鲐鱼多肽的抗氧化活性与抗疲劳作用研究
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摘要
海洋低值鱼类产量高，蛋白质含量丰富，利用低值鱼制备特定的生物活性肽具有重要意义。本文以蛋白质含量丰富的鲐鱼为原料，利用酶解技术、超滤分级技术优化制备抗氧化活性肽，研究其抗氧化活性的稳定性，并采用离子交换层析和凝胶过滤层析技术对目标多肽进行分离纯化，采用体外化学模型和动物模型相结合的方法评价目标肽段的抗氧化活性和抗疲劳功效，初步探讨其抗氧化活性与抗疲劳作用的关系。主要研究结果如下：
1．确定了鲐鱼多肽的最佳酶解工艺：鲐鱼是一种高蛋白（粗蛋白含量为16.40%）、低脂肪的原料，其氨基酸种类丰富，尤其是与抗氧化活性有关的氨基酸，如谷氨酸、亮氨酸、赖氨酸含量较高，是一种制备抗氧化肽的优质原料。采用胰蛋白酶、酸性蛋白酶、木瓜蛋白酶、风味蛋白酶和中性蛋白酶水解鲐鱼鱼肉，综合比较了各水解产物的水解度、氮素回收率、DPPH自由基清除能力以及相对分子质量分布，最终确认中性蛋白酶为最佳用酶。在单因素实验基础上进行响应面优化实验，得到最佳条件为：加酶量1726.85U/g，pH值7.0，酶解温度39.55℃，酶解时间5.5h，液固比25:1。在最优条件下验证其DPPH自由基清除率为78.43%0.32，接近预测值79.19%。
2．鲐鱼多肽的分离纯化技术研究：采用超滤法对鲐鱼多肽(MPH)进行分级得到5个组分，抗氧化活性表明，分子量小于2.5kDa的三个组分(MPH-III，MPH-IV和MPH-V)抗氧化活性较高。离子交换色谱能够依据电荷数量的差异、凝胶色谱根据分子量大小的差异可对不同多肽进行有效的分离。本文采用SPSephadex C-25阳离子交换色谱和Sephadex G-25凝胶色谱依据电荷和分子量对MPHs (<2.5kDa)进行分离纯化研究，通过高效液相色谱分析，发现分子量为1664Da的多肽对DPPH自由基和羟自由基清除能力最强。
3．鲐鱼多肽的稳定性研究：研究发现，鲐鱼低分子量的多肽热稳定性非常好，即使在100℃高温处理后也不会降低其抗氧化活性；冻融次数和紫外线照射对MPHs的DPPH自由基和羟自由基清除能力无显著性影响(P<0.05)；pH值对MPHs抗氧化活性影响较大，随着pH值增大，其DPPH自由基和羟自由基清除能力降低，当pH值为9.2时，MPHs的DPPH自由基清除率减少89%；MPHs对金属离子敏感，特别是Fe2+、Fe3+、Zn2+和Cu2+能显著降低MPHs的抗氧化活性，尤其是Fe2+和Fe3+浓度达到5mM时，其DPPH自由基清除率仅为1.1%和0.6%，Cu2+浓度达到5mM时则完全抑制了MPHs的DPPH自由基清除能力，而K+、Mg2+对MPHs抗氧化活性影响不显著(P<0.05)。
4．鲐鱼多肽的抗疲劳功效评价：对分级得到的鲐鱼多肽MPHs进行了体内抗疲劳功效评价，结果表明：MPHs对小鼠体重和生长无影响，可提高小鼠肝脏系数、脾脏系数和胸腺系数，具有一定的免疫能力；MPHs能显著延长小鼠力竭游泳时间，MPHs低、中、高剂量组分别为对照组的2.8、3.0和4.8倍，其浓度与小鼠游泳时间呈正相关性；与对照组相比，MPHs各剂量组分别增加肝糖原含量33%50%，降低小鼠体内的乳酸、血尿素氮和丙二醛含量14%19%、16%17%和16%31%；，抗氧化物酶体系SOD和GSH-Px的活力分别提高11%15%和9%13%，表明MPHs具有一定的体内抗氧化活性。MPHs各剂量组中GSH-Px的活力与小鼠力竭游泳时间之间的相关系数达到0.912，说明MPHs在具有体内抗氧化活性的同时，还具有抗疲劳作用，两者之间具有很好的相关性。
Marine low-value fishes had high productivity and high protein content, it wouldbe of great importance in processing the low-value fishes into special bioactivepeptides. In our study, the antioxidant active peptides were prepared from mackerelprotein which with high protein content through enzymatic technology andultrafiltration. Their antioxidant activity stabilities were also studied, andion-exchange chromatography and gel filtration chromatography were used toseparate and purify the target peptides. Finally, chemical models in vitro and animalmodels in vivo were used to evaluate the antioxidant activity and antifatigue effect oftarget peptides, and the relationship between antioxidant activity and antifatigue effectof target peptides were discussed. The research results of this study were as follows:
1. Determine the optimum enzymatic hydrolysis technology of mackerel peptides:Mackerel was a material with high protein content (16.40%), low fat content and avariety of amino acids, especially Glu, Lys and Leu which had been reported tocontribute greatly to the potency of antioxidant peptides, had high content. Themackerel was appropriate to prepare antioxidant peptides. Five kinds of proteases, i.e.trypsin, acid protease, papain, flavourzymeand neutrase were used for hydrolysis toproduce the mackerel protein hydrolysate, considering the degree of hydrolysis,nitrogen recovery, DPPH radical scavenging activity and the relative molecularweight distributions, neutrase was the best enzyme for the hydrolysis. On the basis ofsingle factor experiment, the suggested hydrolysis conditions were performed by theresponse surface methodology (RSM): enzyme concentration1726.85U/g,pH7.00,temperature39.55℃, time5.50h and water to substrate at25:1. Themaximum DPPH scavenging activity was78.43%0.32, which was similar to theexperimental value of79.19%.
2. The research on the separation and purification of mackerel peptides: TheMPH was fractionated into five portions using ultrafiltration, the results of antioxidantactivities showed that the MPHs with molecular weight below2.5kDa (MPH-III, MPH-IV and MPH-V) exhibited higher antioxidant activities. Ion-exchangechromatography separated the peptides on the basis of different number of charge andgel filtration chromatography separated the peptides on the different size of molecularweight. Finally, the MPHs (<2.5kDa) was isolated and purified through SP SephadexC-25ion-exchange chromatography and Sephadex G-25gel filtration chromatography,respectively, and the peptide which had the highest DPPH radicals and hydroxylradicals scavenging activities was identified as the molecular weight of1664Da usingHPLC.
3. The stabilities of mackerel peptides: The antioxidant activity stabilities ofMPHs were investigated, and the results showed that the mackerel protein peptideshad good stabilities of temperature even though the high temperature of100℃could not reduce their antioxidant activities. In addition, the freeze-melt frequencyand UV had no significant effect on DPPH radicals and hydroxyl radicals scavengingactivities of MPHs (P<0.05). The MPHs had bad stabilities of pH, the DPPH radicalsand hydroxyl radicals scavenging activities of MPHs were reduced with the pH valuewas increase, when the pH value was9.2, the DPPH scavenging activity wasdecreased by89%. The MPHs was sensitive to metal ions, especially Fe2+、Fe3+、Zn2+and Cu2+could significantly decreased the antioxidant activities of MPHs, especiallywhen the concentrations of Fe2+and Fe3+were5mM, the DPPH scavenging activitieswere only1.1%and0.6%，respectively. while when the Cu2+with concentration of5mM could completely inhibited the DPPH scavenging activity of MPHs, on the otherhand, the K+、Mg2+had no significant effect on the antioxidant activities of MPHs(P<0.05).
4. The evaluation on antifatigue effect of mackerel peptides: the antifatigue effectsof MPHs were evaluated and the results showed that the MPHs had no effect on bodyweight and growth of mice, and could increase the viscera index of mice. The MPHscould significantly prolong the exhaustion swimming time, the MPHs-H, MPHs-Mand MPHs-L groups were4.8-fold,2.8-fold and3.0-fold of the control group, and allMPHs groups were showed a dose-dependent behavior; the liver glycogen level was markedly increased by3335%and the BUN (1617%), LA (1419%) and MDA(1631%) levels were decreased in MPHs groups compared to that of the controlgroup. The activities of SOD and GSH-Px were increased by1115%and913%,respectively, the result showed that the MPHs had in vitro antioxidant activities. Inaddition, the correlation coefficient between the activity of GSH-Px and exhaustionswimming time was0.912, which indicated that the in vivo antioxidant activities ofMPHs had a good relationship with their antifatigue effect.
1．确定了鲐鱼多肽的最佳酶解工艺：鲐鱼是一种高蛋白（粗蛋白含量为16.40%）、低脂肪的原料，其氨基酸种类丰富，尤其是与抗氧化活性有关的氨基酸，如谷氨酸、亮氨酸、赖氨酸含量较高，是一种制备抗氧化肽的优质原料。采用胰蛋白酶、酸性蛋白酶、木瓜蛋白酶、风味蛋白酶和中性蛋白酶水解鲐鱼鱼肉，综合比较了各水解产物的水解度、氮素回收率、DPPH自由基清除能力以及相对分子质量分布，最终确认中性蛋白酶为最佳用酶。在单因素实验基础上进行响应面优化实验，得到最佳条件为：加酶量1726.85U/g，pH值7.0，酶解温度39.55℃，酶解时间5.5h，液固比25:1。在最优条件下验证其DPPH自由基清除率为78.43%0.32，接近预测值79.19%。
2．鲐鱼多肽的分离纯化技术研究：采用超滤法对鲐鱼多肽(MPH)进行分级得到5个组分，抗氧化活性表明，分子量小于2.5kDa的三个组分(MPH-III，MPH-IV和MPH-V)抗氧化活性较高。离子交换色谱能够依据电荷数量的差异、凝胶色谱根据分子量大小的差异可对不同多肽进行有效的分离。本文采用SPSephadex C-25阳离子交换色谱和Sephadex G-25凝胶色谱依据电荷和分子量对MPHs (<2.5kDa)进行分离纯化研究，通过高效液相色谱分析，发现分子量为1664Da的多肽对DPPH自由基和羟自由基清除能力最强。
3．鲐鱼多肽的稳定性研究：研究发现，鲐鱼低分子量的多肽热稳定性非常好，即使在100℃高温处理后也不会降低其抗氧化活性；冻融次数和紫外线照射对MPHs的DPPH自由基和羟自由基清除能力无显著性影响(P<0.05)；pH值对MPHs抗氧化活性影响较大，随着pH值增大，其DPPH自由基和羟自由基清除能力降低，当pH值为9.2时，MPHs的DPPH自由基清除率减少89%；MPHs对金属离子敏感，特别是Fe2+、Fe3+、Zn2+和Cu2+能显著降低MPHs的抗氧化活性，尤其是Fe2+和Fe3+浓度达到5mM时，其DPPH自由基清除率仅为1.1%和0.6%，Cu2+浓度达到5mM时则完全抑制了MPHs的DPPH自由基清除能力，而K+、Mg2+对MPHs抗氧化活性影响不显著(P<0.05)。
4．鲐鱼多肽的抗疲劳功效评价：对分级得到的鲐鱼多肽MPHs进行了体内抗疲劳功效评价，结果表明：MPHs对小鼠体重和生长无影响，可提高小鼠肝脏系数、脾脏系数和胸腺系数，具有一定的免疫能力；MPHs能显著延长小鼠力竭游泳时间，MPHs低、中、高剂量组分别为对照组的2.8、3.0和4.8倍，其浓度与小鼠游泳时间呈正相关性；与对照组相比，MPHs各剂量组分别增加肝糖原含量33%50%，降低小鼠体内的乳酸、血尿素氮和丙二醛含量14%19%、16%17%和16%31%；，抗氧化物酶体系SOD和GSH-Px的活力分别提高11%15%和9%13%，表明MPHs具有一定的体内抗氧化活性。MPHs各剂量组中GSH-Px的活力与小鼠力竭游泳时间之间的相关系数达到0.912，说明MPHs在具有体内抗氧化活性的同时，还具有抗疲劳作用，两者之间具有很好的相关性。
Marine low-value fishes had high productivity and high protein content, it wouldbe of great importance in processing the low-value fishes into special bioactivepeptides. In our study, the antioxidant active peptides were prepared from mackerelprotein which with high protein content through enzymatic technology andultrafiltration. Their antioxidant activity stabilities were also studied, andion-exchange chromatography and gel filtration chromatography were used toseparate and purify the target peptides. Finally, chemical models in vitro and animalmodels in vivo were used to evaluate the antioxidant activity and antifatigue effect oftarget peptides, and the relationship between antioxidant activity and antifatigue effectof target peptides were discussed. The research results of this study were as follows:
1. Determine the optimum enzymatic hydrolysis technology of mackerel peptides:Mackerel was a material with high protein content (16.40%), low fat content and avariety of amino acids, especially Glu, Lys and Leu which had been reported tocontribute greatly to the potency of antioxidant peptides, had high content. Themackerel was appropriate to prepare antioxidant peptides. Five kinds of proteases, i.e.trypsin, acid protease, papain, flavourzymeand neutrase were used for hydrolysis toproduce the mackerel protein hydrolysate, considering the degree of hydrolysis,nitrogen recovery, DPPH radical scavenging activity and the relative molecularweight distributions, neutrase was the best enzyme for the hydrolysis. On the basis ofsingle factor experiment, the suggested hydrolysis conditions were performed by theresponse surface methodology (RSM): enzyme concentration1726.85U/g,pH7.00,temperature39.55℃, time5.50h and water to substrate at25:1. Themaximum DPPH scavenging activity was78.43%0.32, which was similar to theexperimental value of79.19%.
2. The research on the separation and purification of mackerel peptides: TheMPH was fractionated into five portions using ultrafiltration, the results of antioxidantactivities showed that the MPHs with molecular weight below2.5kDa (MPH-III, MPH-IV and MPH-V) exhibited higher antioxidant activities. Ion-exchangechromatography separated the peptides on the basis of different number of charge andgel filtration chromatography separated the peptides on the different size of molecularweight. Finally, the MPHs (<2.5kDa) was isolated and purified through SP SephadexC-25ion-exchange chromatography and Sephadex G-25gel filtration chromatography,respectively, and the peptide which had the highest DPPH radicals and hydroxylradicals scavenging activities was identified as the molecular weight of1664Da usingHPLC.
3. The stabilities of mackerel peptides: The antioxidant activity stabilities ofMPHs were investigated, and the results showed that the mackerel protein peptideshad good stabilities of temperature even though the high temperature of100℃could not reduce their antioxidant activities. In addition, the freeze-melt frequencyand UV had no significant effect on DPPH radicals and hydroxyl radicals scavengingactivities of MPHs (P<0.05). The MPHs had bad stabilities of pH, the DPPH radicalsand hydroxyl radicals scavenging activities of MPHs were reduced with the pH valuewas increase, when the pH value was9.2, the DPPH scavenging activity wasdecreased by89%. The MPHs was sensitive to metal ions, especially Fe2+、Fe3+、Zn2+and Cu2+could significantly decreased the antioxidant activities of MPHs, especiallywhen the concentrations of Fe2+and Fe3+were5mM, the DPPH scavenging activitieswere only1.1%and0.6%，respectively. while when the Cu2+with concentration of5mM could completely inhibited the DPPH scavenging activity of MPHs, on the otherhand, the K+、Mg2+had no significant effect on the antioxidant activities of MPHs(P<0.05).
4. The evaluation on antifatigue effect of mackerel peptides: the antifatigue effectsof MPHs were evaluated and the results showed that the MPHs had no effect on bodyweight and growth of mice, and could increase the viscera index of mice. The MPHscould significantly prolong the exhaustion swimming time, the MPHs-H, MPHs-Mand MPHs-L groups were4.8-fold,2.8-fold and3.0-fold of the control group, and allMPHs groups were showed a dose-dependent behavior; the liver glycogen level was markedly increased by3335%and the BUN (1617%), LA (1419%) and MDA(1631%) levels were decreased in MPHs groups compared to that of the controlgroup. The activities of SOD and GSH-Px were increased by1115%and913%,respectively, the result showed that the MPHs had in vitro antioxidant activities. Inaddition, the correlation coefficient between the activity of GSH-Px and exhaustionswimming time was0.912, which indicated that the in vivo antioxidant activities ofMPHs had a good relationship with their antifatigue effect.
引文
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