喷射蒸煮制备米糠、碎米蛋白及其功能性研究
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摘要
大米加工副产物产量巨大，利用率低。其中的大米蛋白以其营养性、低过敏性以及抗肿瘤活性等独特属性，近年来受到广泛的关注与应用。然而商业化的大米蛋白产品，尤其是来源于米糠的蛋白产品，产量仍然有限。其原因在于蛋白的组成复杂、溶解性差，缺乏合适可行的加工方法对蛋白进行提取与改性。本论文针对蛋白在提取过程中存在的问题以及功能特性上的不利因素，分别以米糠与碎米为原料，首次采用喷射蒸煮处理对热稳定化米糠蛋白进行制备，并对蛋白的结构与性质进行表征，探讨了喷射蒸煮在难溶蛋白结构改变与功能改善上的作用机制；其次，利用酶辅助的物理手段同步制备低变性与高消化性能的碎米蛋白，并揭示该蛋白高消化性能的原因。本文的主要结论如下：
1、采用喷射蒸煮结合淀粉酶预处理提高热稳定米糠蛋白的提取率和纯度，评估了米糠分离蛋白的生化特性与功能特性。单一的喷射蒸煮处理能有效提高蛋白的提取率，且随温度的升高提取率相应提高，但蛋白纯度显著下降；但是淀粉酶的预处理可以显著提高蛋白的提取率与纯度分别为45～50%与72～74%。蛋白的亚基及分子量变化表明喷射蒸煮处理能引起米糠中难溶的蛋白聚集体分离，同时增加可溶性聚集体的含量。二硫键含量与表面疏水性的增加表明这些可溶性的聚集体主要是由共价结合的分子内与分子间二硫键及非共价结合的表面疏水作用力形成的。此外，蛋白接枝度数据、SDS-PAGE糖染色电泳图谱、糖荧光光谱分析表明在喷射蒸煮过程中，米糠蛋白与米糠中的还原糖还会发生湿热接枝反应，生成糖基化产物，是蛋白理化特性改变的原因之一。
2、对喷射蒸煮结合酶处理制备的热稳定米糠分离蛋白的功能特性与消化特性的分析表明：蛋白的溶解性与pH的关系呈典型的U型曲线，在等电点pH3.5～4.0处的溶解度最低，在pH＞6.0的中性与碱性条件下溶解性要显著高于酸性条件；与前人提取的米糠蛋白相比，喷射蒸煮结合酶处理制备的蛋白具有更好的发泡性能、持水性与持油性。对热稳定米糠分离蛋白制备的乳液进行分析，表明该蛋白具有较强的乳化性能，且该乳液在中性pH条件下具有较强的耐热性能，但不具备抗冻的能力。体外胃蛋白酶和胰蛋白酶模拟消化过程表明，处理后制备的热稳定米糠分离蛋白具有与天然米糠分离蛋白类似的消化率，其中可溶性氮释放量达到77.9%，处理不影响米糠分离蛋白的体外消化性能。
3、采用添加还原糖的方式，以期验证在喷射蒸煮过程中发生的糖基化反应。喷射蒸煮处理有助于米糠中的蛋白与还原糖发生糖基化反应，且添加葡萄糖的糖基化反应程度要高于乳糖。添加糖接枝后制备的蛋白具有良好的溶解性与乳化性，糖基化反应是造成这些变化的主要原因，但对蛋白的消化性能影响不大。因此，喷射蒸煮可以作为一种新型的湿热接枝技术应用到难溶蛋白的提取与改性上。
4、采用酶辅助微射流技术制备回收率与纯度分别为81.87%与87.89%的碎米蛋白，并对酶辅助微射流蛋白(EM-RP)与传统的碱法提取蛋白(AE-RP)的蛋白组成、溶解性、结构与消化性能进行比较。研究表明EM-RP蛋白的主要组成为谷蛋白，具有低的蛋白溶解度与更天然的结构，而在AE-RP蛋白中除谷蛋白外，还有大量的醇溶蛋白与球蛋白。蛋白组成的不同影响蛋白中的氨基酸组成，其中AE-RP蛋白中富含亮氨酸，谷氨酸/谷氨酰胺，芳香族氨基酸与带电氨基酸。EM-RP蛋白具有更好的胃蛋白酶-胰蛋白酶体外消化性能，其原因在于富含易消化的谷蛋白。尽管在碱提过程中有部分蛋白变性水解，但是醇溶蛋白仍然影响AE-RP蛋白的消化性能。
5、系统研究了大米蛋白与大米淀粉的相互作用对淀粉糊化特性的影响，并且对大米蛋白进行脱酰胺改性。添加EM-RP蛋白会降低大米淀粉的糊化晗值、峰值黏度、崩解值和回生值，但对起始的糊化温度和峰值温度无明显影响；流变的数据也表明，在低蛋白浓度下，随着蛋白含量的增加，糊化液的黏度与剪切力相应提高，但当蛋白浓度进一步增加（＞10%）时（相当于大米中原有的蛋白含量），糊化液的黏度与剪切力反而下降。喷射蒸煮是一种可行的蛋白脱酰胺手段，但是对蛋白的结构改善并不明显。随着酸浓度的增加，蛋白的脱酰胺程度相应增加；但在相同酸浓度下，随着蛋白含量的增加，脱酰胺度反而下降。通过该法制备的脱酰胺蛋白的最佳工艺条件为1%蛋白浓度、0.4mol/LHCL浓度，且产品水解度较低，脱酰胺度可达25%。
The production of rice by-products is huge, but the value is low. Rice protein has beenfound to be of nutritional, hypoallergenic and having anti-cancer activity for food applicationsand rice protein products has been in demand in recent years. However, due to the complexityof protein composition and difficulties in the processing, low protein extraction and purityhave been recognized, which greatly limit its application in food industry. In this work,hydrothermal cooking (HTC) was firstly used to extraction and modification of protein fromheat-stabilized rice bran and broken rice kernels. Protein extraction, structure and functionproperties were described in the HTC process, and the mechanism induced by HTC wasinvestigated. The broken rice protein with low denaturation and high digestibility wasprepared by enzyme-assisted microfluidization, and the reason of high digestibility was alsoinvestigated. Main results are as follows:
1. Hydrothermal cooking (HTC) combined with amylase pretreatment (AP) was used toimprove protein extraction from heat-stabilized rice bran. The physicochemical andemulsifying properties of rice bran protein isolate (RPI) were evaluated. HTC alonesignificantly increased extraction yield, while protein purity was decreased. In contrast, HTCcombined with AP significantly improved both extraction yield and protein purity (about45-50%and72-74%, respectively). Electrophoresis and size exclusion chromatographyprofiles indicated that HTC led to the dissociation of insoluble protein aggregates in rice bran,with subsequent increase of soluble aggregates in RPI, linked by non-covalent (e.g.,hydrophobic interaction) and covalent bonds (disulfide bond). This result was evidenced bythe increased disulfide bond contents and surface hydrophobicity of RPI. In addition, the dateof DG, fluorescence spectrum and electrophoresis stained by PSA indicated that HTC led tothe conjugations of protein and reducing sugar in rice bran, which led to the change inphysical and chemical characteristics of the protein.
2. HTC-prepared RPI exhibited excellent functional properties and in vitro digestibilityproperty. All protein samples exhibited typical U-shaped solubility-pH profiles, with theminimum at pH3.5–4.5and gradually increasing at pH values below3.0and above6.0. Foamcapacity and stability, the water holding capacity (WHC) and the oil holding capacity (OHC)in these proteins were superior to previous products. In addition, HTC-prepared RPI exhibitedexcellent emulsifying property. The emulsion is stable under neutral pH after heat treatment,but is unstable after freeze-thaw processing. In vitro digestion model, heat-stabilized rice bran protein showed similar digestibility with un-stabilized rice bran protein after HTC treatmentand the soluble nitrogen release was77.9%.
3. In order to verify the glycosylation reaction occurred in the HTC process, reducingsugar was added in heat-stabilized rice bran. HTC treatment is an effective technique in theenhancement of the conjugation of protein and reducing sugar in heat-stabilized rice bran, andglycosylation reaction extent of lactose is higher than that of the glucose. Glycosylationreaction products exhibited excellent solubility emulsifying property, but have no change invitro digestion. Therefore, HTC can be applied in extraction andmodification of the insoluble protein as a new liquid heat glycosylation reaction technique.
4. High protein recovery (81.87%) and purity (87.89%) were obtained from broken riceby enzyme-assisted microfluidization. The protein composition, solubility, structuralproperties, and in vitro digestibility of rice proteins prepared by enzyme-assistedmicrofluidization (EM-RP) and alkaline extraction (AE-RP) were compared. EM-RP wasmainly composed of glutelin with low solubility and native structure, whereas large quantitiesof prolamin and globulin appeared in the AE-RP except glutelin. Glutamic acid/glutamine,leucine, aromatic and charged amino acids were rich in the AE-RP because of differentprotein composition. Compared to AE-RP, EM-RP showed higher digestibility due to therichness of glutelin (an easy-to-digest protein), as evidenced by higher nitrogen release duringpepsin-trypsin digestion. The presence of prolamin (an indigestible protein) in AE-RPdecreased protein digestibility although alkaline extraction improved its hydrolysis.
5. The interaction of between rice protein and rice starch and the protein effect onstarch pasting properties were studied systemically and acidic deamidation of rice protein wasprepared by HTC.The gelatinization, peak viscosity, breakdown and setback of rice starchwere reduced after the addition of EM-RP protein, but the gelatinization temperature and peaktemperature have significantly different. The rheological property of rice starch showedapparent viscosity and shear force increased with increasing the protein content under lowerprotein content (<10%). However, these values will decrease with further increasing theprotein content. HTC treatment was a feasible acidic deamidation of EM-RP rice protein,but had no significant effect on the protein structure. The degree of protein deamidation wascorresponding increasing with acid concentration increasing, but the degree of that wasdecreased with protein content increasing under the same acid concentration. The optimaldeamidation conditions were0.4mol/L HCl,1%rice protein,90s for reaction time and140℃under HTC treatment with the25%deamidation and low degree of hydrolysis levels.
1、采用喷射蒸煮结合淀粉酶预处理提高热稳定米糠蛋白的提取率和纯度，评估了米糠分离蛋白的生化特性与功能特性。单一的喷射蒸煮处理能有效提高蛋白的提取率，且随温度的升高提取率相应提高，但蛋白纯度显著下降；但是淀粉酶的预处理可以显著提高蛋白的提取率与纯度分别为45～50%与72～74%。蛋白的亚基及分子量变化表明喷射蒸煮处理能引起米糠中难溶的蛋白聚集体分离，同时增加可溶性聚集体的含量。二硫键含量与表面疏水性的增加表明这些可溶性的聚集体主要是由共价结合的分子内与分子间二硫键及非共价结合的表面疏水作用力形成的。此外，蛋白接枝度数据、SDS-PAGE糖染色电泳图谱、糖荧光光谱分析表明在喷射蒸煮过程中，米糠蛋白与米糠中的还原糖还会发生湿热接枝反应，生成糖基化产物，是蛋白理化特性改变的原因之一。
2、对喷射蒸煮结合酶处理制备的热稳定米糠分离蛋白的功能特性与消化特性的分析表明：蛋白的溶解性与pH的关系呈典型的U型曲线，在等电点pH3.5～4.0处的溶解度最低，在pH＞6.0的中性与碱性条件下溶解性要显著高于酸性条件；与前人提取的米糠蛋白相比，喷射蒸煮结合酶处理制备的蛋白具有更好的发泡性能、持水性与持油性。对热稳定米糠分离蛋白制备的乳液进行分析，表明该蛋白具有较强的乳化性能，且该乳液在中性pH条件下具有较强的耐热性能，但不具备抗冻的能力。体外胃蛋白酶和胰蛋白酶模拟消化过程表明，处理后制备的热稳定米糠分离蛋白具有与天然米糠分离蛋白类似的消化率，其中可溶性氮释放量达到77.9%，处理不影响米糠分离蛋白的体外消化性能。
3、采用添加还原糖的方式，以期验证在喷射蒸煮过程中发生的糖基化反应。喷射蒸煮处理有助于米糠中的蛋白与还原糖发生糖基化反应，且添加葡萄糖的糖基化反应程度要高于乳糖。添加糖接枝后制备的蛋白具有良好的溶解性与乳化性，糖基化反应是造成这些变化的主要原因，但对蛋白的消化性能影响不大。因此，喷射蒸煮可以作为一种新型的湿热接枝技术应用到难溶蛋白的提取与改性上。
4、采用酶辅助微射流技术制备回收率与纯度分别为81.87%与87.89%的碎米蛋白，并对酶辅助微射流蛋白(EM-RP)与传统的碱法提取蛋白(AE-RP)的蛋白组成、溶解性、结构与消化性能进行比较。研究表明EM-RP蛋白的主要组成为谷蛋白，具有低的蛋白溶解度与更天然的结构，而在AE-RP蛋白中除谷蛋白外，还有大量的醇溶蛋白与球蛋白。蛋白组成的不同影响蛋白中的氨基酸组成，其中AE-RP蛋白中富含亮氨酸，谷氨酸/谷氨酰胺，芳香族氨基酸与带电氨基酸。EM-RP蛋白具有更好的胃蛋白酶-胰蛋白酶体外消化性能，其原因在于富含易消化的谷蛋白。尽管在碱提过程中有部分蛋白变性水解，但是醇溶蛋白仍然影响AE-RP蛋白的消化性能。
5、系统研究了大米蛋白与大米淀粉的相互作用对淀粉糊化特性的影响，并且对大米蛋白进行脱酰胺改性。添加EM-RP蛋白会降低大米淀粉的糊化晗值、峰值黏度、崩解值和回生值，但对起始的糊化温度和峰值温度无明显影响；流变的数据也表明，在低蛋白浓度下，随着蛋白含量的增加，糊化液的黏度与剪切力相应提高，但当蛋白浓度进一步增加（＞10%）时（相当于大米中原有的蛋白含量），糊化液的黏度与剪切力反而下降。喷射蒸煮是一种可行的蛋白脱酰胺手段，但是对蛋白的结构改善并不明显。随着酸浓度的增加，蛋白的脱酰胺程度相应增加；但在相同酸浓度下，随着蛋白含量的增加，脱酰胺度反而下降。通过该法制备的脱酰胺蛋白的最佳工艺条件为1%蛋白浓度、0.4mol/LHCL浓度，且产品水解度较低，脱酰胺度可达25%。
The production of rice by-products is huge, but the value is low. Rice protein has beenfound to be of nutritional, hypoallergenic and having anti-cancer activity for food applicationsand rice protein products has been in demand in recent years. However, due to the complexityof protein composition and difficulties in the processing, low protein extraction and purityhave been recognized, which greatly limit its application in food industry. In this work,hydrothermal cooking (HTC) was firstly used to extraction and modification of protein fromheat-stabilized rice bran and broken rice kernels. Protein extraction, structure and functionproperties were described in the HTC process, and the mechanism induced by HTC wasinvestigated. The broken rice protein with low denaturation and high digestibility wasprepared by enzyme-assisted microfluidization, and the reason of high digestibility was alsoinvestigated. Main results are as follows:
1. Hydrothermal cooking (HTC) combined with amylase pretreatment (AP) was used toimprove protein extraction from heat-stabilized rice bran. The physicochemical andemulsifying properties of rice bran protein isolate (RPI) were evaluated. HTC alonesignificantly increased extraction yield, while protein purity was decreased. In contrast, HTCcombined with AP significantly improved both extraction yield and protein purity (about45-50%and72-74%, respectively). Electrophoresis and size exclusion chromatographyprofiles indicated that HTC led to the dissociation of insoluble protein aggregates in rice bran,with subsequent increase of soluble aggregates in RPI, linked by non-covalent (e.g.,hydrophobic interaction) and covalent bonds (disulfide bond). This result was evidenced bythe increased disulfide bond contents and surface hydrophobicity of RPI. In addition, the dateof DG, fluorescence spectrum and electrophoresis stained by PSA indicated that HTC led tothe conjugations of protein and reducing sugar in rice bran, which led to the change inphysical and chemical characteristics of the protein.
2. HTC-prepared RPI exhibited excellent functional properties and in vitro digestibilityproperty. All protein samples exhibited typical U-shaped solubility-pH profiles, with theminimum at pH3.5–4.5and gradually increasing at pH values below3.0and above6.0. Foamcapacity and stability, the water holding capacity (WHC) and the oil holding capacity (OHC)in these proteins were superior to previous products. In addition, HTC-prepared RPI exhibitedexcellent emulsifying property. The emulsion is stable under neutral pH after heat treatment,but is unstable after freeze-thaw processing. In vitro digestion model, heat-stabilized rice bran protein showed similar digestibility with un-stabilized rice bran protein after HTC treatmentand the soluble nitrogen release was77.9%.
3. In order to verify the glycosylation reaction occurred in the HTC process, reducingsugar was added in heat-stabilized rice bran. HTC treatment is an effective technique in theenhancement of the conjugation of protein and reducing sugar in heat-stabilized rice bran, andglycosylation reaction extent of lactose is higher than that of the glucose. Glycosylationreaction products exhibited excellent solubility emulsifying property, but have no change invitro digestion. Therefore, HTC can be applied in extraction andmodification of the insoluble protein as a new liquid heat glycosylation reaction technique.
4. High protein recovery (81.87%) and purity (87.89%) were obtained from broken riceby enzyme-assisted microfluidization. The protein composition, solubility, structuralproperties, and in vitro digestibility of rice proteins prepared by enzyme-assistedmicrofluidization (EM-RP) and alkaline extraction (AE-RP) were compared. EM-RP wasmainly composed of glutelin with low solubility and native structure, whereas large quantitiesof prolamin and globulin appeared in the AE-RP except glutelin. Glutamic acid/glutamine,leucine, aromatic and charged amino acids were rich in the AE-RP because of differentprotein composition. Compared to AE-RP, EM-RP showed higher digestibility due to therichness of glutelin (an easy-to-digest protein), as evidenced by higher nitrogen release duringpepsin-trypsin digestion. The presence of prolamin (an indigestible protein) in AE-RPdecreased protein digestibility although alkaline extraction improved its hydrolysis.
5. The interaction of between rice protein and rice starch and the protein effect onstarch pasting properties were studied systemically and acidic deamidation of rice protein wasprepared by HTC.The gelatinization, peak viscosity, breakdown and setback of rice starchwere reduced after the addition of EM-RP protein, but the gelatinization temperature and peaktemperature have significantly different. The rheological property of rice starch showedapparent viscosity and shear force increased with increasing the protein content under lowerprotein content (<10%). However, these values will decrease with further increasing theprotein content. HTC treatment was a feasible acidic deamidation of EM-RP rice protein,but had no significant effect on the protein structure. The degree of protein deamidation wascorresponding increasing with acid concentration increasing, but the degree of that wasdecreased with protein content increasing under the same acid concentration. The optimaldeamidation conditions were0.4mol/L HCl,1%rice protein,90s for reaction time and140℃under HTC treatment with the25%deamidation and low degree of hydrolysis levels.
引文
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