高压脉冲电场下蛋白与淀粉混合凝胶机理及特性研究
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摘要
本文以肌原纤维蛋白和葛根作为研究对象，通过分析在高压脉冲电场作用下，电场强度、脉冲数、pH、盐、以及SH基和S-S分子间内部交换反应等因素对肌原纤维蛋白和葛根淀粉混合凝胶的强度、硬度、弹性、保水性等的影响，探索该混合凝胶的形成机理。同时以凝胶制品的钙生物利用率和抗氧化能力为主要指标，考察凝胶制品的功能特性。
本文充分考虑影响凝胶形成的各种因素，从实验中获得真实可靠的数据，筛选出加工工艺参数，建立了肌原纤维蛋白与葛根淀粉在高压脉冲电场下混合凝胶的技术平台。主要包括以下5个方面：
（1）用L8（27）正交实验研究了葛根淀粉浓度、pH、离子强度、金属离子对纯葛根淀粉硬度等质构特性的影响。研究了浓度配比、pH和离子强度对肌原纤维蛋白与葛根淀粉混合凝胶保水性和质构特性的影响。结果显示：不同因素对葛根淀粉凝胶硬度的影响程度顺序为：K+＞离子强度＞Ca2+＞葛根淀粉浓度＞pH＞Mg2+；肌原纤维蛋白与葛根淀粉的混合凝胶的硬度等均比纯葛根淀粉和纯蛋白高，保水性比葛根淀粉略低。影响肌原纤维蛋白与葛根淀粉混合凝胶硬度的主要因素依次为：浓度配比＞离子强度＞pH。
（2）以肌原纤维蛋白为研究对象，在电场强度15~55kV/cm、脉冲数2~8个、水浴温度50~90℃范围内，通过单因素试验考察各因素对肌原纤维蛋白硬度、弹性和保水性的影响。研究结果表明：肌原纤维蛋白经高压脉冲电场作用后，所形成凝胶的性质有显著变化。与未受高压脉冲电场处理的对照样相比，凝胶硬度、弹性和保水性在电场强度15~25kV/cm时降低，在电场强度35kV/cm时显著提高，45kV/cm时又降低。在电场强度35kV/cm条件下，凝胶硬度、弹性和保水性随着脉冲数的增加而增强，但后期增幅变化不显著。凝胶硬度和弹性在水浴温度为80℃时最佳，保水性在水浴温度为70℃时最佳。
（3）以肌原纤维蛋白为研究对象，将不同浓度的葛粉添加其中，混合溶液经高压脉冲电场预处理后，再经水浴加热处理形成混合凝胶，测定其凝胶强度。通过单因素试验和正交分析试验，认为在场强30kV/cm，脉冲数6的条件下，经高压脉冲电场预处理后，肌原纤维蛋白（质量分数5%）和葛粉（质量分数5%）混合溶液在80℃水浴加热30min，可以形成较好的混合凝胶。各因素对混合凝胶强度影响程度由大至小依次为电场强度、脉冲数、葛根淀粉质量分数。结果表明，高压脉冲电场预处理能够促进肌原纤维蛋白和葛粉形成良好的混合凝胶。
（4）为研究高压脉冲电场辅助处理对总黄酮提取效果的影响，以葛根为试材，找出了其最佳的处理技术参数。在乙醇浓度、电场强度、脉冲数和料液比4个单因素试验的基础上，采用L9（34）正交试验来优化工艺参数。试验结果表明，料液比是最主要的影响因素，其次分别是电场强度、脉冲数和乙醇浓度。在料液比为1∶40，电场强度为20kV/cm，脉冲数为8，乙醇浓度为75%的条件下葛根总黄酮的提取率最高，达到5.12%，比传统提取法提高了2.5倍以上。试验结果表明，高压脉冲电场辅助提取技术在葛根总黄酮提取工艺中具有较高的应用价值，可作为一种新型工艺技术进行推广。
（5）以D-半乳糖致衰老的小鼠为模型，将三种不同的凝胶分别携带相同剂量的葛根总黄酮喂养小白鼠，研究不同凝胶对致衰老小鼠体内抗氧化能力的影响。试验中，将凝胶分为A、B、C三组。其中A组为高压脉冲电场处理下肌原纤维蛋白和葛根淀粉混合凝胶，B组为肌原纤维蛋白和葛根淀粉混合热凝胶，C组为肌原纤维蛋白凝胶。结果表明：与对照组相比，喂养含有葛根总黄酮提取物凝胶的D-半乳糖致衰老小鼠血清、肝脏、脑组织中的SOD、GSH-Px、CAT、T-AOC活力明显上升，MDA含量明显减小，且差异显著。证实葛根总黄酮能显著提高亚急性衰老小鼠体内抗氧化酶活性。综合考虑各组间的变化，抗氧化效果为：A组＞B组＞C组＞对照组，即喂食高压脉冲电场处理后的肌原纤维蛋白和葛根淀粉混合凝胶对小白鼠的抗氧化效果最好。
本研究结果一定程度上阐明了高压脉冲电场作用对蛋白凝胶以及蛋白-淀粉混合凝胶的影响机理，为继续深入研究提供了学术上重要的参考价值。
In this paper, myofibrillar protein and pueraria were choosed as the object ofstudy. Under the processing of high-voltage pulsed electric field, the influence onrheological property, microstructure, texture property and water-retaining property ofmixed gel of myofibrillar protein and pueraria starch was analyzed based on thedifferent experimental condition of electric field intensity, pulse count, pH, salt, andinternal exchange reaction between SH and S-S molecule is analyzed. Besides, theformation mechanism of the mixed gel was discussed. In addition, functionalcharacteristics of various gel products were investigated by taking the digestibility andoxidation resistance as the main indexes.
In this paper, various factors which influence the gel formation were fullyconsidered. True and reliable data are obtained from experiments, and the processingtechnological parameters are screened. A technology platform for the gel mixingproduction from myofibrillar protein and pueraria starch using high-voltage pulsedelectric field processing was established. The research contents are as follows:
(1) The effect of concentration, pH, ionic strength, and metal ions on gelhardness of pueraria starch were studied using the L8(27) orthogonal testes. Moreover,the effect of pueraria starch proportion, pH and ionic strength on the hardness andwater holding capacity (WHC) of protein-starch mixed gel were also studied. Theresults showed that the order of the factors which affect the hardness of puerariastarch is: K+>ionic strength>Ca2+>pueraria starch content>Mg2+. The hardness ofmixed gels of myofibrils and pueraria starch are higher than that of pure myofibrilsand pure pueraria starch case while the WHC is lower than that of the pueraria starchcase.The order of factors which effect the hardness of myofibrillar protein andpueraria starch mixed gels is: pueraria starch ratios> ionic strength>pH.
(2) The influence of the treating conditions on the hardness, elasticity and water holding capacity of the myofibrillar protein gels were investigated by the single-factorexperiments, in which the ranges of the electric field strength, pulse numbers andheating temperature are15to55kV/cm,2to8and50to90℃, respectively. Theresearches results showed that the treatment of the high intensity pulsed electric fieldcan lead a significant change in the properties of the formed gel. Compared with theuntreated myofibrillar protein, the hardness, elasticity and water holding capacity ofthe HIPEF treated gels decrease when the electric field strength varied from15to25kV/cm, increase significantly when the electric field strength was35kV/cm, anddecrease again when the electric field strength is reached to45kV/cm. Under theelectric field strength of35kV/cm, the hardness, elasticity and water holding capacityof gel increase with the increase of pulse number, but the gradient is graduallydecreased. The gel has the optimal performances of hardness and elasticity, and thewater holding capacity at the heating temperature of80℃and70℃, respectively.
(3) In order to produce the protein-starch gel, the mixed solution with variouspueraria starch content was firstly treated by HIPEF, and then processed by water bathtreatment. The gelling properties of the myofibril protein-pueraria starch mixed gelwere tested using single factor experiments and orthogonal analysis experiments, inwhich the different factors of pueraria starch content, electric intensity, and pulsenumber were studied. The results indicated that the mixed gels have favorable gellingproperties under the conditions of mix solutions with myofibril protein (5%) andpueraria starch (5%),30kv/cm HIPEF pretreatment, pulse number of6and water bathof80℃for30min. Effects degree of factors on the gelling properties of the mixedgels is electric intensity, pulse number and mass fraction of pueraria starch in theorder of decreasing evolutionary.
(4) The effects of the HIPEF-assisted extraction of total flavonoids from puerariawere preliminary investigated, in which the technical parameters were optimized.Four factors including ethanol concentration (35%,45%,55%,65%,75%,85%, and95%), electric field intensity (10,20,30,40,50,60kV/cm), electric pulse number (0,2,4,6,8,10and12) and ratio of material-solvent (1:20,1:30,1:40,1:50,1:60and1:70) were investigated using the L9(34) orthogonal test. The results indicated that theratio of material-solvent is the main factor, followed by electric field intensity, pulsenumber and ethanol concentration. The optimal conditions were as follows:material-solvent ratio of1:40, electric field intensity of20kV/cm, pulse number of8 and ethanol concentration of75%. Under these conditions, the extraction yield of totalflavonoids can reach to5.12%which is higher then that of conventional extractionmethod for1.56%, and the microwave assisted extraction for1.92%. The resultssuggested that the HIPEF-assisted extraction process is feasible, and the extractionyield of total flavonoids from pueraria leaves was higher than that of the conventionalmethod.
(5) The D-galactose induced aging mice were chose as the model and fed withdifferent gels that have the same content of pueraria lobata flavone to study theefficacy of the different gel on the antioxidant capacity in induced aging mice. The gelsamples contain three types of gel (Group A, B, C). The Group A is HIPEF processedmyofibrillar protein and pueraria starch mixed gel; Group B is heating processedmyofibrillar protein and pueraria starch mixed thermal gel; and Group C is myofibrillarprotein gel. The results show that by comparing with the control group, the activitiesof SOD, GSH-Px, CAT, and T-AOC in the serum, liver and brain tissues of the micefed by the gel contents flavone extract are significantly increased as well as thecontent of MDA is dramatically decreased. This result indicates that the flavonoidscontained in pueraria starch have the great effect of improving the activity ofantioxidase in subacute senile mice. Considering the changes among the groups, theantioxidant effect in a descending order is: Group A> Goup B> Group C> controlgroup, i.e. feeding the myofibrillar proteins and pueraria starch mixed gel whichproceeded by HIPEF can obtain the best antioxidant effect in mice.
This study disscussed the mechanisms of the high-voltage pulse electric fieldtreatment on the formation and property of the protein gel and protein-starch mixedgel. The results of this study provide a basis for the further researches in this field.
The results of this study indicated the mechanism of the high-voltage pulseelectric field that plays the role on protein gels and protein gels and protein-starchmixed gel to some extent, which provides an important reference value in theacademic for further research.
本文充分考虑影响凝胶形成的各种因素，从实验中获得真实可靠的数据，筛选出加工工艺参数，建立了肌原纤维蛋白与葛根淀粉在高压脉冲电场下混合凝胶的技术平台。主要包括以下5个方面：
（1）用L8（27）正交实验研究了葛根淀粉浓度、pH、离子强度、金属离子对纯葛根淀粉硬度等质构特性的影响。研究了浓度配比、pH和离子强度对肌原纤维蛋白与葛根淀粉混合凝胶保水性和质构特性的影响。结果显示：不同因素对葛根淀粉凝胶硬度的影响程度顺序为：K+＞离子强度＞Ca2+＞葛根淀粉浓度＞pH＞Mg2+；肌原纤维蛋白与葛根淀粉的混合凝胶的硬度等均比纯葛根淀粉和纯蛋白高，保水性比葛根淀粉略低。影响肌原纤维蛋白与葛根淀粉混合凝胶硬度的主要因素依次为：浓度配比＞离子强度＞pH。
（2）以肌原纤维蛋白为研究对象，在电场强度15~55kV/cm、脉冲数2~8个、水浴温度50~90℃范围内，通过单因素试验考察各因素对肌原纤维蛋白硬度、弹性和保水性的影响。研究结果表明：肌原纤维蛋白经高压脉冲电场作用后，所形成凝胶的性质有显著变化。与未受高压脉冲电场处理的对照样相比，凝胶硬度、弹性和保水性在电场强度15~25kV/cm时降低，在电场强度35kV/cm时显著提高，45kV/cm时又降低。在电场强度35kV/cm条件下，凝胶硬度、弹性和保水性随着脉冲数的增加而增强，但后期增幅变化不显著。凝胶硬度和弹性在水浴温度为80℃时最佳，保水性在水浴温度为70℃时最佳。
（3）以肌原纤维蛋白为研究对象，将不同浓度的葛粉添加其中，混合溶液经高压脉冲电场预处理后，再经水浴加热处理形成混合凝胶，测定其凝胶强度。通过单因素试验和正交分析试验，认为在场强30kV/cm，脉冲数6的条件下，经高压脉冲电场预处理后，肌原纤维蛋白（质量分数5%）和葛粉（质量分数5%）混合溶液在80℃水浴加热30min，可以形成较好的混合凝胶。各因素对混合凝胶强度影响程度由大至小依次为电场强度、脉冲数、葛根淀粉质量分数。结果表明，高压脉冲电场预处理能够促进肌原纤维蛋白和葛粉形成良好的混合凝胶。
（4）为研究高压脉冲电场辅助处理对总黄酮提取效果的影响，以葛根为试材，找出了其最佳的处理技术参数。在乙醇浓度、电场强度、脉冲数和料液比4个单因素试验的基础上，采用L9（34）正交试验来优化工艺参数。试验结果表明，料液比是最主要的影响因素，其次分别是电场强度、脉冲数和乙醇浓度。在料液比为1∶40，电场强度为20kV/cm，脉冲数为8，乙醇浓度为75%的条件下葛根总黄酮的提取率最高，达到5.12%，比传统提取法提高了2.5倍以上。试验结果表明，高压脉冲电场辅助提取技术在葛根总黄酮提取工艺中具有较高的应用价值，可作为一种新型工艺技术进行推广。
（5）以D-半乳糖致衰老的小鼠为模型，将三种不同的凝胶分别携带相同剂量的葛根总黄酮喂养小白鼠，研究不同凝胶对致衰老小鼠体内抗氧化能力的影响。试验中，将凝胶分为A、B、C三组。其中A组为高压脉冲电场处理下肌原纤维蛋白和葛根淀粉混合凝胶，B组为肌原纤维蛋白和葛根淀粉混合热凝胶，C组为肌原纤维蛋白凝胶。结果表明：与对照组相比，喂养含有葛根总黄酮提取物凝胶的D-半乳糖致衰老小鼠血清、肝脏、脑组织中的SOD、GSH-Px、CAT、T-AOC活力明显上升，MDA含量明显减小，且差异显著。证实葛根总黄酮能显著提高亚急性衰老小鼠体内抗氧化酶活性。综合考虑各组间的变化，抗氧化效果为：A组＞B组＞C组＞对照组，即喂食高压脉冲电场处理后的肌原纤维蛋白和葛根淀粉混合凝胶对小白鼠的抗氧化效果最好。
本研究结果一定程度上阐明了高压脉冲电场作用对蛋白凝胶以及蛋白-淀粉混合凝胶的影响机理，为继续深入研究提供了学术上重要的参考价值。
In this paper, myofibrillar protein and pueraria were choosed as the object ofstudy. Under the processing of high-voltage pulsed electric field, the influence onrheological property, microstructure, texture property and water-retaining property ofmixed gel of myofibrillar protein and pueraria starch was analyzed based on thedifferent experimental condition of electric field intensity, pulse count, pH, salt, andinternal exchange reaction between SH and S-S molecule is analyzed. Besides, theformation mechanism of the mixed gel was discussed. In addition, functionalcharacteristics of various gel products were investigated by taking the digestibility andoxidation resistance as the main indexes.
In this paper, various factors which influence the gel formation were fullyconsidered. True and reliable data are obtained from experiments, and the processingtechnological parameters are screened. A technology platform for the gel mixingproduction from myofibrillar protein and pueraria starch using high-voltage pulsedelectric field processing was established. The research contents are as follows:
(1) The effect of concentration, pH, ionic strength, and metal ions on gelhardness of pueraria starch were studied using the L8(27) orthogonal testes. Moreover,the effect of pueraria starch proportion, pH and ionic strength on the hardness andwater holding capacity (WHC) of protein-starch mixed gel were also studied. Theresults showed that the order of the factors which affect the hardness of puerariastarch is: K+>ionic strength>Ca2+>pueraria starch content>Mg2+. The hardness ofmixed gels of myofibrils and pueraria starch are higher than that of pure myofibrilsand pure pueraria starch case while the WHC is lower than that of the pueraria starchcase.The order of factors which effect the hardness of myofibrillar protein andpueraria starch mixed gels is: pueraria starch ratios> ionic strength>pH.
(2) The influence of the treating conditions on the hardness, elasticity and water holding capacity of the myofibrillar protein gels were investigated by the single-factorexperiments, in which the ranges of the electric field strength, pulse numbers andheating temperature are15to55kV/cm,2to8and50to90℃, respectively. Theresearches results showed that the treatment of the high intensity pulsed electric fieldcan lead a significant change in the properties of the formed gel. Compared with theuntreated myofibrillar protein, the hardness, elasticity and water holding capacity ofthe HIPEF treated gels decrease when the electric field strength varied from15to25kV/cm, increase significantly when the electric field strength was35kV/cm, anddecrease again when the electric field strength is reached to45kV/cm. Under theelectric field strength of35kV/cm, the hardness, elasticity and water holding capacityof gel increase with the increase of pulse number, but the gradient is graduallydecreased. The gel has the optimal performances of hardness and elasticity, and thewater holding capacity at the heating temperature of80℃and70℃, respectively.
(3) In order to produce the protein-starch gel, the mixed solution with variouspueraria starch content was firstly treated by HIPEF, and then processed by water bathtreatment. The gelling properties of the myofibril protein-pueraria starch mixed gelwere tested using single factor experiments and orthogonal analysis experiments, inwhich the different factors of pueraria starch content, electric intensity, and pulsenumber were studied. The results indicated that the mixed gels have favorable gellingproperties under the conditions of mix solutions with myofibril protein (5%) andpueraria starch (5%),30kv/cm HIPEF pretreatment, pulse number of6and water bathof80℃for30min. Effects degree of factors on the gelling properties of the mixedgels is electric intensity, pulse number and mass fraction of pueraria starch in theorder of decreasing evolutionary.
(4) The effects of the HIPEF-assisted extraction of total flavonoids from puerariawere preliminary investigated, in which the technical parameters were optimized.Four factors including ethanol concentration (35%,45%,55%,65%,75%,85%, and95%), electric field intensity (10,20,30,40,50,60kV/cm), electric pulse number (0,2,4,6,8,10and12) and ratio of material-solvent (1:20,1:30,1:40,1:50,1:60and1:70) were investigated using the L9(34) orthogonal test. The results indicated that theratio of material-solvent is the main factor, followed by electric field intensity, pulsenumber and ethanol concentration. The optimal conditions were as follows:material-solvent ratio of1:40, electric field intensity of20kV/cm, pulse number of8 and ethanol concentration of75%. Under these conditions, the extraction yield of totalflavonoids can reach to5.12%which is higher then that of conventional extractionmethod for1.56%, and the microwave assisted extraction for1.92%. The resultssuggested that the HIPEF-assisted extraction process is feasible, and the extractionyield of total flavonoids from pueraria leaves was higher than that of the conventionalmethod.
(5) The D-galactose induced aging mice were chose as the model and fed withdifferent gels that have the same content of pueraria lobata flavone to study theefficacy of the different gel on the antioxidant capacity in induced aging mice. The gelsamples contain three types of gel (Group A, B, C). The Group A is HIPEF processedmyofibrillar protein and pueraria starch mixed gel; Group B is heating processedmyofibrillar protein and pueraria starch mixed thermal gel; and Group C is myofibrillarprotein gel. The results show that by comparing with the control group, the activitiesof SOD, GSH-Px, CAT, and T-AOC in the serum, liver and brain tissues of the micefed by the gel contents flavone extract are significantly increased as well as thecontent of MDA is dramatically decreased. This result indicates that the flavonoidscontained in pueraria starch have the great effect of improving the activity ofantioxidase in subacute senile mice. Considering the changes among the groups, theantioxidant effect in a descending order is: Group A> Goup B> Group C> controlgroup, i.e. feeding the myofibrillar proteins and pueraria starch mixed gel whichproceeded by HIPEF can obtain the best antioxidant effect in mice.
This study disscussed the mechanisms of the high-voltage pulse electric fieldtreatment on the formation and property of the protein gel and protein-starch mixedgel. The results of this study provide a basis for the further researches in this field.
The results of this study indicated the mechanism of the high-voltage pulseelectric field that plays the role on protein gels and protein gels and protein-starchmixed gel to some extent, which provides an important reference value in theacademic for further research.
引文
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