小麦胚芽油超临界CO_2萃取、评价及麦胚蛋白修饰改性的研究
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摘要
小麦胚芽是小麦籽粒的生命源泉，虽仅占小麦籽粒的1.4%-3.9%，却蕴含着丰富的营养成分，被誉为“人类天然的营养宝库”。我国小麦总产量位居世界第一，每年可以开发利用的小麦胚芽资源潜藏量高达280-420万吨。但我国对小麦胚芽资源的开发利用起步较晚，长期以来，除少量小麦胚芽用作提取制备小麦胚芽油的原料外，绝大部分被混在麸皮中用作廉价饲料，使得小麦胚芽资源一直处于优质资源低值化利用的境地。
本研究以小麦胚芽为原料，以提高小麦胚芽综合利用价值为目的，采用超临界CO_2萃取技术制取活性成分高的小麦胚芽油，并进行品质分析和生物学评价；通过糖基化反应、发酵等方法对小麦胚芽蛋白进行改性，以期为小麦胚芽蛋白改性探索新的途径。主要研究内容及结果如下：
1.采用超临界CO_2萃取小麦胚芽油，通过单因素实验和响应面实验优化，确定了最佳萃取工艺：原料含水量和粒度分别为4.37%和60-80目，萃取压力为30MPa，萃取温度为40℃，分离温度为50℃，分离压力为5MPa，萃取时间为1.7h，在该条件下小麦胚芽油提取率达到10.38%，油中VE含量为3.19mg/g。小麦胚芽油提取率和油中VE含量之间的相关性并没有达到显著水平，需根据提取目的的不同选择不同的提取工艺。
2.不同提取方式对小麦胚芽油中活性成分VE和总酚类物质含量影响较大。其中超临界CO_2萃取所得油中VE含量较高，其在不同浓度下的DPPH清除率也略高。但不同提取方式对小麦胚芽油的脂肪酸组成及分布、脱脂小麦胚芽粉的氨基酸组成等影响不大。
3.超临界CO_2萃取所得小麦胚芽油在外观、理化指标、氧化稳定性等方面都优于有机溶剂浸提所得油。油中富含多不饱和脂肪酸，达69.73%，其中亚油酸含量更是高达64.82%。Sn-2位脂肪酸分布也以不饱和脂肪酸为主，其中油酸和亚油酸含量约占80%。油品具有小麦胚芽特有的气味，采用HS-SPME-GC-MS方法从中鉴定出44种成分。油中不皂化物含量达4.16%，通过GC-MS共鉴定出了5种主要物质，其中β-谷甾醇相对含量高达60.38%。此外，超临界CO_2萃取所得脱脂小麦胚芽粉中残油量仅为0.96%，水分含量为0.47%，变性程度较低，且蛋白质含量高(34.3%)，氨基酸组成平衡，必需氨基酸含量丰富。
4.小麦胚芽油的摄入不会影响大鼠的正常生长，对心、脾和肾指数无显著影响，但却能显著降低肝指数。与Control组相比，单纯高脂肪模型中，小麦胚芽油的摄入并不会显著提高血清中的总胆固醇、甘油三酯水平以及动脉硬化指数(p＜0.05)；但却能显著提高肝脏中总胆固醇、甘油三酯和直接高密度脂蛋白胆固醇水平(p＜0.05)。两组模型中小麦胚芽油的摄入均能显著增加肝脏中还原型谷胱甘肽含量，提高过氧化氢酶和超氧化物歧化酶活力，并显著降低丙二醛含量(p＜0.05)。
5.采用“酶解+碱溶酸沉”技术制备小麦胚芽蛋白，蛋白质含量达到88.26%，其中清蛋白占19.5%、球蛋白占27.37%、谷蛋白占21.24%、醇溶蛋白占2.94%。小麦胚芽蛋白中必需氨基酸占总氨基酸含量的40.67%，且分布平衡，是一种理想的氨基酸组成模式。与大豆分离蛋白相比，小麦胚芽蛋白的起泡性、持油性较优，但其溶解性、乳化性及乳化稳定性、持水性较差。
6.采用糖基化反应对小麦胚芽蛋白进行改性，选择葡聚糖作为糖基供体。通过单因素实验和正交实验优化，确定了改性的最佳条件。在底物浓度为2%，蛋白：葡聚糖=1:1，pH11.0，温度110℃，时间20min时，改性效果最好，此时接枝度为12.05%，产物溶解性为85.34%。经过糖基化反应改性后，产物的等电点向低pH移动，且溶解性、乳化性及乳化稳定性均有一定程度的提高。通过荧光光谱分析发现，反应产物在激发波长347nm，发射波长422nm处有最大荧光强度，符合糖基化反应产物的荧光特征。氨基酸分析表明该反应主要发生在小麦胚芽蛋白的赖氨酸和精氨酸侧链上。反应产物蛋白结构中α-螺旋数量显著增多。由于羟基的接入，提高了分子间的空间位阻作用，产物热稳定性略有所增强。
7.采用发酵法制备小麦胚芽活性肽。以枯草芽孢杆菌为出发菌株，通过单因素实验和响应面实验优化确定了发酵制备小麦胚芽肽的最优条件：原料粒度60-80目，初始pH6.5，发酵温度31℃，发酵时间48h，接种量8%。在此条件下，发酵产物中肽含量达到8.69mg/mL。通过考察发酵过程中肽生成量与总抗氧化能力的关系，发现产物中肽生成量与抗氧化能力之间显示良好的相关性(R2=0.9911)。该方法制备的小麦胚芽肽具有一定的体外清除自由基(DPPH·、O-2和OH-)的能力，其中对DPPH清除率最高，且清除效果与肽的浓度呈正相关。通过对分子量分布研究发现，分子量在500-1000Da和180-500Da之间的肽对DPPH清除率最高。
Wheat germ is the life source of wheat kernel. Although it makes up about1.4%-3.9%of wheatkernel, it contains highly concentrated nutrients and is considered as “treasure house of naturalnutrition for human”. The total production of wheat kernel in China is the highest in the world andits yield is up to280-420million tons every year. However, the development of wheat germ is late inour country. For a long time, only a small amount of wheat germ is used to product wheat germ oil,while the majority of defatted wheat germ is used for animal feed, because of the wheat bran mixed.Up to now, wheat germ is still in a high quality resource low utilization condition.
The purpose of this study is to enhance the utilization value of wheat germ. We not only focuseson the production and evaluation of wheat germ oil with highly activity components by supercriticalCO_2extraction technology, but also pay attention to the use of defatted wheat germ. In order toexplore new method to modify wheat germ protein, Maillard reaction and fermentation technologywere applied. The main results and conclusions obtained are as follows.
1. The supercritical CO_2extraction technology was applied to extract wheat germ oil. Theoptimal extract condition was obtained through single factor experiments and a Box-Behnken designcombined with response surface methodology. The maximal oil yield of10.38%was achieved underthis optimal condition of wheat germ particle size60-80mesh, water content4.37%, extractionpressure30MPa, extraction temperature40℃, extraction time1.7h, separation pressure5MPa andseparation temperature50℃. The VEcontent was3.19mg/g under this condition. Data showed that apositive correlation was found between oil yield and VEcontent, but the correlation was notsignificant. The extraction condition may be selected according to the aim of extraction.
2. Different extraction methods showed greater impact on the content of activitycomponents (such as VEand total phenols) in wheat germ oil. The oil obtained by supercritical CO_2extraction owned higher content of activity components, such as VE, so the oil showed strongerDPPH radical scavenging ability at different concentration. However, different extraction methodsshowed no impact on the composition of fatty acids and amino acids.
3. The oil obtained by supercritical CO_2extraction showed better quality than the oilobtained by solvent extraction in appearance, physical and chemical index, oxidation stability and soon. The oil obtained was rich in polyunsaturated fatty acid (69.73%), especially in linoleic acid(64.82%). The mainly fatty acids distributed in Sn-2were unsaturated fatty acids, such as oleic acidand linoleic acid (80%). The oil has a unique smell of wheat germ,44volatile compounds wereisolated and identified by HS-SPME-GC-MS. The content of unsaponifiable matters in oil was4.16%, and5compounds were identified by GC-MS. The main content of unsaponifiable matterswas sterol, especially β-sitosterol (64.64%). In addition, the residual oil of defatted wheat germobtained supercritical CO_2extraction was only0.96%and the water content was0.47%. Thedefatted meal was lower denaturation and the content of protein was high (34.3%). The compositionof amino acid was balance and the essential amino acids were abundant.
4. The intakes of wheat germ oil had no significant effect on the normal growth and theheart, spleen and kidney index, but significantly reduced the liver index. Compared with the Controlgroup, in a simple high-fat model, the TC, TG and AI of serum were not significantly increased bythe intakes of wheat germ oil (p＜0.05). However, the TC, TG and HDL-C of liver lipid were significantly increased (p＜0.05). Moreover, the intakes of wheat germ oil can significantlyimproved the concentration of GSH, the activities of SOD and CAT, and reduced the concentrationof MDA in the two different models (p＜0.05).
5.“Enzyme hydrolysis+alkali-soluble and acid precipitation” technology was applied toextract protein from defatted wheat germ. The purity of protein was88.26%, and albumin was19.5%, globulin was27.37%, glutelin was21.24%, prolamine was2.94%. The distribution ofamino acid was balance and the amount of essential amino acids reached40.67%. Compared withsoy protein isolate, although foam properties and oil absorption capacity of wheat germ proteinwere better, its solubility, emulsifying and emulsion stability, as well as water absorption capacitywere poorer.
6. Wheat germ protein was modified by Maillard reaction with Dextran. The optimumcondition was obtained through single factor experiments and orthogonal optimization experiments.Under the condition(substrate concentration of2%, protein: dextran=1:1, pH11.0, temperature110℃, time20min), the DS reached12.05%and NSI was85.34%. After modification, the isoelectricpoint of the product moved to lower pH. The functional properties of conjugates, such as solubility,emulsification activity and emulsification stablility were improved greatly. The conjugates have thestrongest florescence intensity when emission was347nm and excitation was422nm, which inaccordance with the fluorescence characteristics of Maillard reaction product. The amino acidanalysis showed that the content of Lys and Arg was decreased by glycation. In addition, thenumber of α-helix structure was significantly increased in MRPs. Because the introduction ofhydroxy, the intermolecular steric effect was increased and thermal stability was enhanced slightly.
7. Wheat germ peptides were prepared by fermentation with Bacillus Subtilis B1. Thefermentation condition was optimized through single factor experiments and a Box-Behnkendesign combined with response surface methodology. A maximal yield of peptides was achieved8.69mg/mL under optimal conditions: wheat germ particle size60-80mesh, pH6.5, inoculum size8%, fermentation temperature31°C and time48h. A positive correlation (R2=0.9911) was foundbetween the concentration of peptides and total antioxidant capacity. The peptides presented asignificant does-dependent on scavenging activities of DPPH, hydroxyl and superoxide anionradicals, especially on scavenging activities of DPPH in the molecular weight distribution ofpeptides between500-1000Da and180-500Da.
本研究以小麦胚芽为原料，以提高小麦胚芽综合利用价值为目的，采用超临界CO_2萃取技术制取活性成分高的小麦胚芽油，并进行品质分析和生物学评价；通过糖基化反应、发酵等方法对小麦胚芽蛋白进行改性，以期为小麦胚芽蛋白改性探索新的途径。主要研究内容及结果如下：
1.采用超临界CO_2萃取小麦胚芽油，通过单因素实验和响应面实验优化，确定了最佳萃取工艺：原料含水量和粒度分别为4.37%和60-80目，萃取压力为30MPa，萃取温度为40℃，分离温度为50℃，分离压力为5MPa，萃取时间为1.7h，在该条件下小麦胚芽油提取率达到10.38%，油中VE含量为3.19mg/g。小麦胚芽油提取率和油中VE含量之间的相关性并没有达到显著水平，需根据提取目的的不同选择不同的提取工艺。
2.不同提取方式对小麦胚芽油中活性成分VE和总酚类物质含量影响较大。其中超临界CO_2萃取所得油中VE含量较高，其在不同浓度下的DPPH清除率也略高。但不同提取方式对小麦胚芽油的脂肪酸组成及分布、脱脂小麦胚芽粉的氨基酸组成等影响不大。
3.超临界CO_2萃取所得小麦胚芽油在外观、理化指标、氧化稳定性等方面都优于有机溶剂浸提所得油。油中富含多不饱和脂肪酸，达69.73%，其中亚油酸含量更是高达64.82%。Sn-2位脂肪酸分布也以不饱和脂肪酸为主，其中油酸和亚油酸含量约占80%。油品具有小麦胚芽特有的气味，采用HS-SPME-GC-MS方法从中鉴定出44种成分。油中不皂化物含量达4.16%，通过GC-MS共鉴定出了5种主要物质，其中β-谷甾醇相对含量高达60.38%。此外，超临界CO_2萃取所得脱脂小麦胚芽粉中残油量仅为0.96%，水分含量为0.47%，变性程度较低，且蛋白质含量高(34.3%)，氨基酸组成平衡，必需氨基酸含量丰富。
4.小麦胚芽油的摄入不会影响大鼠的正常生长，对心、脾和肾指数无显著影响，但却能显著降低肝指数。与Control组相比，单纯高脂肪模型中，小麦胚芽油的摄入并不会显著提高血清中的总胆固醇、甘油三酯水平以及动脉硬化指数(p＜0.05)；但却能显著提高肝脏中总胆固醇、甘油三酯和直接高密度脂蛋白胆固醇水平(p＜0.05)。两组模型中小麦胚芽油的摄入均能显著增加肝脏中还原型谷胱甘肽含量，提高过氧化氢酶和超氧化物歧化酶活力，并显著降低丙二醛含量(p＜0.05)。
5.采用“酶解+碱溶酸沉”技术制备小麦胚芽蛋白，蛋白质含量达到88.26%，其中清蛋白占19.5%、球蛋白占27.37%、谷蛋白占21.24%、醇溶蛋白占2.94%。小麦胚芽蛋白中必需氨基酸占总氨基酸含量的40.67%，且分布平衡，是一种理想的氨基酸组成模式。与大豆分离蛋白相比，小麦胚芽蛋白的起泡性、持油性较优，但其溶解性、乳化性及乳化稳定性、持水性较差。
6.采用糖基化反应对小麦胚芽蛋白进行改性，选择葡聚糖作为糖基供体。通过单因素实验和正交实验优化，确定了改性的最佳条件。在底物浓度为2%，蛋白：葡聚糖=1:1，pH11.0，温度110℃，时间20min时，改性效果最好，此时接枝度为12.05%，产物溶解性为85.34%。经过糖基化反应改性后，产物的等电点向低pH移动，且溶解性、乳化性及乳化稳定性均有一定程度的提高。通过荧光光谱分析发现，反应产物在激发波长347nm，发射波长422nm处有最大荧光强度，符合糖基化反应产物的荧光特征。氨基酸分析表明该反应主要发生在小麦胚芽蛋白的赖氨酸和精氨酸侧链上。反应产物蛋白结构中α-螺旋数量显著增多。由于羟基的接入，提高了分子间的空间位阻作用，产物热稳定性略有所增强。
7.采用发酵法制备小麦胚芽活性肽。以枯草芽孢杆菌为出发菌株，通过单因素实验和响应面实验优化确定了发酵制备小麦胚芽肽的最优条件：原料粒度60-80目，初始pH6.5，发酵温度31℃，发酵时间48h，接种量8%。在此条件下，发酵产物中肽含量达到8.69mg/mL。通过考察发酵过程中肽生成量与总抗氧化能力的关系，发现产物中肽生成量与抗氧化能力之间显示良好的相关性(R2=0.9911)。该方法制备的小麦胚芽肽具有一定的体外清除自由基(DPPH·、O-2和OH-)的能力，其中对DPPH清除率最高，且清除效果与肽的浓度呈正相关。通过对分子量分布研究发现，分子量在500-1000Da和180-500Da之间的肽对DPPH清除率最高。
Wheat germ is the life source of wheat kernel. Although it makes up about1.4%-3.9%of wheatkernel, it contains highly concentrated nutrients and is considered as “treasure house of naturalnutrition for human”. The total production of wheat kernel in China is the highest in the world andits yield is up to280-420million tons every year. However, the development of wheat germ is late inour country. For a long time, only a small amount of wheat germ is used to product wheat germ oil,while the majority of defatted wheat germ is used for animal feed, because of the wheat bran mixed.Up to now, wheat germ is still in a high quality resource low utilization condition.
The purpose of this study is to enhance the utilization value of wheat germ. We not only focuseson the production and evaluation of wheat germ oil with highly activity components by supercriticalCO_2extraction technology, but also pay attention to the use of defatted wheat germ. In order toexplore new method to modify wheat germ protein, Maillard reaction and fermentation technologywere applied. The main results and conclusions obtained are as follows.
1. The supercritical CO_2extraction technology was applied to extract wheat germ oil. Theoptimal extract condition was obtained through single factor experiments and a Box-Behnken designcombined with response surface methodology. The maximal oil yield of10.38%was achieved underthis optimal condition of wheat germ particle size60-80mesh, water content4.37%, extractionpressure30MPa, extraction temperature40℃, extraction time1.7h, separation pressure5MPa andseparation temperature50℃. The VEcontent was3.19mg/g under this condition. Data showed that apositive correlation was found between oil yield and VEcontent, but the correlation was notsignificant. The extraction condition may be selected according to the aim of extraction.
2. Different extraction methods showed greater impact on the content of activitycomponents (such as VEand total phenols) in wheat germ oil. The oil obtained by supercritical CO_2extraction owned higher content of activity components, such as VE, so the oil showed strongerDPPH radical scavenging ability at different concentration. However, different extraction methodsshowed no impact on the composition of fatty acids and amino acids.
3. The oil obtained by supercritical CO_2extraction showed better quality than the oilobtained by solvent extraction in appearance, physical and chemical index, oxidation stability and soon. The oil obtained was rich in polyunsaturated fatty acid (69.73%), especially in linoleic acid(64.82%). The mainly fatty acids distributed in Sn-2were unsaturated fatty acids, such as oleic acidand linoleic acid (80%). The oil has a unique smell of wheat germ,44volatile compounds wereisolated and identified by HS-SPME-GC-MS. The content of unsaponifiable matters in oil was4.16%, and5compounds were identified by GC-MS. The main content of unsaponifiable matterswas sterol, especially β-sitosterol (64.64%). In addition, the residual oil of defatted wheat germobtained supercritical CO_2extraction was only0.96%and the water content was0.47%. Thedefatted meal was lower denaturation and the content of protein was high (34.3%). The compositionof amino acid was balance and the essential amino acids were abundant.
4. The intakes of wheat germ oil had no significant effect on the normal growth and theheart, spleen and kidney index, but significantly reduced the liver index. Compared with the Controlgroup, in a simple high-fat model, the TC, TG and AI of serum were not significantly increased bythe intakes of wheat germ oil (p＜0.05). However, the TC, TG and HDL-C of liver lipid were significantly increased (p＜0.05). Moreover, the intakes of wheat germ oil can significantlyimproved the concentration of GSH, the activities of SOD and CAT, and reduced the concentrationof MDA in the two different models (p＜0.05).
5.“Enzyme hydrolysis+alkali-soluble and acid precipitation” technology was applied toextract protein from defatted wheat germ. The purity of protein was88.26%, and albumin was19.5%, globulin was27.37%, glutelin was21.24%, prolamine was2.94%. The distribution ofamino acid was balance and the amount of essential amino acids reached40.67%. Compared withsoy protein isolate, although foam properties and oil absorption capacity of wheat germ proteinwere better, its solubility, emulsifying and emulsion stability, as well as water absorption capacitywere poorer.
6. Wheat germ protein was modified by Maillard reaction with Dextran. The optimumcondition was obtained through single factor experiments and orthogonal optimization experiments.Under the condition(substrate concentration of2%, protein: dextran=1:1, pH11.0, temperature110℃, time20min), the DS reached12.05%and NSI was85.34%. After modification, the isoelectricpoint of the product moved to lower pH. The functional properties of conjugates, such as solubility,emulsification activity and emulsification stablility were improved greatly. The conjugates have thestrongest florescence intensity when emission was347nm and excitation was422nm, which inaccordance with the fluorescence characteristics of Maillard reaction product. The amino acidanalysis showed that the content of Lys and Arg was decreased by glycation. In addition, thenumber of α-helix structure was significantly increased in MRPs. Because the introduction ofhydroxy, the intermolecular steric effect was increased and thermal stability was enhanced slightly.
7. Wheat germ peptides were prepared by fermentation with Bacillus Subtilis B1. Thefermentation condition was optimized through single factor experiments and a Box-Behnkendesign combined with response surface methodology. A maximal yield of peptides was achieved8.69mg/mL under optimal conditions: wheat germ particle size60-80mesh, pH6.5, inoculum size8%, fermentation temperature31°C and time48h. A positive correlation (R2=0.9911) was foundbetween the concentration of peptides and total antioxidant capacity. The peptides presented asignificant does-dependent on scavenging activities of DPPH, hydroxyl and superoxide anionradicals, especially on scavenging activities of DPPH in the molecular weight distribution ofpeptides between500-1000Da and180-500Da.
引文
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