH)2 showed that high-pressure treatment can increase extracion yield of the WUAX and content of WUAX in the extract as well. The optimum conditions of extracting WUAX by Ba(OH)2 are as follows: WUS (the water unextractable substance that removed protein and starch):Ba(OH)2 is 1:188(w/v), the extraction temperature is 63°C, the extraction time is 3h, the extraction rate is 31.60%. Variance analysis showed that the yield factors model was significant (P<0.05), and the square of the extraction temperature highly significant (P<0.001). Under optimum conditions, WUAX contents in the extract is 92.09%.
Laccase (LAC) increase the viscosity of AX more obviously comparing with POX/H2O2, LOX/LA and chemical oxidants KBrO3, azodicarbonamide(ADA), benzoyl peroxide (BPO) and (NH4)2S2O8. When added LAC, the average weight molecular weight increased most obviously, which indicated that LAC can catalyze oxidation crosslinking more efficiently. By adding LAC, the FA of AX decreased more, which indicated that more FA involved in the oxidative cross-linking of AX. The free FA was added in the mixture of AX and LAC, the content of free FA in the mixture was measured after 1 h reaction. The results show that the free FA decreased and participated in the oxiditave cross-linking of AX, which means that AX cross-link through FA. When fumaric acid was added in the mixture of AX and LAC, concentrations of FA was almost no change, which indicated that the site of oxidation of AX was not double bond of FA structure. While by adding vanillic acid, FA in the solutions reduced and and with the increasing concentration of the vanillic acid, FA reduces more. To draw a conclusion, the site of oxidative crosslinking of AX was benzene ring and not double bond.
Studies on the effect of WEAX and WUAX on viscoelasticity of gluten by combination of creep tests and dynamic small amplitude oscillatory measurements showed that WEAX and WUAX can significantly increase the viscoelasticity of gluten. When NaCl concentration was 4.0% (w/v), gluten and gluten-WEAX mixtures (G-WEAX) showed the largest viscoelastic. The gluten-WUAX mixtures (G-WUAX) showed the maximum elasticty at 3.0% (w/v) NaCl concentration and the maximum viscosity at 4.0% (w/v) NaCl concentration.The viscoelasticity of gluten, G-WEAX and G-WUAX reached the maximum at 50% water level. The free sulfhydryl of LMWG obviously reduced by adding WEAX and WUAX, which is one of the reason that the viscoelasticity gluten was increased by adding WEAX and WUAX.
The fluorescence anisotropy measurements of gliadin and glutenin by adding AX showed that anisotropy value of glutenin significantly increased when AX was add in, indicating that AX interacted with glutenin.
The ineractions of gluten proteins and G-AX determined by 13C NMR spectrum showed that the glutenin absorption peaks at chemical shift (δ) 136.5 and 128.8ppm (both were the absorption peaks of benzene ring) shifted to 137.6ppm and 129.9ppm, respectively by adding WEAX and shifted to 137.8ppm and130.1ppm, respectively by adding WUAX. For gliadin, there was no chemical shift by adding WEAX or WUAX. The results indicated that AX interacted with glutenin by covalently bond FA to tyrosine of glutenin.
Studies on the effect of LAC, POX/H2O2 and LOX/LA on dough rheological properties showed that LAC most significantly improved the ordinary and the whole meal dough viscoelasticity and more significantly improved the viscoelasticity of the latter. The results of synthetically evaluation of the quality of ordinary bread and whole meal bread showed that the volume of whole meal bread is less than ordinary bread. When added to LOX and LAC, the volume of whole meal bread was significantly increased, and more obviously increased by adding LAC. When added by LAC, the average score of organizational structure of the bread section, mouth feeling and flaver of the whole meal bread increased more than that of the control, which means LAC can obviously improve texture of whole meal bread.
Elasticity and hardness measurements showed that by adding LOX and LAC hardness of ordinary bread and whole meal bread reduced and elasticity increased comparing with control with LAC leading to more hardness reduce and elasticity increase. When adding LAC, changes of hardness and water content of whole meal bread and ordinary bread was the least.
从全麦粉中提取纯化得到纯度较高的WEAX,相对重均分子质量为414,459。其单糖组成为阿拉伯糖42.60%,木糖51.55%,葡萄糖3.89%,甘露糖1.96%。FA含量较低为0.25%。
采用Ba(OH)2提取WUAX,优化了提取工艺。碱提WUAX预处理结果发现高压蒸煮法可提高WUAX的提取率及提取物中WUAX的含量。Ba(OH)2提取WUAX的最佳工艺条件为:料液比1:188,提取温度63℃,提取时间3h,提取率为31.60%;方差分析结果显示各因素对提取率影响的模型显著(P<0.05),提取温度的平方项对提取率的影响高度显著(P<0.001);最佳工艺条件下得到的提取物中WUAX含量为92.09%。
采用毛细管黏度法测定漆酶(laccase,LAC)与氧化酶体系POX/H2O2体系、LOX/LA体系及化学氧化剂KBrO3、ADA、BPO及过硫酸铵对AX粘度的影响,结果表明LAC较其他两种氧化酶体系和四种化学氧化剂更有效的作用于AX,使AX黏度明显增加。添加氧化酶前后,WEAX和WUAX的重均相对分子质量分布测定结果表明,加入氧化酶后WEAX和WUAX的重均相对分子质量均增加,且WUAX增加更明显。加入LAC时,重均相对分子质量增加最为明显。这说明LAC催化AX氧化交联能力较强。加入LAC后,AX中阿魏酸(Ferulic acid,FA)含量降低较明显,证明在LAC的作用下,更多的FA参与了氧化交联。在AX和LAC混合体系中加入游离的FA,反应1h后经反相高效液相色谱法(RP-HPLC)测定混合物中游离FA的含量,结果表明,游离的FA,在LAC的作用下,参与形成聚合物,因此WEAX在LAC的作用下可通过FA产生氧化交联。在AX和LAC混合体系中加入富马酸时,WEAX及WUAX溶液中FA浓度与未加入富马酸时FA浓度下降速度差别不大。说明富马酸不参与LAC催化的AX的氧化交联反应。而加入香草酸时,溶液中FA较未加入时降低速度明显加快。且随香草酸浓度的增大,FA降低速度加快。说明加入香草酸对AX的氧化交联反应有影响。进而证明了AX的氧化交联中心为酚羟基而非双键。
采用蠕变试验与小振幅振荡动态流变性质的测定相结合,研究WEAX和WUAX对面筋蛋白黏弹性的影响,结果表明面筋蛋白(gluten)、面筋蛋白与WEAX混合体系(G-WEAX)及面筋蛋白与WUAX的混合体系(G-WUAX)当NaCl浓度为4.0%(w/v)时,面筋蛋白、G-WEAX黏弹性最大;G-WUAX在NaCl浓度为3.0%(w/v)时弹性最大,在NaCl浓度为4.0%(w/v)时黏度最大。当加水量为50%时,面筋蛋白及G-WEAX和G-WUAX混合体黏弹性最佳。WEAX和WUAX可显著提高面筋蛋白的黏弹性。加入WEAX和WUAX后,面筋蛋白的LMWG中自由巯基的数量明显减少,这是面筋蛋白黏弹性增大的原因之一。
采用荧光各向异性法研究了AX对醇溶蛋白和麦谷蛋白荧光各向异性的影响,结果表明,麦谷蛋白中加入AX,其各向异性值显著提高,说明AX与麦谷蛋白分之间存在相互作用。
采用13C NMR法研究AX和面筋蛋白的相互作用,结果表明麦谷蛋白中加入WEAX时化学位移δ为136.5和128.8ppm的吸收峰(归属于芳香烃类化合物),分别向低场强方向移动至137.6ppm和129.9ppm;当加入WUAX时,化学位移δ为136.5和128.8ppm的吸收峰也分别向低场强方向移动至137.8ppm和130.1ppm。对于醇溶蛋白,当加入WEAX或WUAX时,未发现化学位移的变化。结果表明AX与面筋蛋白的相互作用主要是由AX分子侧链上的阿魏酸与麦谷蛋白分子上的酪氨酸基团相互作用引起的。
采用小振幅动态流变试验测定面团的黏弹性,面包体积测定、感官评定法与质构测定法相结合对面包品质进行综合评价,并对面包进行了贮存试验,结果表明LAC可提高普通面团和全麦面团的黏弹性,对全麦面团的作用更强。全麦面包的比容普遍小于普通面包,全麦面包中加入LOX和LAC能显著增加全麦面包的比容,并以LAC提高比容最为明显。当加入LAC时,评分员对全麦面包切面的组织结构、口感的评分的平均值较对照面包提高较大,说明LAC对全麦面包质构的改善作用最明显,且能改善全麦面包的风味。质构测定结果表明当加入LOX和LAC的普通面包硬度较对照面包减小。加入LAC的面包弹性增大和硬度降低较明显。说明LAC对全麦面包质构的改善作用最强。面包贮存过程中面包心硬度的变化和面包中水分含量的变化测定表明,加入LAC的全麦面包经贮存7天后硬度变化较小,水分下降速度较全麦面包对照面包明显减慢,说明LAC可明显延长面包的老化时间。
Whole meal foods are rich in dietary fiber, vitamin, minerals and various phytonutrients. Regular consumption of whole meal food will reduce the incidence of multiple chronic diseases. Whole meal bread as the staple food in western countries, is the best way to supplement the dietary fiber and a variety of nutrients. In our country, the coarse structure of whole meal bread leads to poor acceptance of the consumer and tremendous restrictions of the consumption. Gluten protein, arabinoxylan (AX) and their interactions have great influence on rheological properties of the whole meal dough and quality of the whole meal bread. The texture of whole meal bread can be improved and national health can be siginificantly enhanced by studying the functional properties of AX, which has important theoretical and practical value.
In this paper, whole wheat flour was used as material, the extraction process of water unextractable arabinoxylan (WUAX) was optimized, and the high purity water extractable arabinoxylan (WEAX) and WUAX was obtained. The oxidative gelation mechanism of WEAX and WUAX as well as the interaction between gluten protein and AX was studied.
The relative weight-average molecular weight of extracted WEAX is 414, 459 and WEAX is composed of 42.60% of arabinose, 51.55% of xylose, 3.89% of glucose and 1.96% of mannose. WEAX contains 0.25% of ferulic acid (FA), which play an important role in the functional properties of WEAX. The pretreatment results of extraction of WUAX by Ba(OH)2 showed that high-pressure treatment can increase extracion yield of the WUAX and content of WUAX in the extract as well. The optimum conditions of extracting WUAX by Ba(OH)2 are as follows: WUS (the water unextractable substance that removed protein and starch):Ba(OH)2 is 1:188(w/v), the extraction temperature is 63°C, the extraction time is 3h, the extraction rate is 31.60%. Variance analysis showed that the yield factors model was significant (P<0.05), and the square of the extraction temperature highly significant (P<0.001). Under optimum conditions, WUAX contents in the extract is 92.09%.
Laccase (LAC) increase the viscosity of AX more obviously comparing with POX/H2O2, LOX/LA and chemical oxidants KBrO3, azodicarbonamide(ADA), benzoyl peroxide (BPO) and (NH4)2S2O8. When added LAC, the average weight molecular weight increased most obviously, which indicated that LAC can catalyze oxidation crosslinking more efficiently. By adding LAC, the FA of AX decreased more, which indicated that more FA involved in the oxidative cross-linking of AX. The free FA was added in the mixture of AX and LAC, the content of free FA in the mixture was measured after 1 h reaction. The results show that the free FA decreased and participated in the oxiditave cross-linking of AX, which means that AX cross-link through FA. When fumaric acid was added in the mixture of AX and LAC, concentrations of FA was almost no change, which indicated that the site of oxidation of AX was not double bond of FA structure. While by adding vanillic acid, FA in the solutions reduced and and with the increasing concentration of the vanillic acid, FA reduces more. To draw a conclusion, the site of oxidative crosslinking of AX was benzene ring and not double bond.
Studies on the effect of WEAX and WUAX on viscoelasticity of gluten by combination of creep tests and dynamic small amplitude oscillatory measurements showed that WEAX and WUAX can significantly increase the viscoelasticity of gluten. When NaCl concentration was 4.0% (w/v), gluten and gluten-WEAX mixtures (G-WEAX) showed the largest viscoelastic. The gluten-WUAX mixtures (G-WUAX) showed the maximum elasticty at 3.0% (w/v) NaCl concentration and the maximum viscosity at 4.0% (w/v) NaCl concentration.The viscoelasticity of gluten, G-WEAX and G-WUAX reached the maximum at 50% water level. The free sulfhydryl of LMWG obviously reduced by adding WEAX and WUAX, which is one of the reason that the viscoelasticity gluten was increased by adding WEAX and WUAX.
The fluorescence anisotropy measurements of gliadin and glutenin by adding AX showed that anisotropy value of glutenin significantly increased when AX was add in, indicating that AX interacted with glutenin.
The ineractions of gluten proteins and G-AX determined by 13C NMR spectrum showed that the glutenin absorption peaks at chemical shift (δ) 136.5 and 128.8ppm (both were the absorption peaks of benzene ring) shifted to 137.6ppm and 129.9ppm, respectively by adding WEAX and shifted to 137.8ppm and130.1ppm, respectively by adding WUAX. For gliadin, there was no chemical shift by adding WEAX or WUAX. The results indicated that AX interacted with glutenin by covalently bond FA to tyrosine of glutenin.
Studies on the effect of LAC, POX/H2O2 and LOX/LA on dough rheological properties showed that LAC most significantly improved the ordinary and the whole meal dough viscoelasticity and more significantly improved the viscoelasticity of the latter. The results of synthetically evaluation of the quality of ordinary bread and whole meal bread showed that the volume of whole meal bread is less than ordinary bread. When added to LOX and LAC, the volume of whole meal bread was significantly increased, and more obviously increased by adding LAC. When added by LAC, the average score of organizational structure of the bread section, mouth feeling and flaver of the whole meal bread increased more than that of the control, which means LAC can obviously improve texture of whole meal bread.
Elasticity and hardness measurements showed that by adding LOX and LAC hardness of ordinary bread and whole meal bread reduced and elasticity increased comparing with control with LAC leading to more hardness reduce and elasticity increase. When adding LAC, changes of hardness and water content of whole meal bread and ordinary bread was the least.
引文
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