蛋清抗氧化肽活性及作用机理研究
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摘要
本研究是国家“863”项目计划项目(2007AA10Z329)——“蛋清抗氧化肽与降压肽的生物制备技术”研究课题子课题的一部分。
本研究主要对中试条件下生产的蛋清肽进行抗氧化生物活性评价并探讨其机理。建立了蛋清肽体外抗氧化活性多指标标准化评价体系,以DPPH自由基清除率、超氧阴离子自由基清除率、还原力吸光度值及油脂过氧化值四个指标评价蛋清肽的体外抗氧化活性;以受试小鼠血清学指标及皮肤组织中羟脯氨酸含量为指标评价蛋清肽在体内清除自由基及抑制脂质过氧化的能力;以受试小鼠的生产性能指标考察蛋清肽对小鼠的营养学功能;以脏器系数评价蛋清肽对受试小鼠免疫功能的影响;利用HPLC凝胶过滤色谱测定经碱性蛋白酶和风味蛋白酶双酶酶解制得的蛋清高F值寡肽的分子量分布范围。
Research on the antioxidant activity and the mechanism of peptides derived from egg white protein is attached to a branch of the“863”-plan called Biont Preparative Technique of Antioxidant Peptides and Underpressure Peptides.This paper develop five main kinds of work. The conclusions are listed below.
1.It could be found when study on the antioxidant activity in vitro that:
(1) It could be found when study on the effect of ethanol concentration on the DPPH free radical scaving activity of egg white protein hydrolysates and high Fisher ratio oligopeptide derived from egg white protein that: white protein hydrolysates and high Fisher ratio oligopeptide derived from egg white protein are are more suitable to 50% ethanol.
(2)It could be found the superoxide anion free radical scaving activity of egg white protein hydrolysates and high Fisher ratio oligopeptide derived from egg white protein showed that: the superoxide anion scaving activity of egg white protein hydrolysates shows such sequence listed: 1h> 2h> 3h> 4h. There is a good positive correlation between the dose of high Fisher ratio oligopeptide and superoxide anion free radical scaving activity.
( 3 ) It could be found the reducing power of egg white protein hydrolysates and high Fisher ratio oligopeptide derived from egg white protein showed that: the reducing power of egg white protein hydrolysates shows such sequence listed: 1h> 2h> 3h> 4h. There is a good positive correlation between the dose of high Fisher ratio oligopeptide with reducing power.
(4)It could be found the POV value of egg white protein hydrolysates and high Fisher ratio oligopeptide derived from egg white protein showed that: the POV value of egg white protein hydrolysates shows such sequence listed: 1h> 2h> 3h> 4h. There is a good positive correlation between the dose of high Fisher ratio oligopeptide with POV value.
(5)It could be draw the conclusion that egg white protein hydrolysates shows such sequence listed :1h> 2h> 3h> 4h in four items of evaluating system in vitro. There are good positive correlations between the dose of high Fisher ratio oligopeptide with the four items.
After all, the egg white protein hydrolysates could scavenge free radicals, enhance reducing power and inhibition lipid peroxidation. It had great antioxidant activity in vitro.
2 It could be found when study on the antioxidant activity in vivo that:
(1)4.5mg/(g·bw·d) proteolysis sampleⅠcan restrain the process of lipid peroxidation and protect skin from free radical.the experiment result for 30d by intragastric administration discerned that macro-proteolysis sample by alkaline proteinase enzymolysis 2h have the ability of restraining the process lipid peroxidation.
(2)4.5mg/(g·bw·d) proteolysis sampleⅡcan protect skin from free radical ,doubtfully,.the experiment result for 30d by intragastric administration discern that micromolecule high Fisher ratio oligopeptide from egg white protein by ambi-proteinase enzymolysis have the ability of cleaning free radical.
(3)In vivo antioxidant activity of positive control bean oligopeptide is high than proteolysis sampleⅠandⅡfrom egg white protein
(4)Different dose of high Fisher ratio oligopeptide from egg white protein can raise the content of SOD、GSH-Px、HYP while cut down the content of MDA by mouse vivo experiment for 30d by intragastric administration, especially 18mg/(g·bw·d) was significant above-mentioned four number index . (P<0.05或P<0.01)。
( 5 ) In the consideration of egg albumen peptide effected theγ- aminobutyric acid neurotransmitter in mouse brain tissue ,we found 0.9mg/(g·bw·d) egg albumen high F value oligopeptide was significantly degrade theγ- aminobutyric acid content(P<0.05);while 90mg/(g·bw·d) egg albumen high F value oligopeptide was significantly increase theγ- aminobutyric acid content(P<0.05).
3. Study of suffered mouse production trait by egg albumen peptide ,we found that :
(1) The egg albumen proteolysis obtained from one time enzymolysis and the egg albumen high F value oligopeptide from double enzymolysis used to treat with the weight, the average day increasing weight of 30d mouse ,we found that there was no significantly effects.
(2) With the treatment of the egg albumen peptide and the positive control sample ,the the average day increasing weight of 30d mouse was no significantly effect , overall showed the tendency of“decrease—increase ---decrease”,the average ingestion appetite of each experiment group was lower than the intragastric administration distilled water; Compared to controlling group , the intragastric administration 30d 4.5mg/(g·bw·d),the average day ingestion appetite for mouse by the high F value egg albumen oligopeptide was significantly increased, the other oligopeptide density were out of the treatment by 30d ; take a wide view effect of egg albumen peptide treating with the average day ingestion appetite for the 90d mouse ,except 4.5mg/(g·bw·d)egg albumen proteolysis from one time enzymolysis for four hours, all the treatments could significantly decrease the average day ingestion appetite of the mouse.
(3)The intake of suitable egg albumen peptide could increase the module of thymus and spleen ,but the exorbitancy high dose could increase the burden of organ, leading to the significantly change of other organ .
4.Molecular weight distribution of high Fisher ratio oligopeptide
(1)The range of molecular weight of high Fisher ratio oligopeptide that was centring production was analysis to by HPLC gelatum chromatographic. the experiment determine the best technical conditions as follows: mobile phase ratio is aethylnitrile:water: trifluoroacetic acid=10:90:0.1、flow rate is 0.5mL/min、to enter sample amount is 1μL、sample concentration is 0.2mg/mL、detect wave length is 220nm and column temperature is 30℃.
(2)Under the best chromatographic condition,it could be seen that the retention time of Gly-Gly-Gly standard preparation was 20.50min, the retention time of Gly-Gly–Tyr-Arg standard preparation was 21.63min and the retention time of Bacitracine standard preparation was 20.75min. It revealed the linear correlation of molecular weight logarithm of standard preparations and there retention time. The regression equation was y=-0.5018x+13.545 and the correlation coefficient was 0.9941showed in regression curve.
(3)Under the best chromatographic condition, high Fisher ratio oligopeptide was analysis by chromatographic gelatum. molecular weight of sample can be calculated according to retention time of sample and regression equation: y=-0.5018x+13.545.The molecular weight distribution of 61.95%high Fisher ratio oligopeptide derived from egg white protein ranged from 189.93~599.62Da. it was made of 2～5 amino acids. The portion of free aminoacid of the molecular weight is 82.47Da was 10.422%
本研究主要对中试条件下生产的蛋清肽进行抗氧化生物活性评价并探讨其机理。建立了蛋清肽体外抗氧化活性多指标标准化评价体系,以DPPH自由基清除率、超氧阴离子自由基清除率、还原力吸光度值及油脂过氧化值四个指标评价蛋清肽的体外抗氧化活性;以受试小鼠血清学指标及皮肤组织中羟脯氨酸含量为指标评价蛋清肽在体内清除自由基及抑制脂质过氧化的能力;以受试小鼠的生产性能指标考察蛋清肽对小鼠的营养学功能;以脏器系数评价蛋清肽对受试小鼠免疫功能的影响;利用HPLC凝胶过滤色谱测定经碱性蛋白酶和风味蛋白酶双酶酶解制得的蛋清高F值寡肽的分子量分布范围。
Research on the antioxidant activity and the mechanism of peptides derived from egg white protein is attached to a branch of the“863”-plan called Biont Preparative Technique of Antioxidant Peptides and Underpressure Peptides.This paper develop five main kinds of work. The conclusions are listed below.
1.It could be found when study on the antioxidant activity in vitro that:
(1) It could be found when study on the effect of ethanol concentration on the DPPH free radical scaving activity of egg white protein hydrolysates and high Fisher ratio oligopeptide derived from egg white protein that: white protein hydrolysates and high Fisher ratio oligopeptide derived from egg white protein are are more suitable to 50% ethanol.
(2)It could be found the superoxide anion free radical scaving activity of egg white protein hydrolysates and high Fisher ratio oligopeptide derived from egg white protein showed that: the superoxide anion scaving activity of egg white protein hydrolysates shows such sequence listed: 1h> 2h> 3h> 4h. There is a good positive correlation between the dose of high Fisher ratio oligopeptide and superoxide anion free radical scaving activity.
( 3 ) It could be found the reducing power of egg white protein hydrolysates and high Fisher ratio oligopeptide derived from egg white protein showed that: the reducing power of egg white protein hydrolysates shows such sequence listed: 1h> 2h> 3h> 4h. There is a good positive correlation between the dose of high Fisher ratio oligopeptide with reducing power.
(4)It could be found the POV value of egg white protein hydrolysates and high Fisher ratio oligopeptide derived from egg white protein showed that: the POV value of egg white protein hydrolysates shows such sequence listed: 1h> 2h> 3h> 4h. There is a good positive correlation between the dose of high Fisher ratio oligopeptide with POV value.
(5)It could be draw the conclusion that egg white protein hydrolysates shows such sequence listed :1h> 2h> 3h> 4h in four items of evaluating system in vitro. There are good positive correlations between the dose of high Fisher ratio oligopeptide with the four items.
After all, the egg white protein hydrolysates could scavenge free radicals, enhance reducing power and inhibition lipid peroxidation. It had great antioxidant activity in vitro.
2 It could be found when study on the antioxidant activity in vivo that:
(1)4.5mg/(g·bw·d) proteolysis sampleⅠcan restrain the process of lipid peroxidation and protect skin from free radical.the experiment result for 30d by intragastric administration discerned that macro-proteolysis sample by alkaline proteinase enzymolysis 2h have the ability of restraining the process lipid peroxidation.
(2)4.5mg/(g·bw·d) proteolysis sampleⅡcan protect skin from free radical ,doubtfully,.the experiment result for 30d by intragastric administration discern that micromolecule high Fisher ratio oligopeptide from egg white protein by ambi-proteinase enzymolysis have the ability of cleaning free radical.
(3)In vivo antioxidant activity of positive control bean oligopeptide is high than proteolysis sampleⅠandⅡfrom egg white protein
(4)Different dose of high Fisher ratio oligopeptide from egg white protein can raise the content of SOD、GSH-Px、HYP while cut down the content of MDA by mouse vivo experiment for 30d by intragastric administration, especially 18mg/(g·bw·d) was significant above-mentioned four number index . (P<0.05或P<0.01)。
( 5 ) In the consideration of egg albumen peptide effected theγ- aminobutyric acid neurotransmitter in mouse brain tissue ,we found 0.9mg/(g·bw·d) egg albumen high F value oligopeptide was significantly degrade theγ- aminobutyric acid content(P<0.05);while 90mg/(g·bw·d) egg albumen high F value oligopeptide was significantly increase theγ- aminobutyric acid content(P<0.05).
3. Study of suffered mouse production trait by egg albumen peptide ,we found that :
(1) The egg albumen proteolysis obtained from one time enzymolysis and the egg albumen high F value oligopeptide from double enzymolysis used to treat with the weight, the average day increasing weight of 30d mouse ,we found that there was no significantly effects.
(2) With the treatment of the egg albumen peptide and the positive control sample ,the the average day increasing weight of 30d mouse was no significantly effect , overall showed the tendency of“decrease—increase ---decrease”,the average ingestion appetite of each experiment group was lower than the intragastric administration distilled water; Compared to controlling group , the intragastric administration 30d 4.5mg/(g·bw·d),the average day ingestion appetite for mouse by the high F value egg albumen oligopeptide was significantly increased, the other oligopeptide density were out of the treatment by 30d ; take a wide view effect of egg albumen peptide treating with the average day ingestion appetite for the 90d mouse ,except 4.5mg/(g·bw·d)egg albumen proteolysis from one time enzymolysis for four hours, all the treatments could significantly decrease the average day ingestion appetite of the mouse.
(3)The intake of suitable egg albumen peptide could increase the module of thymus and spleen ,but the exorbitancy high dose could increase the burden of organ, leading to the significantly change of other organ .
4.Molecular weight distribution of high Fisher ratio oligopeptide
(1)The range of molecular weight of high Fisher ratio oligopeptide that was centring production was analysis to by HPLC gelatum chromatographic. the experiment determine the best technical conditions as follows: mobile phase ratio is aethylnitrile:water: trifluoroacetic acid=10:90:0.1、flow rate is 0.5mL/min、to enter sample amount is 1μL、sample concentration is 0.2mg/mL、detect wave length is 220nm and column temperature is 30℃.
(2)Under the best chromatographic condition,it could be seen that the retention time of Gly-Gly-Gly standard preparation was 20.50min, the retention time of Gly-Gly–Tyr-Arg standard preparation was 21.63min and the retention time of Bacitracine standard preparation was 20.75min. It revealed the linear correlation of molecular weight logarithm of standard preparations and there retention time. The regression equation was y=-0.5018x+13.545 and the correlation coefficient was 0.9941showed in regression curve.
(3)Under the best chromatographic condition, high Fisher ratio oligopeptide was analysis by chromatographic gelatum. molecular weight of sample can be calculated according to retention time of sample and regression equation: y=-0.5018x+13.545.The molecular weight distribution of 61.95%high Fisher ratio oligopeptide derived from egg white protein ranged from 189.93~599.62Da. it was made of 2～5 amino acids. The portion of free aminoacid of the molecular weight is 82.47Da was 10.422%
引文
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