牛肉预煮液中肌肽的分离纯化及功能性研究
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摘要
肌肽是一种广泛存在于动物肌肉中的天然的二肽,具有抗氧化、与金属离子螯合、清除自由基和供氢、供电子能力等活性,在动物肌肉组织中具有较高的含量。牛肉预煮液是在牛肉加工过程中产生的副产品,在过去一直作为废液被倒掉而没有引起相关厂家的重视。本试验预煮液中肌肽含量进行了检测,并分别使用液相和凝胶过滤色谱对肌肽进行了分离,最后对肌肽的抗氧化性进行了测定。
通过对牛肉预煮液进行超滤等处理后,使用高效液相色谱法对预煮液中肌肽进行了初步检测,发现牛肉中肌肽的含量较高,其含量可达4530.72 mg／kg,在预煮液中含量也较高,达到1510.24 mg／L。通过加标实验,进一步确定了该峰就是肌肽。使用液相收集目标峰,收集液相中样品的目标峰,进行检测,检测结果可以看出收集得到的肌肽纯度较高,达到95%以上,可以用于制备。
酶解处理对肉中肌肽含量有一定的影响,实验发现,使用木瓜蛋白酶酶解处理可以较显著的提高肉中肌肽的含量,但使用菠萝蛋白酶处理对肌肽的含量影响不大。
肌肽对超氧阴离子有清除作用,但肌肽浓度增大到50 mmol／L时,抑制作用逐渐减弱,粗提液的也具有一定程度的抑制作用,但抑制作用低于同浓度的肌肽标准液。随肌肽标准液浓度增加,肌肽对羟自由基的清除率也随之增加,1mmol／L时清除率达到17.94%,表明肌肽在低浓度对·OH就有显著的清除能力,随肌肽浓度增加对-OH的清除能力快速增长,20 mmol／L达到71.04%,肌肽浓度继续增大时,清除率增长变慢。
1 mmol／L肌肽标准液对猪肉匀浆中丙二醛的形成量就能起到显著的抑制作用(P<0.05),此时对体系中脂类氧化的抑制率即达到12%,5 mmol／L肌肽对体系中脂类氧化的抑制率达15%,20 mmol／L肌肽对体系中脂类氧化的抑制率达47%,表明不同浓度的肌肽均能对猪肉匀浆中脂类氧化起显著的抑制作用,其中随浓度的增加效果增强。与BHT和α-生育酚相比,肌肽对TBARS的抑制能力较高,并且由于肌肽是动物体内的内源性二肽,对人体没有危害,也不会造成二次污染,因此作为冷鲜肉的保鲜剂不但效果良好而且没有危害性。
肌肽标品对DPPH有显著的清除能力(p<0.05),随着浓度的增大,清除率不断增大,并且不同浓度的肌肽标品对DPPH的清楚效果差异性显著。肌肽粗提液对DPPH具有很强的清除效果,但其清除能力远远高于肌肽纯品,经过去离子处理的肌肽粗提液对DPPH的清除作用高于肌肽粗提液,具体原因尚不清楚。
肌肽粗提液的抗氧化率弱于肌肽纯品,肌肽纯品的抗氧化率弱于Vc,而Vc的抗氧化率弱于茶多酚。随着肌肽粗提物溶液浓度的递增,抑菌圈也成递减趋势,当粗提物溶液浓度达到Vc溶液浓度4倍时,它们的抑菌圈大小接近,抗氧化性表观抗氧化率接近。
肌肽可以延长冷鲜肉的保存时间,添加了肌肽的冷鲜肉保藏时间由不添加保鲜剂的9天增加到15天,其效果要优于抗坏血酸,肌肽粗提液也可以延长冷鲜肉的贮藏时间。
Carnosine belongs to a natural di-peptide which exists widely in animal muscle. It has anti-oxidation, compound metal ions, cleanup free radicals and supply hydrogen and electron, which has a high content in animal muscle tissue. Pre-cooked beef juice is the byproduct in the course of beef production processing, which has often been discarded as a useless liquid and does not draw attention to the relevant manufacturers up to now. In this study, purification and determination of carnosine from pre-cooking beef juice was implemented by using Gel filtering chromatography and HPLC, at last the antioxidant carnosine were determined.
Firstly, retention time of carnosine peak on C18 column was determined and compared with standard sample by HPLC method. Final, carnosine peak of actual sample was verified by a series of tests spiked. Carnosine content in pre-cooked beef juice ultra-filtrated achieve to 1510.24 mg/L, namely, carnosine of beef is 4530.72 mg/kg. The target peak from sample ultra-filtrated was collected and detected using HPLC, which showed higher purity of carnosine up to 95%, can be used in the preparation.
Papain was found that it could significantly improve carnosine content in beef during the zymohydrolysis, in a contrast, treatment samples of bromelain were not notably effect on the content of carnosine.
Carnosine is able to clear the superoxide anion, but its inhibition is gradually weakened with carnosine concentration increasing to 50 mmol/L. the extraction sample also has a certain degree of inhibition, but inhibition is lower than the same concentration of standard solution of carnosine. And the clearance rate of carnosine for hydroxyl radical is increasing with increase of the concentration of carnosine, the clearance rate is to 17.94% while 1 mmol/L carnosine, scavenging capacity of carnosine for·OH is significantly rapid growing to reach 71.04% while concentrations of carnosine increased 20 mmol/L, but carnosine concentrations continued to increase, the clear increase in the rate of change slow.
1 mmol/L standard solution of carnosine is able to play a significant inhibition (P <0.05) TBARS formation in pork homogenate, and the inhibition of lipid oxidation rate could reach 12%,5 mmol/L carnosine on lipid oxidation system inhibition rate of was 15%,20 mmol/L carnosine on lipid oxidation system suppression rate reached to 47%. These attributes indicate that concentration of carnosine was significantly inhibited on lipid oxidation of pork homogenates the effect of which increased with the effect of enhanced concentration. And BHT and a-tocopherol compared to carnosine inhibited the ability of higher TBARS and carnosine because animals are the two endogenous peptides, there is no harm to humans, it will not cause secondary pollution, so as Cold fresh meat Preservative effect of not only good and no harm.
Standard of carnosine has significant DPPH scavenging capacity (p<0.05), and clearance rate increasing with concentration increasing, and the different concentration solution of standard carnosine was significant difference each other for clear DPPH. Pre-cooked beef juice ultra-filtrated has a strong effect on the removal of DPPH, but the capacity is much higher than the standard carnosine. Pre-cooked beef juice by ion exchange column obtains better effect of DPPH scavenging than the pre-cooked beef juice ultra-filtrated.
Antioxidation of pre-cooked beef juice ultra-filtrated is lower than that of pure carnosine, but standard carnosine is less than Vc, and Vc is weaker than polyphenols according to these determination results of the several reagents antioxidation. Inhibition zone reduces as the increasing of carnosine extracts solution concentration. These observed numbers of the inhibition zone and the apparent antioxidant antioxidant are respectively non-difference when the concentration of crude extract solution grows up to 4 times concentration of Vc solution.
In the condition of 4±0.5℃and preservative film packaging, carnosine standard solution is able to prolong preservation time of chilling meat, and storage time of chilling pork adding carnosine is stored from 9 days to 15 days, which is better than the control chilled pork and the dealing samples (storage time:13d) with ascorbic acid dealing. However, pre-cooked juice ultra-filtrated is also able to prolong storage time of the chilled pork up to 11 d.
通过对牛肉预煮液进行超滤等处理后,使用高效液相色谱法对预煮液中肌肽进行了初步检测,发现牛肉中肌肽的含量较高,其含量可达4530.72 mg／kg,在预煮液中含量也较高,达到1510.24 mg／L。通过加标实验,进一步确定了该峰就是肌肽。使用液相收集目标峰,收集液相中样品的目标峰,进行检测,检测结果可以看出收集得到的肌肽纯度较高,达到95%以上,可以用于制备。
酶解处理对肉中肌肽含量有一定的影响,实验发现,使用木瓜蛋白酶酶解处理可以较显著的提高肉中肌肽的含量,但使用菠萝蛋白酶处理对肌肽的含量影响不大。
肌肽对超氧阴离子有清除作用,但肌肽浓度增大到50 mmol／L时,抑制作用逐渐减弱,粗提液的也具有一定程度的抑制作用,但抑制作用低于同浓度的肌肽标准液。随肌肽标准液浓度增加,肌肽对羟自由基的清除率也随之增加,1mmol／L时清除率达到17.94%,表明肌肽在低浓度对·OH就有显著的清除能力,随肌肽浓度增加对-OH的清除能力快速增长,20 mmol／L达到71.04%,肌肽浓度继续增大时,清除率增长变慢。
1 mmol／L肌肽标准液对猪肉匀浆中丙二醛的形成量就能起到显著的抑制作用(P<0.05),此时对体系中脂类氧化的抑制率即达到12%,5 mmol／L肌肽对体系中脂类氧化的抑制率达15%,20 mmol／L肌肽对体系中脂类氧化的抑制率达47%,表明不同浓度的肌肽均能对猪肉匀浆中脂类氧化起显著的抑制作用,其中随浓度的增加效果增强。与BHT和α-生育酚相比,肌肽对TBARS的抑制能力较高,并且由于肌肽是动物体内的内源性二肽,对人体没有危害,也不会造成二次污染,因此作为冷鲜肉的保鲜剂不但效果良好而且没有危害性。
肌肽标品对DPPH有显著的清除能力(p<0.05),随着浓度的增大,清除率不断增大,并且不同浓度的肌肽标品对DPPH的清楚效果差异性显著。肌肽粗提液对DPPH具有很强的清除效果,但其清除能力远远高于肌肽纯品,经过去离子处理的肌肽粗提液对DPPH的清除作用高于肌肽粗提液,具体原因尚不清楚。
肌肽粗提液的抗氧化率弱于肌肽纯品,肌肽纯品的抗氧化率弱于Vc,而Vc的抗氧化率弱于茶多酚。随着肌肽粗提物溶液浓度的递增,抑菌圈也成递减趋势,当粗提物溶液浓度达到Vc溶液浓度4倍时,它们的抑菌圈大小接近,抗氧化性表观抗氧化率接近。
肌肽可以延长冷鲜肉的保存时间,添加了肌肽的冷鲜肉保藏时间由不添加保鲜剂的9天增加到15天,其效果要优于抗坏血酸,肌肽粗提液也可以延长冷鲜肉的贮藏时间。
Carnosine belongs to a natural di-peptide which exists widely in animal muscle. It has anti-oxidation, compound metal ions, cleanup free radicals and supply hydrogen and electron, which has a high content in animal muscle tissue. Pre-cooked beef juice is the byproduct in the course of beef production processing, which has often been discarded as a useless liquid and does not draw attention to the relevant manufacturers up to now. In this study, purification and determination of carnosine from pre-cooking beef juice was implemented by using Gel filtering chromatography and HPLC, at last the antioxidant carnosine were determined.
Firstly, retention time of carnosine peak on C18 column was determined and compared with standard sample by HPLC method. Final, carnosine peak of actual sample was verified by a series of tests spiked. Carnosine content in pre-cooked beef juice ultra-filtrated achieve to 1510.24 mg/L, namely, carnosine of beef is 4530.72 mg/kg. The target peak from sample ultra-filtrated was collected and detected using HPLC, which showed higher purity of carnosine up to 95%, can be used in the preparation.
Papain was found that it could significantly improve carnosine content in beef during the zymohydrolysis, in a contrast, treatment samples of bromelain were not notably effect on the content of carnosine.
Carnosine is able to clear the superoxide anion, but its inhibition is gradually weakened with carnosine concentration increasing to 50 mmol/L. the extraction sample also has a certain degree of inhibition, but inhibition is lower than the same concentration of standard solution of carnosine. And the clearance rate of carnosine for hydroxyl radical is increasing with increase of the concentration of carnosine, the clearance rate is to 17.94% while 1 mmol/L carnosine, scavenging capacity of carnosine for·OH is significantly rapid growing to reach 71.04% while concentrations of carnosine increased 20 mmol/L, but carnosine concentrations continued to increase, the clear increase in the rate of change slow.
1 mmol/L standard solution of carnosine is able to play a significant inhibition (P <0.05) TBARS formation in pork homogenate, and the inhibition of lipid oxidation rate could reach 12%,5 mmol/L carnosine on lipid oxidation system inhibition rate of was 15%,20 mmol/L carnosine on lipid oxidation system suppression rate reached to 47%. These attributes indicate that concentration of carnosine was significantly inhibited on lipid oxidation of pork homogenates the effect of which increased with the effect of enhanced concentration. And BHT and a-tocopherol compared to carnosine inhibited the ability of higher TBARS and carnosine because animals are the two endogenous peptides, there is no harm to humans, it will not cause secondary pollution, so as Cold fresh meat Preservative effect of not only good and no harm.
Standard of carnosine has significant DPPH scavenging capacity (p<0.05), and clearance rate increasing with concentration increasing, and the different concentration solution of standard carnosine was significant difference each other for clear DPPH. Pre-cooked beef juice ultra-filtrated has a strong effect on the removal of DPPH, but the capacity is much higher than the standard carnosine. Pre-cooked beef juice by ion exchange column obtains better effect of DPPH scavenging than the pre-cooked beef juice ultra-filtrated.
Antioxidation of pre-cooked beef juice ultra-filtrated is lower than that of pure carnosine, but standard carnosine is less than Vc, and Vc is weaker than polyphenols according to these determination results of the several reagents antioxidation. Inhibition zone reduces as the increasing of carnosine extracts solution concentration. These observed numbers of the inhibition zone and the apparent antioxidant antioxidant are respectively non-difference when the concentration of crude extract solution grows up to 4 times concentration of Vc solution.
In the condition of 4±0.5℃and preservative film packaging, carnosine standard solution is able to prolong preservation time of chilling meat, and storage time of chilling pork adding carnosine is stored from 9 days to 15 days, which is better than the control chilled pork and the dealing samples (storage time:13d) with ascorbic acid dealing. However, pre-cooked juice ultra-filtrated is also able to prolong storage time of the chilled pork up to 11 d.
引文
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