蛋清活性蛋白高压脉冲电场提取及功能特性研究
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摘要
本文以鸡蛋蛋清为研究对象,以高压脉冲电场技术为主要处理手段,与膜分离、离子交换色谱分离技术相结合,对鸡蛋蛋清中主要活性蛋白溶菌酶、卵转铁蛋白和卵白蛋白进行分离制备,并对其生理活性和功能特性的影响进行研究,以达到提高分离效果,增强生物活性,探讨高压脉冲电场对生物大分子作用机理的研究目的。本论文经过大量的实验研究,获得了合理的活性蛋白的分离制备条件,并通过与非高压脉冲电场处理的对照组比较,证明高压脉冲电场能够提高活性蛋白的得率,并对溶菌酶和卵转铁蛋白的抑菌活性有一定的提高。同时结合荧光光谱技术也证明了高压脉冲电场对多种不同条件(浓度、pH值、离子强度、温度和储藏时间)下溶菌酶、卵转铁蛋白的活性和卵白蛋白功能特性具有一定影响,为丰富高压脉冲电场对不同介质条件下活性蛋白的提取以及生物活性变化的探究提供重要依据。
Eggs are nutritious and abundant in substance of biological activity. Nowadays, it has proved that the functions like disease prevention, anti-bacteria, anti-virus, anti-cancer, immunity are relevant to the chemical ingredients in eggs, which declares the significance of eggs in people’s well-being. The active substance has widely aroused the circle’s attention and to some extent, an industrialized operation has been formed. Lysozyme, ovotransferrin (OVT), anti-biotin proteins, lgy, lecithin in eggs have been used in disease prevention and control. The active substance in eggs must be properly used in medicine, health food and food fortification in the future.
Poultry breeding is one of China’s advantages. The output of eggs ranks first in the world. According to the data from FAO, the total output of eggs was 54 million tons in 2003, 23.37 of which were from China accounting for 43% of the total. The per-capita quantity is 18.8kg in China, larger than the world’s 8.9kg. Hens’eggs outnumber others in China, accounting for 82% of the total. The plentiful supply of eggs provides a good condition for egg processing and at the same time, stricter processing conditions are required. However, the efficiency of egg processing is low and there isn’t a big choice among the product varieties. Highly processed egg products are expected to be developed to make the best use of active ingredients in eggs. To enhance the additional value of eggs is a wise way to promote the development of hen breeding. Use of egg white mainly involves in lysozyme, OVT and anti-biotin proteins due to their high biological properties.
PEF (pulsed electric fields) has been recognized as one of the most advanced sterilization techniques and there is no doubt that it is a great innovation based on traditional ways. Furthermore, it embraces many subjects like electronics, chemistry, microbiology, physics, engineering etc., and is a typical cross-subject. It offers a new way for life science research (eg: cell fusion, ions incorporating into cells, extraction of cell contents) to solve some problems crying out for solutions.
There have been a lot of reports on PEF sterilizing and inactivating enzymes, but very few involve in biological activation and extraction of active substance. The present research markedly concentrates on PEF applied in extraction of lysozyme, OVT, and albumin. The research not only shows a great value of application but significance of creativity.
In the research, albumin of egg white was used as raw material. The viscosity of the albumin was decreased by means of PEF; Membrane Separation Technology and Ion Exchange Chromatography Separation Technology were used to extract albumin of egg white, OVT and lysozyme. The purity and yield were compared and analyzed. Based on the experimental data, in depth and systematic research was carried out on the change of biological activity of ovalbumin, OVT and lysozyme. Factors causing the change were analyzed and the mechanism of PEF affecting biological macromolecules were discussed. The research contributes to the theory and reference of PEF involved in the storage and production of protein foods.
Research on the extraction of active ovalbumin by PEF and its functional features were conducted, and the results are shown as follows:
(1) Both the PEF processing and homogenization have the effect of degrading the viscosity of egg white, and the optimal craft parameters were that : homogenization 30Mpa, electric field intensity 25 Kv/cm.On the basis of the main factors such as the homogenization pressure ( Z1), electric field intensity ( Z2) and pulsed number ( Z3), the quadratic orthogonal rotation combination design equation was obtained: Y=132.93055-10.7102 Z1+12.48609Z2+3.476075Z3+0.2126 Z1 Z2-0.03537 Z1 Z3-0.011789 Z2 Z3+0.13098 Z12-0.333156 Z22-0.10488 Z32,which provides a foundation for theoretical support for the application of production.
(2) Research on the production of albumin by means of salting out.60% saturated solution of ammonium is the optimal condition for the recovery of the albumin. While in the control group, (the sample of which wasn’t treated with PEF) the recovery rate was 9.88% lower and the purity wasn’t as high as that in the experiment group.
(3)By means of Q-Sepharose F.F Ino Exchange Chromatography(IEC), lysozyme and OVT were extracted. The results indicate that the purity and recovery rate of OVT were markedly affected by pH and the amount of sample injected. When purity was constant, the affecting intensity varied as follows: pH>amount of sample>concentration of Tris-HCl. The optimal combination of parameters is: pH:9.0; Tris-HCl: 10mmol/L; sample: 150mL. When the recovery rate was constant, the affecting intensity varied as follows: amount of sample> pH> concentration of Tris-HCl. The optimal combination of parameters is: pH:8.5; concentration of Tris-HCl: 30mmol/L; amount of sample: 100mL.
(4)To check up on the reliability, verification experiment was conducted under the optimal condition for OVT extraction. The purity of lysozyme and OVT was 98% and 95% respectively which was nearly the same to that in the control group. The recovery rate after washing, however, was different, 5.23% and 9.26% more than that of the control group for lysozyme and OVT respectively.
(5) Extraction of lysozyme, OVT and ovalbumin by percolation film after PEF treatment. Comparing to the result of control group, the purity hardly changed, but the recovery rate was more than that of the control group. Respectively, the rate of lysozyme, OVT and ovalbumin increased 11.4%、15.9% and 18.2%
(6) The influence of PEF on the bacteriostasis capacity of lysozyme showed that the activity of lysozyme after ion exchange increased by 20.51% than that of the control group, the lysozyme after ultrafiltration increased by 12.12%, in general, the bacteriostasis of the former was higher than the latter. With the increase of the PEF intensity, the bacteriostasis capacity of different concentration of lysozyme changed up and down, and when the concentration increased the activity of lysozyme that inhibited colibacillary increased with the enhancement of PEF intensity. The activity came to the maximum when the intensity reached 25kv/cm, and then the activity declined with the increase of PEF intensity. The pH value and temperature had little influence on bacteriostasis capacity with the increase of PEF intensity. As NaCl concentration raised, the activity increased and then declined as time passed by, on the other hand, the activity declined with a longer storage time. Less pulse was beneficial to the bacteriostasis capacity of lysozyme, however, after the pulse exceeded 6 the increase of pulse would depressed the capacity. By fluorescence analysis, it indicated that PEF intensity had some effect on the structure of lysozyme, but in the approved scale, the influence could be ignored.
(7)Research on the influence of PEF on bacteriostasis activity of self-made ovotransferrin (OVT) showed that minimal inhibitory concentration (MIC) of to E.coli was 2.0 mg/ml. The activity of self-made OVT treated with PEF was little higher than that of Sigma product, which also indicated that the increase of the PEF intensity and the pulse could be helpful to improve the bacteriostasis activity of OVT. The activity increased with the increase of OVT concentration, and declined with the increase of pH and ion concentration. The bacteriostasis activity of OVT increased by 28.6% compared with control group when the pulse came to 10. The temperature between 15℃to 45℃had a little influence on the bacteriostasis activity, however, when the temperature varied from 45℃to 45℃the bacteriostasis activity reduced 44.4% because of the denaturation of OVT that was accelerated by PEF.
(8)Affect of PEF on ovalbumin (OVA) functional properties was studied. The increase of intensity and pulse didn’t affect the solubility of ovalbumin markedly when there were less PEF intensity and pulse, but the solubility decreased markedly as the PEF exceeded 30kV/cm and the pulse exceeded 12. The PEF intensity played a very important role in the aspect of OVA solubility than that of the pulse viewed to the decrease extent. Around isoelectric point (pH=6), the decrease of the solubility was obvious, while under alkaline condition (pH=8) the solubility hardly varied. The solubility recovered gradually as time gone on, and then it returned to its original level after 24 hours.
The emulsifibility and stability, foaming capacity and stability increased as the PEF increased, when the PEF intensity reached 30kV/cm, they attained the maximum, and then decreased as time gone. The foam capacity and foam stability increased to some extent at alkaline condition (pH=8), but decreased markedly at acid condition (pH=6) and 30kV/cm of PEF intensity. The emulsifibility and stability, foaming capacity and stability declined as time gone, when the OVA (treated by PEF) was stood still for 10h or 24h at 4℃, respectively.
Through detection on hydrophobicity of OVA by spectrofluorophotometer, the relative fluorescence intensity reached the maximum when the pulse intensity approached to 30kV/cm. Placed for a while, the intensity declined gradually, which indicated that the reaction was a reversible change. In the meanwhile, the pulse didn’t affect the hydrophobicity of OVA obviously.
In summary, by means of PEF, the viscosity of egg white dropped, the molecular structure was loosened, and the extract rates of lysozyme, OVT, ovalbumin all increased. Furthermore, PEF affected the biological activity and functional property to different extent. The research possesses highly academic value and will enhance economic returns for enterprises eventually.
The research is only exploratory focusing on PEF affecting the extract of main active proteins in egg white and the functional property of them. The research on the functional principle of PEF affecting biomacromolecules is being expected.
Eggs are nutritious and abundant in substance of biological activity. Nowadays, it has proved that the functions like disease prevention, anti-bacteria, anti-virus, anti-cancer, immunity are relevant to the chemical ingredients in eggs, which declares the significance of eggs in people’s well-being. The active substance has widely aroused the circle’s attention and to some extent, an industrialized operation has been formed. Lysozyme, ovotransferrin (OVT), anti-biotin proteins, lgy, lecithin in eggs have been used in disease prevention and control. The active substance in eggs must be properly used in medicine, health food and food fortification in the future.
Poultry breeding is one of China’s advantages. The output of eggs ranks first in the world. According to the data from FAO, the total output of eggs was 54 million tons in 2003, 23.37 of which were from China accounting for 43% of the total. The per-capita quantity is 18.8kg in China, larger than the world’s 8.9kg. Hens’eggs outnumber others in China, accounting for 82% of the total. The plentiful supply of eggs provides a good condition for egg processing and at the same time, stricter processing conditions are required. However, the efficiency of egg processing is low and there isn’t a big choice among the product varieties. Highly processed egg products are expected to be developed to make the best use of active ingredients in eggs. To enhance the additional value of eggs is a wise way to promote the development of hen breeding. Use of egg white mainly involves in lysozyme, OVT and anti-biotin proteins due to their high biological properties.
PEF (pulsed electric fields) has been recognized as one of the most advanced sterilization techniques and there is no doubt that it is a great innovation based on traditional ways. Furthermore, it embraces many subjects like electronics, chemistry, microbiology, physics, engineering etc., and is a typical cross-subject. It offers a new way for life science research (eg: cell fusion, ions incorporating into cells, extraction of cell contents) to solve some problems crying out for solutions.
There have been a lot of reports on PEF sterilizing and inactivating enzymes, but very few involve in biological activation and extraction of active substance. The present research markedly concentrates on PEF applied in extraction of lysozyme, OVT, and albumin. The research not only shows a great value of application but significance of creativity.
In the research, albumin of egg white was used as raw material. The viscosity of the albumin was decreased by means of PEF; Membrane Separation Technology and Ion Exchange Chromatography Separation Technology were used to extract albumin of egg white, OVT and lysozyme. The purity and yield were compared and analyzed. Based on the experimental data, in depth and systematic research was carried out on the change of biological activity of ovalbumin, OVT and lysozyme. Factors causing the change were analyzed and the mechanism of PEF affecting biological macromolecules were discussed. The research contributes to the theory and reference of PEF involved in the storage and production of protein foods.
Research on the extraction of active ovalbumin by PEF and its functional features were conducted, and the results are shown as follows:
(1) Both the PEF processing and homogenization have the effect of degrading the viscosity of egg white, and the optimal craft parameters were that : homogenization 30Mpa, electric field intensity 25 Kv/cm.On the basis of the main factors such as the homogenization pressure ( Z1), electric field intensity ( Z2) and pulsed number ( Z3), the quadratic orthogonal rotation combination design equation was obtained: Y=132.93055-10.7102 Z1+12.48609Z2+3.476075Z3+0.2126 Z1 Z2-0.03537 Z1 Z3-0.011789 Z2 Z3+0.13098 Z12-0.333156 Z22-0.10488 Z32,which provides a foundation for theoretical support for the application of production.
(2) Research on the production of albumin by means of salting out.60% saturated solution of ammonium is the optimal condition for the recovery of the albumin. While in the control group, (the sample of which wasn’t treated with PEF) the recovery rate was 9.88% lower and the purity wasn’t as high as that in the experiment group.
(3)By means of Q-Sepharose F.F Ino Exchange Chromatography(IEC), lysozyme and OVT were extracted. The results indicate that the purity and recovery rate of OVT were markedly affected by pH and the amount of sample injected. When purity was constant, the affecting intensity varied as follows: pH>amount of sample>concentration of Tris-HCl. The optimal combination of parameters is: pH:9.0; Tris-HCl: 10mmol/L; sample: 150mL. When the recovery rate was constant, the affecting intensity varied as follows: amount of sample> pH> concentration of Tris-HCl. The optimal combination of parameters is: pH:8.5; concentration of Tris-HCl: 30mmol/L; amount of sample: 100mL.
(4)To check up on the reliability, verification experiment was conducted under the optimal condition for OVT extraction. The purity of lysozyme and OVT was 98% and 95% respectively which was nearly the same to that in the control group. The recovery rate after washing, however, was different, 5.23% and 9.26% more than that of the control group for lysozyme and OVT respectively.
(5) Extraction of lysozyme, OVT and ovalbumin by percolation film after PEF treatment. Comparing to the result of control group, the purity hardly changed, but the recovery rate was more than that of the control group. Respectively, the rate of lysozyme, OVT and ovalbumin increased 11.4%、15.9% and 18.2%
(6) The influence of PEF on the bacteriostasis capacity of lysozyme showed that the activity of lysozyme after ion exchange increased by 20.51% than that of the control group, the lysozyme after ultrafiltration increased by 12.12%, in general, the bacteriostasis of the former was higher than the latter. With the increase of the PEF intensity, the bacteriostasis capacity of different concentration of lysozyme changed up and down, and when the concentration increased the activity of lysozyme that inhibited colibacillary increased with the enhancement of PEF intensity. The activity came to the maximum when the intensity reached 25kv/cm, and then the activity declined with the increase of PEF intensity. The pH value and temperature had little influence on bacteriostasis capacity with the increase of PEF intensity. As NaCl concentration raised, the activity increased and then declined as time passed by, on the other hand, the activity declined with a longer storage time. Less pulse was beneficial to the bacteriostasis capacity of lysozyme, however, after the pulse exceeded 6 the increase of pulse would depressed the capacity. By fluorescence analysis, it indicated that PEF intensity had some effect on the structure of lysozyme, but in the approved scale, the influence could be ignored.
(7)Research on the influence of PEF on bacteriostasis activity of self-made ovotransferrin (OVT) showed that minimal inhibitory concentration (MIC) of to E.coli was 2.0 mg/ml. The activity of self-made OVT treated with PEF was little higher than that of Sigma product, which also indicated that the increase of the PEF intensity and the pulse could be helpful to improve the bacteriostasis activity of OVT. The activity increased with the increase of OVT concentration, and declined with the increase of pH and ion concentration. The bacteriostasis activity of OVT increased by 28.6% compared with control group when the pulse came to 10. The temperature between 15℃to 45℃had a little influence on the bacteriostasis activity, however, when the temperature varied from 45℃to 45℃the bacteriostasis activity reduced 44.4% because of the denaturation of OVT that was accelerated by PEF.
(8)Affect of PEF on ovalbumin (OVA) functional properties was studied. The increase of intensity and pulse didn’t affect the solubility of ovalbumin markedly when there were less PEF intensity and pulse, but the solubility decreased markedly as the PEF exceeded 30kV/cm and the pulse exceeded 12. The PEF intensity played a very important role in the aspect of OVA solubility than that of the pulse viewed to the decrease extent. Around isoelectric point (pH=6), the decrease of the solubility was obvious, while under alkaline condition (pH=8) the solubility hardly varied. The solubility recovered gradually as time gone on, and then it returned to its original level after 24 hours.
The emulsifibility and stability, foaming capacity and stability increased as the PEF increased, when the PEF intensity reached 30kV/cm, they attained the maximum, and then decreased as time gone. The foam capacity and foam stability increased to some extent at alkaline condition (pH=8), but decreased markedly at acid condition (pH=6) and 30kV/cm of PEF intensity. The emulsifibility and stability, foaming capacity and stability declined as time gone, when the OVA (treated by PEF) was stood still for 10h or 24h at 4℃, respectively.
Through detection on hydrophobicity of OVA by spectrofluorophotometer, the relative fluorescence intensity reached the maximum when the pulse intensity approached to 30kV/cm. Placed for a while, the intensity declined gradually, which indicated that the reaction was a reversible change. In the meanwhile, the pulse didn’t affect the hydrophobicity of OVA obviously.
In summary, by means of PEF, the viscosity of egg white dropped, the molecular structure was loosened, and the extract rates of lysozyme, OVT, ovalbumin all increased. Furthermore, PEF affected the biological activity and functional property to different extent. The research possesses highly academic value and will enhance economic returns for enterprises eventually.
The research is only exploratory focusing on PEF affecting the extract of main active proteins in egg white and the functional property of them. The research on the functional principle of PEF affecting biomacromolecules is being expected.
引文
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