蛋清肽制备及其生物功能性研究
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摘要
本文优化了蛋清抗氧化肽的制备工艺。选用胃蛋白酶酶解鸡蛋清,通过对加酶量、底物浓度、酶解时间、pH值等进行单因素分析,然后进行正交试验,优化得到胃蛋白酶酶解鸡蛋清制备抗氧化肽的最佳工艺条件为：酶解温度37℃、pH值2.0、底物浓度3%、加酶量9000 U/g、酶解时间5 h。
采用膜过滤对对制备获得的蛋清肽粗品进行超滤分级,将蛋清肽粗品按照分子量范围分为<1 KDa、1-2 KDa、2-5 KDa及>5 KDa四种蛋清肽,并对四种蛋清肽进行自由基清除能力测定。结果表明,分子量范围为2-5 KDa蛋清肽自由基清除能力最强,其清除羟基自由基的能力较蛋清肽粗品提高了1倍。并且,正交试验优化得到分子截留量为2 KDa、5 KDa超滤膜分级制备蛋清肽使膜效能达到最大的超滤条件均为：压力0.2 MPa、浓度2%、pH 6.0、温度25℃。
采用五种体外方法综合评价了蛋清肽粗品及2-5 KDa蛋清肽的抗氧化能力,试验结果表明,蛋清肽具有较强的羟基自由基清除能力、超氧阴离子清除能力、DPPH自由基清除能力以及还原能力,并且能够有效抑制油脂氧化。与蛋清肽粗品相比较,分子量范围为2-5 KDa蛋清肽,当浓度为5 mg/mL时,其对·OH-、O2-·清除能力以及还原能力分别提高了114.08%、39.1%及261.9%。
蛋清肽体内抗氧化能力及抗疲劳能力测定结果表明,灌胃蛋清肽样品组小鼠具有很强的耐疲劳能力与体内抗氧化能力。与空白对照组相比,灌胃原肽组及2-5 KDa高剂量组的小鼠体重分别提高了116.1%与108.1%。较对照组,灌胃2-5 KDa蛋清肽高剂量组小鼠的体内抗氧化指标如全血总抗氧化能力、谷胱甘肽酶活分别提高了1.9倍、64.3%；血浆丙二醛含量降低了66.5%。对疲劳指标,较对照组,灌胃2-5 KDa蛋清肽高剂量组小鼠的力竭游泳时间、肌糖原贮存量分别提高了2.8倍、40.8%；血尿氮与肝乳酸含量分别降低了40.9%与41.1%。
为了了解蛋清肽的实际应用价值,本文对蛋清肽的稳定性及应用理化指标进行了测定。分别考察了蛋清肽的贮藏稳定性、加热稳定性、酸碱稳定性以及保护剂对蛋清肽稳定性的影响。结果表明：蛋清肽在低温下比较稳定,4℃保存90 d后其抗氧化能力没有显著的降低。高温处理不利于蛋清肽生物活性的保持,且其在酸性条件下比较稳定；另外,保护剂的添加有利于蛋清肽溶解度的提高,但对羟基自由基清除能力没有增强作用。蛋清肽不但具有较强的起泡能力,乳化能力还表现出了极强的吸水、吸油能力以及持水能力。蛋清肽的这些性质均有利于其在食品行业及化妆品行业中应用。
In this paper, the technology of preparation antioxidant peptide of egg white was optimized.at first, and then pepsin was used as the hydrolytic enzyme. The enzymatic reaction conditions including enzyme concentration, substrate concentration, reaction time and pH value was studied successively by single factor experiment. The optimum enzymolysis condition of egg white was obtained with pepsin hydrolysis temperature 37℃, pH 2.0, substrate concentration 3%, dosage of enzyme 9000 U/g, and reaction time 5h.
Membrane filtration technology was used for separating egg white crude hydrolyzate. According to molecular weight, egg white peptide was divided into four parts, including <1 KDa,1-2 KDa,2-5 KDa and>5 KDa. Then the antioxidant activity of the four egg white peptide were Evaluated, and the peptide with the molecular weight between 2-5 KDa showed the strongest radical scavenging activity. Compared with the crude egg white peptide, the antioxidant activity of egg white peptide with molecular weight 2-5 KDa increased by about 100%. The results of orthogonal test suggested that:the optimum operation conditions of the membrane with molecular interception of 5 KDa and 2 KDa was pressure 0.2 MPa, feed concentration 2%, pH 6.0, and at 35℃.
Five in vitro methods were used to assess egg white peptides'antioxidant ability, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging, hydroxyl radical scavenging, superoxide anion-scavenging, reducing power and lipid peroxidation inhibition. The results showed that egg white peptide possessed strong antioxidant capacity and inhibition lipid oxidation capacity. Compared with the crude egg white peptide, the hydroxyl radical scavenging, superoxide anion-scavenging and reducing power of egg white peptide with molecular weight 2-5 KDa were increased 114.08%, 39.1% and 261.9%, separately.
Subsequently, in vivo antioxidant capacity and resistance to fatigue of egg white peptide were determined by animal experiments. Compared with the control group of blank, the groups fed egg white peptide showed strong resistance to fatigue, and oxidative indicators in the mouse also showed that egg peptide peptide possessed in vivo antioxidant capacity; The body weight of groups feding crude and 2-5 KDa egg white peptide increased by 116.1% and 108.1%, repetively. And the body antioxidant indicators such as total antioxidant capacity of whole blood, glutathione activity of the group mice fed the high dose egg white peptide with 2-5 KDa increased by 119% and 64.3%, repetively, and MDA levels of the plasma decreased in 66.5%. For the fatigue index, exhaustive swimming time, muscle glycogen storage contentncreased by 119% and 40.8%, repetively, urine nitrogen and liver lactate content decreased by 40.9% and 41.1%, repetively.
In order to understand the practical application value of the egg white peptide, the text also evaluated its stability and application of physical and chemical indexes. The storage stability, heat stability, pH stability and the effects of protection agents on the stability of the egg white peptide were determined. The results suggested that egg white peptide was more stable at low temperature, and the antioxidant capacity changed little after keeping for 90d at 4℃. High temperature is not conducive to maintaining biological activity of egg white peptide, and it relatively stable under acidic conditions. Moreover, the addition of protective agents in favor of increased the solubility of egg white peptide, but these had no effect on enhancing hydroxyl radical scavenging acticity. Egg white peptide has good foaming capacity, emulsifying capacity, water adsorption, water-holding capacity and oil absorption. The results all provided a foundation for its using in food and cosmetic industry.
采用膜过滤对对制备获得的蛋清肽粗品进行超滤分级,将蛋清肽粗品按照分子量范围分为<1 KDa、1-2 KDa、2-5 KDa及>5 KDa四种蛋清肽,并对四种蛋清肽进行自由基清除能力测定。结果表明,分子量范围为2-5 KDa蛋清肽自由基清除能力最强,其清除羟基自由基的能力较蛋清肽粗品提高了1倍。并且,正交试验优化得到分子截留量为2 KDa、5 KDa超滤膜分级制备蛋清肽使膜效能达到最大的超滤条件均为：压力0.2 MPa、浓度2%、pH 6.0、温度25℃。
采用五种体外方法综合评价了蛋清肽粗品及2-5 KDa蛋清肽的抗氧化能力,试验结果表明,蛋清肽具有较强的羟基自由基清除能力、超氧阴离子清除能力、DPPH自由基清除能力以及还原能力,并且能够有效抑制油脂氧化。与蛋清肽粗品相比较,分子量范围为2-5 KDa蛋清肽,当浓度为5 mg/mL时,其对·OH-、O2-·清除能力以及还原能力分别提高了114.08%、39.1%及261.9%。
蛋清肽体内抗氧化能力及抗疲劳能力测定结果表明,灌胃蛋清肽样品组小鼠具有很强的耐疲劳能力与体内抗氧化能力。与空白对照组相比,灌胃原肽组及2-5 KDa高剂量组的小鼠体重分别提高了116.1%与108.1%。较对照组,灌胃2-5 KDa蛋清肽高剂量组小鼠的体内抗氧化指标如全血总抗氧化能力、谷胱甘肽酶活分别提高了1.9倍、64.3%；血浆丙二醛含量降低了66.5%。对疲劳指标,较对照组,灌胃2-5 KDa蛋清肽高剂量组小鼠的力竭游泳时间、肌糖原贮存量分别提高了2.8倍、40.8%；血尿氮与肝乳酸含量分别降低了40.9%与41.1%。
为了了解蛋清肽的实际应用价值,本文对蛋清肽的稳定性及应用理化指标进行了测定。分别考察了蛋清肽的贮藏稳定性、加热稳定性、酸碱稳定性以及保护剂对蛋清肽稳定性的影响。结果表明：蛋清肽在低温下比较稳定,4℃保存90 d后其抗氧化能力没有显著的降低。高温处理不利于蛋清肽生物活性的保持,且其在酸性条件下比较稳定；另外,保护剂的添加有利于蛋清肽溶解度的提高,但对羟基自由基清除能力没有增强作用。蛋清肽不但具有较强的起泡能力,乳化能力还表现出了极强的吸水、吸油能力以及持水能力。蛋清肽的这些性质均有利于其在食品行业及化妆品行业中应用。
In this paper, the technology of preparation antioxidant peptide of egg white was optimized.at first, and then pepsin was used as the hydrolytic enzyme. The enzymatic reaction conditions including enzyme concentration, substrate concentration, reaction time and pH value was studied successively by single factor experiment. The optimum enzymolysis condition of egg white was obtained with pepsin hydrolysis temperature 37℃, pH 2.0, substrate concentration 3%, dosage of enzyme 9000 U/g, and reaction time 5h.
Membrane filtration technology was used for separating egg white crude hydrolyzate. According to molecular weight, egg white peptide was divided into four parts, including <1 KDa,1-2 KDa,2-5 KDa and>5 KDa. Then the antioxidant activity of the four egg white peptide were Evaluated, and the peptide with the molecular weight between 2-5 KDa showed the strongest radical scavenging activity. Compared with the crude egg white peptide, the antioxidant activity of egg white peptide with molecular weight 2-5 KDa increased by about 100%. The results of orthogonal test suggested that:the optimum operation conditions of the membrane with molecular interception of 5 KDa and 2 KDa was pressure 0.2 MPa, feed concentration 2%, pH 6.0, and at 35℃.
Five in vitro methods were used to assess egg white peptides'antioxidant ability, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging, hydroxyl radical scavenging, superoxide anion-scavenging, reducing power and lipid peroxidation inhibition. The results showed that egg white peptide possessed strong antioxidant capacity and inhibition lipid oxidation capacity. Compared with the crude egg white peptide, the hydroxyl radical scavenging, superoxide anion-scavenging and reducing power of egg white peptide with molecular weight 2-5 KDa were increased 114.08%, 39.1% and 261.9%, separately.
Subsequently, in vivo antioxidant capacity and resistance to fatigue of egg white peptide were determined by animal experiments. Compared with the control group of blank, the groups fed egg white peptide showed strong resistance to fatigue, and oxidative indicators in the mouse also showed that egg peptide peptide possessed in vivo antioxidant capacity; The body weight of groups feding crude and 2-5 KDa egg white peptide increased by 116.1% and 108.1%, repetively. And the body antioxidant indicators such as total antioxidant capacity of whole blood, glutathione activity of the group mice fed the high dose egg white peptide with 2-5 KDa increased by 119% and 64.3%, repetively, and MDA levels of the plasma decreased in 66.5%. For the fatigue index, exhaustive swimming time, muscle glycogen storage contentncreased by 119% and 40.8%, repetively, urine nitrogen and liver lactate content decreased by 40.9% and 41.1%, repetively.
In order to understand the practical application value of the egg white peptide, the text also evaluated its stability and application of physical and chemical indexes. The storage stability, heat stability, pH stability and the effects of protection agents on the stability of the egg white peptide were determined. The results suggested that egg white peptide was more stable at low temperature, and the antioxidant capacity changed little after keeping for 90d at 4℃. High temperature is not conducive to maintaining biological activity of egg white peptide, and it relatively stable under acidic conditions. Moreover, the addition of protective agents in favor of increased the solubility of egg white peptide, but these had no effect on enhancing hydroxyl radical scavenging acticity. Egg white peptide has good foaming capacity, emulsifying capacity, water adsorption, water-holding capacity and oil absorption. The results all provided a foundation for its using in food and cosmetic industry.
引文
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