超声波辅助酶解谷朊粉制备抗氧化肽的研究
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摘要
本课题研究了碱性蛋白酶酶解谷朊粉,以提高酶解产物的抗氧化能力作为最终目标,使用超声波作为酶解辅助手段,考察了超声波功率、频率、酶解时间对酶解产物抗氧化活性的影响,通过单因素和正交试验筛选出最佳工艺参数为:超声波功率(400w),低频(25kHz)、酶解时间30min。在最佳酶解条件下(温度50℃,pH9.0),亚铁离子螯合能力IC50值为0.513mg/mL,当蛋白质含量为2.4mg/mL时,酶解产物的还原能力为0.385,ABTS自由基清除能力相当于1.40mmol/L Trolox,且对亚油酸自动氧化抑制效果显著(P<0.05)。
研究了超滤处理方式富集酶解产物中的抗氧化组分的效果,结果表明:分子量在5kDa以下的组分抗氧化能力最强,当浓度为0.2和0.4mg/mL时,亚铁离子螯合能力是超滤前样品的2倍左右;对亚油酸自氧化的抑制能力在36h后比其它分子量片段的组分效果明显;而当浓度为2.4mg/mL时,TEAC值达到1.45mmol/L Trolox;并且由高效液相色谱法对其相对分子质量进行分析发现,分子量在2kDa以下的组分含量占了90.81%,推测抗氧化肽主要集中在2kDa以下。
检测分析了谷朊粉酶解产物(WGH)及5kDa超滤膜超滤样(WGHU-5)对D-半乳糖诱导衰老模型小鼠抗氧化能力的影响,结果表明:四组实验组都可以提高D-半乳糖致衰老模型中小鼠血浆、心脏、脑、肝脏和肾组织的SOD(超氧化物歧化酶)、T-AOC(总抗氧化能力)、GSH-PX(谷胱甘肽过氧化物酶)的活性,而降低其体内MDA(丙二醛)含量,WGHU-5高剂量组效果最为显著(P<0.01),说明WGH具有明显的防衰老抗氧化作用。
采用DA201-C型大孔吸附树脂对谷朊粉抗氧化肽进行分级筛选。静态吸附实验发现大孔吸附树脂对谷朊粉抗氧化肽吸附很快,在30min时基本达到吸附平衡,75%乙醇静态解吸率在30min时可以达到70%。动态洗脱中,采用乙醇梯度洗脱效果良好,可以将谷朊粉抗氧化肽很好的分级分离,乙醇梯度洗脱组各组分的抗氧化能力随着疏水性的增加先增加后减小。75%乙醇洗脱组分的抗氧化能力最高,在蛋白质浓度为0.4mg/mL时,亚铁离子螯合能力达到84.13%,TEAC值为0.36mmol/L Trolox,除盐率达到93.56%。。
采用质谱分析了产物中两个主要组分的相对相对分子质量为951和1349Da。MALDI-TOF-TOF MS结合串联质谱仪(TOF MS/MS)的方法得出:951Da肽段的一级序列为Ser-Arg-Tyr-Asp-Ala-Ile-Arg-Ala(简称为SRYDAIRA);1349Da肽段的一级序列为Tyr-Pro-Leu-Glu-Ala-Ala-Gly-Asp-Thr-Lys-Arg-Glu(简称为YPLEAAGDTKRE)。
Wheat gluten was hydrolyzed by Alcalase 2.4L under ultrasound. Different antioxidant assays in vitro were employed to evaluate the antioxidant activities of the wheat gluten hydrolysate (WGH) obtained. The best ultrasonic parameters were the maximum ultrasonic power (400w) and low-frequency (25kHz) under the conditions of hydrolysis 30min. Under optimal conditions (temperature 50℃, pH9.0), WGHL (WGH which was obtained by low frequency ultrasound) exhibited the strongest antioxidant activities, with an IC50 value of 0.513mg/mL for ferrous iron-chelating activity, as well as a high and dose-dependent reducing power. In the linoleic acid system, a longer induction time indicated a significant decrease of lipid peroxidation. In addition, WGH also exhibited notable ABTS radical scavenging activity.
Then, the results of ultrafiltration showed that WGHU-5 (WGH with the molecular weight below 5kDa) possessed the highest antioxidant activity. When the concentration of 0.2-0.4 mg/mL, the ferrous ion chelating activity of WGHU-5 was twice as much as WGH. The oxidation of linoleic acid was significantly inhibited by the addition of WGH (P<0.05) after 36h than WGH. When the concentration of 4mg/mL, TEAC value reached 1.45mmol/L Trolox. And the high-performance liquid chromatography analysis of its molecular weight revealed that the fractions below 2kDa accounted for the majority. It can be conjectured that antioxidant peptides fractions were concentrated in 2kDa. In order to investigate the effect of WGH and WGHU-5 on D-galactose induced aging model mice. The mouse were divided into normal control group, D-galactose model group, WGH low dose group, WGH high dose group, WGHU-5 low dose group and WGHU-5 high dose group. The results show that high-dose group WGHU-5 can significantly improve SOD, T-AOC, GSH- PX activity (P<0.01) of plasma, heart, brain, liver and kidney tissue, but reduce the MDA (MDA) content. It indicated that wheat gluten hydrolysates had obvious antioxidant effect.
In order to get better quality products, the antioxidant peptide was fractionated by ethanol elution from a macroporous adsorption resin (DA201-C). It was found that adsorption reach equilibrium in 30min, and static desorption rate can reach 70% in 30min using the basic 75% ethanol. WGH can be well separated by using gradient elution ethanol. The basic inorganic salt ions are washed out at the end of elution. The antioxidant capacity of each component increased with the hydrophobicity and then decreased. 75% ethanol fractions exhibited the highest antioxidant activity, at a concentration of 0.4mg/mL, the ferrous ion chelating activity was 84.13%, TEAC value of 0.36mmol/L Trolox,and desalination rate reached 93.56%.
The fraction was then identified by MALDI-TOF-TOF MS/MS. Their molecular weights were 951Da and 1349Da respectively. The amino acid sequences were Ser-Arg-Tyr-Asp-Ala-Ile-Arg-Ala(SRYDAIRA) and Tyr-Pro-Leu-Glu-Ala-Ala-Gly-Asp-Thr-Lys- Arg-Glu(YPLEAAGDTKRE).
研究了超滤处理方式富集酶解产物中的抗氧化组分的效果,结果表明:分子量在5kDa以下的组分抗氧化能力最强,当浓度为0.2和0.4mg/mL时,亚铁离子螯合能力是超滤前样品的2倍左右;对亚油酸自氧化的抑制能力在36h后比其它分子量片段的组分效果明显;而当浓度为2.4mg/mL时,TEAC值达到1.45mmol/L Trolox;并且由高效液相色谱法对其相对分子质量进行分析发现,分子量在2kDa以下的组分含量占了90.81%,推测抗氧化肽主要集中在2kDa以下。
检测分析了谷朊粉酶解产物(WGH)及5kDa超滤膜超滤样(WGHU-5)对D-半乳糖诱导衰老模型小鼠抗氧化能力的影响,结果表明:四组实验组都可以提高D-半乳糖致衰老模型中小鼠血浆、心脏、脑、肝脏和肾组织的SOD(超氧化物歧化酶)、T-AOC(总抗氧化能力)、GSH-PX(谷胱甘肽过氧化物酶)的活性,而降低其体内MDA(丙二醛)含量,WGHU-5高剂量组效果最为显著(P<0.01),说明WGH具有明显的防衰老抗氧化作用。
采用DA201-C型大孔吸附树脂对谷朊粉抗氧化肽进行分级筛选。静态吸附实验发现大孔吸附树脂对谷朊粉抗氧化肽吸附很快,在30min时基本达到吸附平衡,75%乙醇静态解吸率在30min时可以达到70%。动态洗脱中,采用乙醇梯度洗脱效果良好,可以将谷朊粉抗氧化肽很好的分级分离,乙醇梯度洗脱组各组分的抗氧化能力随着疏水性的增加先增加后减小。75%乙醇洗脱组分的抗氧化能力最高,在蛋白质浓度为0.4mg/mL时,亚铁离子螯合能力达到84.13%,TEAC值为0.36mmol/L Trolox,除盐率达到93.56%。。
采用质谱分析了产物中两个主要组分的相对相对分子质量为951和1349Da。MALDI-TOF-TOF MS结合串联质谱仪(TOF MS/MS)的方法得出:951Da肽段的一级序列为Ser-Arg-Tyr-Asp-Ala-Ile-Arg-Ala(简称为SRYDAIRA);1349Da肽段的一级序列为Tyr-Pro-Leu-Glu-Ala-Ala-Gly-Asp-Thr-Lys-Arg-Glu(简称为YPLEAAGDTKRE)。
Wheat gluten was hydrolyzed by Alcalase 2.4L under ultrasound. Different antioxidant assays in vitro were employed to evaluate the antioxidant activities of the wheat gluten hydrolysate (WGH) obtained. The best ultrasonic parameters were the maximum ultrasonic power (400w) and low-frequency (25kHz) under the conditions of hydrolysis 30min. Under optimal conditions (temperature 50℃, pH9.0), WGHL (WGH which was obtained by low frequency ultrasound) exhibited the strongest antioxidant activities, with an IC50 value of 0.513mg/mL for ferrous iron-chelating activity, as well as a high and dose-dependent reducing power. In the linoleic acid system, a longer induction time indicated a significant decrease of lipid peroxidation. In addition, WGH also exhibited notable ABTS radical scavenging activity.
Then, the results of ultrafiltration showed that WGHU-5 (WGH with the molecular weight below 5kDa) possessed the highest antioxidant activity. When the concentration of 0.2-0.4 mg/mL, the ferrous ion chelating activity of WGHU-5 was twice as much as WGH. The oxidation of linoleic acid was significantly inhibited by the addition of WGH (P<0.05) after 36h than WGH. When the concentration of 4mg/mL, TEAC value reached 1.45mmol/L Trolox. And the high-performance liquid chromatography analysis of its molecular weight revealed that the fractions below 2kDa accounted for the majority. It can be conjectured that antioxidant peptides fractions were concentrated in 2kDa. In order to investigate the effect of WGH and WGHU-5 on D-galactose induced aging model mice. The mouse were divided into normal control group, D-galactose model group, WGH low dose group, WGH high dose group, WGHU-5 low dose group and WGHU-5 high dose group. The results show that high-dose group WGHU-5 can significantly improve SOD, T-AOC, GSH- PX activity (P<0.01) of plasma, heart, brain, liver and kidney tissue, but reduce the MDA (MDA) content. It indicated that wheat gluten hydrolysates had obvious antioxidant effect.
In order to get better quality products, the antioxidant peptide was fractionated by ethanol elution from a macroporous adsorption resin (DA201-C). It was found that adsorption reach equilibrium in 30min, and static desorption rate can reach 70% in 30min using the basic 75% ethanol. WGH can be well separated by using gradient elution ethanol. The basic inorganic salt ions are washed out at the end of elution. The antioxidant capacity of each component increased with the hydrophobicity and then decreased. 75% ethanol fractions exhibited the highest antioxidant activity, at a concentration of 0.4mg/mL, the ferrous ion chelating activity was 84.13%, TEAC value of 0.36mmol/L Trolox,and desalination rate reached 93.56%.
The fraction was then identified by MALDI-TOF-TOF MS/MS. Their molecular weights were 951Da and 1349Da respectively. The amino acid sequences were Ser-Arg-Tyr-Asp-Ala-Ile-Arg-Ala(SRYDAIRA) and Tyr-Pro-Leu-Glu-Ala-Ala-Gly-Asp-Thr-Lys- Arg-Glu(YPLEAAGDTKRE).
引文
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