扫频超声预处理对玉米醇溶蛋白特性及其酶法制备ACE抑制肽的影响
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摘要
玉米醇溶蛋白为玉米淀粉加工工业的主要副产物,因其含有大量的非极性氨基酸,不溶于水,此外还缺乏色氨酸和赖氨酸,营养价值低,在食品工业中的应用受到限制。国内外研究表明,玉米醇溶蛋白酶解物具有良好的ACE抑制活性,但是传统的制备方法存在酶解时间长、酶的使用效率低和产物转化率低等缺点。本论文旨在通过扫频超声波预处理改善玉米醇溶蛋白酶解制备ACE抑制肽的效率和产品活性。基此,本文初步研究了扫频超声场的性质；系统研究了扫频超声处理后,玉米醇溶蛋白溶解度、巯基含量、表面疏水性等理化性质,荧光光谱、红外光谱、圆二色谱等分子结构特征,以及表面形貌、粒径等蛋白颗粒表面微观结构的变化；探讨了经超声处理后玉米醇溶蛋白制备ACE抑制肽的酶解特性；研究了超声预处理后反应热动力学特性的变化。主要研究结论如下：
(1)关于扫频超声场的声学性质,高速摄像法和碘释放法的研究表明,40kHz的超声振板产生的超声空化产额最高,观察到的超声空化现象最为明显,且扫频模式的效果优于定频模式。
(2)关于扫频超声对玉米醇溶蛋白理化性质影响的研究发现,一定的扫频超声处理能够显著提高玉米醇溶蛋白的水溶性和疏水性、促使肽链伸展、改变其转变温度,上述参数的变化说明超声可以被用于促使蛋白向着有利于酶解反应的方向发展,尤其是疏水性的增加,意味着超声波引起了蛋白内部疏水性氨基酸外露,更有利于蛋白内部疏水性基团与酶的接触,从而可制备出高活性的ACE抑制肽。
(3)关于扫频超声对玉米醇溶蛋白分子结构特性的影响,紫外和荧光发射光谱分析发现,超声波改变了蛋白的结构,处理后表现出荧光猝灭现象,说明超声波使玉米醇溶蛋白多肽链发生了不同程度的伸展；红外光谱和圆二色谱分析表明,超声波处理后玉米醇溶蛋白α-螺旋结构的含量逐渐增加,p-折叠和无规则卷曲变化无规律,表明扫频超声处理改变了包括氢键在内的维持玉米醇溶蛋白二级结构的作用力,从而使四种二级结构的含量发生了变化。
(4)关于扫频超声对玉米醇溶蛋白颗粒微观结构的影响,原子力显微镜测试结果表明,超声波使得玉米醇溶蛋白良好的网状结构被破坏断裂,导致很多蛋白聚集体变得分散,并出现了许多游离球状蛋白颗粒；玉米醇溶蛋白颗粒变小,且蛋白更加均匀；电子显微镜分析表明,与对照相比,超声波使玉米醇溶蛋白颗粒表面更加粗糙,甚至出现小的空洞,表明超声导致蛋白颗粒疏松,蛋白酶容易进入玉米醇溶蛋白颗粒内部,有利于疏水性氨基酸的释放；粒径检测发现,较高频率的超声使蛋白分子粒径分布向大的粒径方向迁移,表明扫频超声预处理引起了玉米醇溶蛋白分子的极化；破坏了维持蛋白分子四级结构的作用力,导致了蛋白亚基解离和疏水性基团的外露；两极分化的亚基相互吸引通过非共价键重新形成更大的蛋白聚集体。
(5)关于扫频超声对玉米醇溶蛋白酶解特性影响的研究发现,定频和扫频两种模式的超声波均能引起玉米醇溶蛋白水解度的显著提高,且能不同程度地改变玉米醇溶蛋白酶解液的ACE抑制率；其中40+2kHz扫频超声波预处理玉米醇溶蛋白时,其水解度最高提高了21.7%,而其酶解液的ACE抑制率最高达到了46.1%,比对照组抑制率提高1.33倍；40±2kHz/68±2kHz组合双频组合超声波预处理后,酶解液的抑制活性最高为43.7%,与未超声对照组相比提高了1.21倍。酶解产物分子量分布及氨基酸分析进一步表明,超声波预处理后酶解产物的小分子量肽链增多,疏水性氨基酸含量和支链氨基酸含量提高,这些都与酶解产物ACE抑制率的提高有关。
(6)关于扫频超声对玉米醇溶蛋白酶解热动力学参数的影响。单频扫频超声波和双频扫频式超声波分别使玉米醇溶蛋白的Ea从5.84kJ/mol降到4.70和5.74kJ/mol;使AH从46.05kJ/mol降到36.55和45.20kJ/mol。说明扫频超声预处理均有利于蛋白酶解催化反应的进行,使反应更容易。
Zein is a co-product of the corn starch industry. It is insoluble in water due to high proportion of nonpolar amino acid residues. In addition, the notable absence of tryptophane and lysine in zein accounts for its lower nutritional value and limits its application in food industry. Zein hydrolysate showed a strong ACE-inhibitory activity. However, traditional enzymolysis has many disadvantages, such as long enzymolysis time, low utilization rate of the enzyme, and low conversion rate of the substrate. The objective of this study was to enhance the efficiency and productivity by preparation of ACE-inhibitory peptides on enzymatic from zein with sweeping frequency ultrasond (SFU) pretreatment. The characteristics of ultrasonic field of water was studied first The objective of this research was to observe the impact of SFU pretreatment on physicochemical properties of zein, were precisely studied by the methods of solubility, surface hydrophobicity and free sulfhydryl content. The structure of zein after SFU pretreatment was investigated by the methods of CD and FT-IR spectra and change of surface morphology, particle size was also invested to assess the enzymatic properties of zein for preparation of ACE inhibitory peptides after ultrasonic pretreatment. Thermodynamic parameters of zein changed with different sweeping frequency ultrasonic treatments. It proved that ultrasonic could promote enzymatic hydrolysis. The main achievements are summarized as follows:
(1) Characteristics of ultrasonic field of water were studied by high speed photography and iodine release method.The ultrasounic cavitation yield at40kHz was remained highest for the range of ultrasounic frequency and the cavitation phenomenon observed for40kHz by high speed photography also more obvious than others The effect ultrasounic cavitation for sweeping model is better than fixed model.
(2) Effects of SFU pretreatment on physicochemical properties of zein was studied. After SFU pretreatment significant increase was observed in increased in solubility and surface hydrophobicity which indicates the polypeptide chains of zein extended and denaturation temperatures were changed. The change for some ultrasonic parameters indicated that ultrasonic pretreatment was beneficial to protein hydrolysis reaction. Specialy an increase for surface hydrophobicity indicated ultrasonic pretreatment help the enzyme to attack the interior of zein aggregate easily which resulted in the release of hydrophobic amino acids from zein aggregate. These changes play an important role in improvement of ACE-inhibitory activity of hydrolysates.
(4) Effects of SFU pretreatment on physical microstructure of zein were studied. Microstructure analysis by AFM revealed that upon SFU pretreatment, the fine meshwork structure of zein was ruptured and there were many dissociated protein globules. As compared to control the particle size of ultrasound-pretreated zein was smaller as well as more homogeneous. Analysis of SEM showed that compared with the smooth surface of untreated zein, the surface structures of the ultrasonic-treated zein became rough and their specific surface area was larger. Interestingly, several micro-holes appeared in the zein following ultrasound pretreatment. The results suggested that ultrasound might loosen the tissue of zein aggregate, and help the enzyme to attack the interior of zein aggregate easily which resulted in the release of hydrophobic amino acids from zein aggregate. The particles size of zein increased upon the higher ultrasounic frequency showed sweeping frequency ultrasounic pretreatment induced polarization of zein and dissociation of sub-units and molecular unfolding; changed molecular conformation and caused hydrophobic groups and free sulfhydryls buried inside the molecules to expose.
(5) Effect of SFU pretreatment on enzymatic characteristics of zein was studied. Significant increase was observed in degree of hydrolysates of zein and hydrolysates of ACE inhibition.The degree of hydrolysates of zein was significantly increased under the sweeping ultrasonic and fixed ultrasonic pretreatment, and hydrolysates of ACE inhibition were also increased. Compared to control, the40±2-kHz SFU pretreatment increased by21.7%and hydrolysate ACE-inhibitory activity was46.1%representing by1.33times increase over the control. Hydrolysate ACE-inhibitory activity was43.7%after dual sweeping frequency ultrasounic pretreatment (40±2kHz/68±2kHz) pretreatment, representing by1.21times increase over the control. Amino acid composition and molecular weight profile revealed that hydrolysates of sweeping frequency ultrasounic pretreatment zein had a higher relative concentration of low molecular weight peptides, hydrophobic amino acid and branched-chain amino acid as compared to control. These changes play an important role in improvement of ACE-inhibitory activity of hydrolysates
(6) Analyzing results of thermodynamic parameters for zein showed that the values of Ea, AH, AS and AG could be decreased after sweeping frequency ultrasonic pretreatment. Single frequency sweeping and dual frequency ultrasonic pretreatment decreased thermodynamic parameters Ea from5.84kJ/mol to4.70and5.74kJ/mol;ΔH from46.05kJ/mol to36.55and45.20kJ/mol.It indicated the digestion of zein become more facility and benefit to release more activity ACE-inhibitory peptides.
(1)关于扫频超声场的声学性质,高速摄像法和碘释放法的研究表明,40kHz的超声振板产生的超声空化产额最高,观察到的超声空化现象最为明显,且扫频模式的效果优于定频模式。
(2)关于扫频超声对玉米醇溶蛋白理化性质影响的研究发现,一定的扫频超声处理能够显著提高玉米醇溶蛋白的水溶性和疏水性、促使肽链伸展、改变其转变温度,上述参数的变化说明超声可以被用于促使蛋白向着有利于酶解反应的方向发展,尤其是疏水性的增加,意味着超声波引起了蛋白内部疏水性氨基酸外露,更有利于蛋白内部疏水性基团与酶的接触,从而可制备出高活性的ACE抑制肽。
(3)关于扫频超声对玉米醇溶蛋白分子结构特性的影响,紫外和荧光发射光谱分析发现,超声波改变了蛋白的结构,处理后表现出荧光猝灭现象,说明超声波使玉米醇溶蛋白多肽链发生了不同程度的伸展；红外光谱和圆二色谱分析表明,超声波处理后玉米醇溶蛋白α-螺旋结构的含量逐渐增加,p-折叠和无规则卷曲变化无规律,表明扫频超声处理改变了包括氢键在内的维持玉米醇溶蛋白二级结构的作用力,从而使四种二级结构的含量发生了变化。
(4)关于扫频超声对玉米醇溶蛋白颗粒微观结构的影响,原子力显微镜测试结果表明,超声波使得玉米醇溶蛋白良好的网状结构被破坏断裂,导致很多蛋白聚集体变得分散,并出现了许多游离球状蛋白颗粒；玉米醇溶蛋白颗粒变小,且蛋白更加均匀；电子显微镜分析表明,与对照相比,超声波使玉米醇溶蛋白颗粒表面更加粗糙,甚至出现小的空洞,表明超声导致蛋白颗粒疏松,蛋白酶容易进入玉米醇溶蛋白颗粒内部,有利于疏水性氨基酸的释放；粒径检测发现,较高频率的超声使蛋白分子粒径分布向大的粒径方向迁移,表明扫频超声预处理引起了玉米醇溶蛋白分子的极化；破坏了维持蛋白分子四级结构的作用力,导致了蛋白亚基解离和疏水性基团的外露；两极分化的亚基相互吸引通过非共价键重新形成更大的蛋白聚集体。
(5)关于扫频超声对玉米醇溶蛋白酶解特性影响的研究发现,定频和扫频两种模式的超声波均能引起玉米醇溶蛋白水解度的显著提高,且能不同程度地改变玉米醇溶蛋白酶解液的ACE抑制率；其中40+2kHz扫频超声波预处理玉米醇溶蛋白时,其水解度最高提高了21.7%,而其酶解液的ACE抑制率最高达到了46.1%,比对照组抑制率提高1.33倍；40±2kHz/68±2kHz组合双频组合超声波预处理后,酶解液的抑制活性最高为43.7%,与未超声对照组相比提高了1.21倍。酶解产物分子量分布及氨基酸分析进一步表明,超声波预处理后酶解产物的小分子量肽链增多,疏水性氨基酸含量和支链氨基酸含量提高,这些都与酶解产物ACE抑制率的提高有关。
(6)关于扫频超声对玉米醇溶蛋白酶解热动力学参数的影响。单频扫频超声波和双频扫频式超声波分别使玉米醇溶蛋白的Ea从5.84kJ/mol降到4.70和5.74kJ/mol;使AH从46.05kJ/mol降到36.55和45.20kJ/mol。说明扫频超声预处理均有利于蛋白酶解催化反应的进行,使反应更容易。
Zein is a co-product of the corn starch industry. It is insoluble in water due to high proportion of nonpolar amino acid residues. In addition, the notable absence of tryptophane and lysine in zein accounts for its lower nutritional value and limits its application in food industry. Zein hydrolysate showed a strong ACE-inhibitory activity. However, traditional enzymolysis has many disadvantages, such as long enzymolysis time, low utilization rate of the enzyme, and low conversion rate of the substrate. The objective of this study was to enhance the efficiency and productivity by preparation of ACE-inhibitory peptides on enzymatic from zein with sweeping frequency ultrasond (SFU) pretreatment. The characteristics of ultrasonic field of water was studied first The objective of this research was to observe the impact of SFU pretreatment on physicochemical properties of zein, were precisely studied by the methods of solubility, surface hydrophobicity and free sulfhydryl content. The structure of zein after SFU pretreatment was investigated by the methods of CD and FT-IR spectra and change of surface morphology, particle size was also invested to assess the enzymatic properties of zein for preparation of ACE inhibitory peptides after ultrasonic pretreatment. Thermodynamic parameters of zein changed with different sweeping frequency ultrasonic treatments. It proved that ultrasonic could promote enzymatic hydrolysis. The main achievements are summarized as follows:
(1) Characteristics of ultrasonic field of water were studied by high speed photography and iodine release method.The ultrasounic cavitation yield at40kHz was remained highest for the range of ultrasounic frequency and the cavitation phenomenon observed for40kHz by high speed photography also more obvious than others The effect ultrasounic cavitation for sweeping model is better than fixed model.
(2) Effects of SFU pretreatment on physicochemical properties of zein was studied. After SFU pretreatment significant increase was observed in increased in solubility and surface hydrophobicity which indicates the polypeptide chains of zein extended and denaturation temperatures were changed. The change for some ultrasonic parameters indicated that ultrasonic pretreatment was beneficial to protein hydrolysis reaction. Specialy an increase for surface hydrophobicity indicated ultrasonic pretreatment help the enzyme to attack the interior of zein aggregate easily which resulted in the release of hydrophobic amino acids from zein aggregate. These changes play an important role in improvement of ACE-inhibitory activity of hydrolysates.
(4) Effects of SFU pretreatment on physical microstructure of zein were studied. Microstructure analysis by AFM revealed that upon SFU pretreatment, the fine meshwork structure of zein was ruptured and there were many dissociated protein globules. As compared to control the particle size of ultrasound-pretreated zein was smaller as well as more homogeneous. Analysis of SEM showed that compared with the smooth surface of untreated zein, the surface structures of the ultrasonic-treated zein became rough and their specific surface area was larger. Interestingly, several micro-holes appeared in the zein following ultrasound pretreatment. The results suggested that ultrasound might loosen the tissue of zein aggregate, and help the enzyme to attack the interior of zein aggregate easily which resulted in the release of hydrophobic amino acids from zein aggregate. The particles size of zein increased upon the higher ultrasounic frequency showed sweeping frequency ultrasounic pretreatment induced polarization of zein and dissociation of sub-units and molecular unfolding; changed molecular conformation and caused hydrophobic groups and free sulfhydryls buried inside the molecules to expose.
(5) Effect of SFU pretreatment on enzymatic characteristics of zein was studied. Significant increase was observed in degree of hydrolysates of zein and hydrolysates of ACE inhibition.The degree of hydrolysates of zein was significantly increased under the sweeping ultrasonic and fixed ultrasonic pretreatment, and hydrolysates of ACE inhibition were also increased. Compared to control, the40±2-kHz SFU pretreatment increased by21.7%and hydrolysate ACE-inhibitory activity was46.1%representing by1.33times increase over the control. Hydrolysate ACE-inhibitory activity was43.7%after dual sweeping frequency ultrasounic pretreatment (40±2kHz/68±2kHz) pretreatment, representing by1.21times increase over the control. Amino acid composition and molecular weight profile revealed that hydrolysates of sweeping frequency ultrasounic pretreatment zein had a higher relative concentration of low molecular weight peptides, hydrophobic amino acid and branched-chain amino acid as compared to control. These changes play an important role in improvement of ACE-inhibitory activity of hydrolysates
(6) Analyzing results of thermodynamic parameters for zein showed that the values of Ea, AH, AS and AG could be decreased after sweeping frequency ultrasonic pretreatment. Single frequency sweeping and dual frequency ultrasonic pretreatment decreased thermodynamic parameters Ea from5.84kJ/mol to4.70and5.74kJ/mol;ΔH from46.05kJ/mol to36.55and45.20kJ/mol.It indicated the digestion of zein become more facility and benefit to release more activity ACE-inhibitory peptides.
引文
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