猪皮胶原蛋白酶解液的抗氧化活性及其分离纯化研究
摘要
本论文以猪皮为原料提取胶原蛋白,用菠萝蛋白酶、木瓜蛋白酶、Alcalase2.4L碱性蛋白酶通过酶解制备胶原蛋白酶解产物,以水解度(DH)和超氧阴离子自由基清除率为指标,根据单因素和正交试验优化酶解条件；在此基础上,对最佳酶解条件下的胶原蛋白酶解产物的抗氧化性进行了研究,同时探讨了胶原蛋白酶解产物抗氧化性的稳定性；利用超滤、离子交换色谱以及凝胶过滤色谱等系列分离纯化技术从最佳酶解条件下的猪皮胶原蛋白酶解产物中分离纯化抗氧化活性肽。
菠萝蛋白酶的水解条件优化结果：时间5h、温度45℃、pH3.5、酶与底物比4000 U/g、底物浓度6%,此时水解度为5.25%,O2-·清除率为44.49%；木瓜蛋白酶的水解条件优化结果：时间6h、温度70℃、pH5.5、酶与底物比6250U/g、底物浓度6%,此时水解度为13.28%,O2-·清除率为41.97%；Alcalase的水解条件优化结果：时间4h、温度55。C、pH7.5、酶与底物比6000 U/g、底物浓度4%,此时水解度为9.55%,O2-·清除率为60.11%。Alcalase酶解后的产物抑制O2-·自由基活性最强。
用Alcalase最佳酶解工艺水解猪皮胶原所得酶解液的体外抗氧化活性测定结果显示：在10 mg/mL-50 mg/mL浓度范围内,该酶解液对·OH具有一定的清除作用,·OH最大清除率为56.38%, IC50=40.99mg/mL; DPPH·最大清除率为95.06%,IC50=3.89mg/mL; O2-·最大清除率为65.89%,IC50=16.43mg/mL;同时该酶解液具有一定的还原能力和抗脂质过氧化能力。总体而言,猪皮胶原蛋白酶解液采用不同的抗氧化活性检测方法进行检测都表现出一定的体外抗氧化效果。
猪皮胶原蛋白酶解产物抗氧化性对热(100℃,10min)、冷冻(-18℃,24h)基本稳定,且在酸性环境中的抗氧化性优于在碱性环境中。
猪皮胶原酶解液经截流分子量为lOkDa、5kDa、3kDa、2kDa的超滤膜分离后,得到了大于lOkDa、5kDa-10kDa、3kDa-5kDa、2kDa-3 kDa和小于2kDa的5个分子量肽段的组分,其中,分子量<2 kD的组分清除O2-·活性最高,多肽浓度为2.24 mg/mL时,清除率为41.11%,IC50值为2.85mg/mL。猪皮胶原蛋白酶解物经过SP-Sephadex C-25离子交换层析得到了7个新组分P1-P7。其中第1个峰P1峰的O2-·清除活性最高,多肽浓度为0.85 mg/mL时,清除率为46.30%,组分P1清除O2-·的IC50值为1.24 mg/mL。组分P1经过凝胶SephadexG-25层析柱后得到2个峰,其中第2个峰P1-B峰的O2-·清除活性最高,多肽浓度为0.9mg/mL时,清除率为49.43%,IC50值为0.98mg/mL。
Collagen was extracted from pig skin, the process of preparing of collagen hydrolysis from pig skin by Bromelain, Papain,Alcalase were studied in the present paper.The factors of effecting process of hydrolysis were investigated by determining the hydrolysis degree of collagen hydrolysis and the scavenging capacities of the hydrolysates on superoxide free radical.Enzymatic hydrolysis conditions were optimized through single factors and orthogonal experiment.The antioxygenic property of collagen hydrolysis was studied by five analytical methods.The stability of antioxygenic property was also evaluated.Peptides derived from porcine collagen hydrolysates were separated by ultrafiltration(UF) and consecutive chromatographic methods including ion exchange chromatography(IEC),and gel filtration chromatography(GFC).
The optimum hydrolysis conditions of Bromelain were determined:pH3.5, hydrolyzing temperature 45℃,ratio of Bromelain 4000 U/g, concentration of substrate 6% and total hydrolysis time 5 h.Under these conditions the hydrolysis degree of collagen protein could reach 5.25%,while the scavenging capacities of the hydrolysates on superoxide free radical could reach 44.49%; the optimum hydrolysis conditions of Papain were determined:pH5.5,hydrolyzing temperature 70℃,ratio of Papain 6250 U/g, concentration of substrate 6% and total hydrolysis time 6 h.Under these conditions the hydrolysis degree of collagen protein could reach 13.28%,while the scavenging capacities of the hydrolysates on superoxide free radical could reach 41.97%; the optimum hydrolysis conditions of Alcalase were determined:pH7.5, hydrolyzing temperature 55℃,ratio of Alcalase 6000 U/g, concentration of substrate 4% and total hydrolysis time 4 h,under these conditions the hydrolysis degree of collagen protein could reach 9.55%,while the scavenging capacities of the hydrolysates on superoxide free radical could reach 60.11%. The hydrolysates hydrolyzed by Alcalase showed the highest superoxide radical scavenging ability.
The antioxidant properties of collagen hydrolysates were determined in vitro. In a certain concentration range(10 mg/mL-50 mg/mL),the results showed that the enzymic hydrolysates of collagen had scavenging ability on hydroxyl radical, the maximum scavenging rates to·OH free radical were 56.38%,IC5o=40.99mg/mL; the maximum scavenging rates to DPPH·free radical were 95.06%, IC50=3.89mg/mL;the maximum scavenging rates to O2-·free radical was 65.89%,IC50 was 16.43mg/mL;the collagen hydrolysates also had certain reductive ability; moreover, the collagen hydrolysates could inhibit the lipid peroxidation.Generally speaking,the collagen hydrolysate showed certain antioxidatative activities with five different analytical methods in vitro.
The antioxygenic property of collagen hydrolysates were stabile to heat and freezing,furthermore,the antioxygenic property in acidic condition was better than in alkaline condition.
The peptides with superoxide radical scavenging activity were isolated and purified from collagen hydrolysates.Four different UF centrifuge tubes having a range of molecular weight cut-offs(MWCO)of 10,5,3 and 2kDa were used to fractionated the enzymic hydrolysates.The portion<2 kDa showed the highest superoxide radical scavenging ability, the maximum scavenging rates to O2-·free radical was 41.11% when the peptide concentration was 2.24 mg/mL, its IC50 value was2.85mg/mL.The collagen hydrolysates were load onto SP-Sephadex C-25 ion exchange column for further purification and seven peaks were eluted.The first peak P1 exhibited higher superoxide radical scavenging ability, the maximum scavenging rates to O2-·free radical was 46.30% when the peptide concentration was 0.85 mg/mL,IC50 value was 1.24 mg/mL.The P1 portion was loaded onto Sephadex G-25 gel filtration column for further isolation.Two peaks were eluted and the second peak P1-B showed the highest superoxide radical scavenging ability, the maximum scavenging rates to O2-·free radical was 49.43% when the peptide concentration was 0.9 mg/mL,IC50 value was 0.98mg/mL
菠萝蛋白酶的水解条件优化结果：时间5h、温度45℃、pH3.5、酶与底物比4000 U/g、底物浓度6%,此时水解度为5.25%,O2-·清除率为44.49%；木瓜蛋白酶的水解条件优化结果：时间6h、温度70℃、pH5.5、酶与底物比6250U/g、底物浓度6%,此时水解度为13.28%,O2-·清除率为41.97%；Alcalase的水解条件优化结果：时间4h、温度55。C、pH7.5、酶与底物比6000 U/g、底物浓度4%,此时水解度为9.55%,O2-·清除率为60.11%。Alcalase酶解后的产物抑制O2-·自由基活性最强。
用Alcalase最佳酶解工艺水解猪皮胶原所得酶解液的体外抗氧化活性测定结果显示：在10 mg/mL-50 mg/mL浓度范围内,该酶解液对·OH具有一定的清除作用,·OH最大清除率为56.38%, IC50=40.99mg/mL; DPPH·最大清除率为95.06%,IC50=3.89mg/mL; O2-·最大清除率为65.89%,IC50=16.43mg/mL;同时该酶解液具有一定的还原能力和抗脂质过氧化能力。总体而言,猪皮胶原蛋白酶解液采用不同的抗氧化活性检测方法进行检测都表现出一定的体外抗氧化效果。
猪皮胶原蛋白酶解产物抗氧化性对热(100℃,10min)、冷冻(-18℃,24h)基本稳定,且在酸性环境中的抗氧化性优于在碱性环境中。
猪皮胶原酶解液经截流分子量为lOkDa、5kDa、3kDa、2kDa的超滤膜分离后,得到了大于lOkDa、5kDa-10kDa、3kDa-5kDa、2kDa-3 kDa和小于2kDa的5个分子量肽段的组分,其中,分子量<2 kD的组分清除O2-·活性最高,多肽浓度为2.24 mg/mL时,清除率为41.11%,IC50值为2.85mg/mL。猪皮胶原蛋白酶解物经过SP-Sephadex C-25离子交换层析得到了7个新组分P1-P7。其中第1个峰P1峰的O2-·清除活性最高,多肽浓度为0.85 mg/mL时,清除率为46.30%,组分P1清除O2-·的IC50值为1.24 mg/mL。组分P1经过凝胶SephadexG-25层析柱后得到2个峰,其中第2个峰P1-B峰的O2-·清除活性最高,多肽浓度为0.9mg/mL时,清除率为49.43%,IC50值为0.98mg/mL。
Collagen was extracted from pig skin, the process of preparing of collagen hydrolysis from pig skin by Bromelain, Papain,Alcalase were studied in the present paper.The factors of effecting process of hydrolysis were investigated by determining the hydrolysis degree of collagen hydrolysis and the scavenging capacities of the hydrolysates on superoxide free radical.Enzymatic hydrolysis conditions were optimized through single factors and orthogonal experiment.The antioxygenic property of collagen hydrolysis was studied by five analytical methods.The stability of antioxygenic property was also evaluated.Peptides derived from porcine collagen hydrolysates were separated by ultrafiltration(UF) and consecutive chromatographic methods including ion exchange chromatography(IEC),and gel filtration chromatography(GFC).
The optimum hydrolysis conditions of Bromelain were determined:pH3.5, hydrolyzing temperature 45℃,ratio of Bromelain 4000 U/g, concentration of substrate 6% and total hydrolysis time 5 h.Under these conditions the hydrolysis degree of collagen protein could reach 5.25%,while the scavenging capacities of the hydrolysates on superoxide free radical could reach 44.49%; the optimum hydrolysis conditions of Papain were determined:pH5.5,hydrolyzing temperature 70℃,ratio of Papain 6250 U/g, concentration of substrate 6% and total hydrolysis time 6 h.Under these conditions the hydrolysis degree of collagen protein could reach 13.28%,while the scavenging capacities of the hydrolysates on superoxide free radical could reach 41.97%; the optimum hydrolysis conditions of Alcalase were determined:pH7.5, hydrolyzing temperature 55℃,ratio of Alcalase 6000 U/g, concentration of substrate 4% and total hydrolysis time 4 h,under these conditions the hydrolysis degree of collagen protein could reach 9.55%,while the scavenging capacities of the hydrolysates on superoxide free radical could reach 60.11%. The hydrolysates hydrolyzed by Alcalase showed the highest superoxide radical scavenging ability.
The antioxidant properties of collagen hydrolysates were determined in vitro. In a certain concentration range(10 mg/mL-50 mg/mL),the results showed that the enzymic hydrolysates of collagen had scavenging ability on hydroxyl radical, the maximum scavenging rates to·OH free radical were 56.38%,IC5o=40.99mg/mL; the maximum scavenging rates to DPPH·free radical were 95.06%, IC50=3.89mg/mL;the maximum scavenging rates to O2-·free radical was 65.89%,IC50 was 16.43mg/mL;the collagen hydrolysates also had certain reductive ability; moreover, the collagen hydrolysates could inhibit the lipid peroxidation.Generally speaking,the collagen hydrolysate showed certain antioxidatative activities with five different analytical methods in vitro.
The antioxygenic property of collagen hydrolysates were stabile to heat and freezing,furthermore,the antioxygenic property in acidic condition was better than in alkaline condition.
The peptides with superoxide radical scavenging activity were isolated and purified from collagen hydrolysates.Four different UF centrifuge tubes having a range of molecular weight cut-offs(MWCO)of 10,5,3 and 2kDa were used to fractionated the enzymic hydrolysates.The portion<2 kDa showed the highest superoxide radical scavenging ability, the maximum scavenging rates to O2-·free radical was 41.11% when the peptide concentration was 2.24 mg/mL, its IC50 value was2.85mg/mL.The collagen hydrolysates were load onto SP-Sephadex C-25 ion exchange column for further purification and seven peaks were eluted.The first peak P1 exhibited higher superoxide radical scavenging ability, the maximum scavenging rates to O2-·free radical was 46.30% when the peptide concentration was 0.85 mg/mL,IC50 value was 1.24 mg/mL.The P1 portion was loaded onto Sephadex G-25 gel filtration column for further isolation.Two peaks were eluted and the second peak P1-B showed the highest superoxide radical scavenging ability, the maximum scavenging rates to O2-·free radical was 49.43% when the peptide concentration was 0.9 mg/mL,IC50 value was 0.98mg/mL
引文
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