蛋黄卵磷脂高压脉冲电场提取关键技术及增强记忆功能评价
摘要
本论文得到了吉林省科技厅农业重点项目《禽蛋高附加值活性物质提取及其功能食品开发》(项目编号:20050202-3)和吉林大学农学部科研启动基金《高压脉冲电场提取蛋黄卵磷脂的研究》(2006018)的资助,已被确定为吉林省科学技术成果(成果证书编号:2008317)。
蛋黄卵磷脂是一种制作保健品和药用辅料的优良原料,由于目前其提取方法普遍存在效率低、产品稳定性差等问题,还不能实现大规模工业化生产,满足不了社会的需求。我国作为产蛋大国,其深加工水平较低的现状也亟需改善。PEF技术作为一种非热加工高新技术,具有适合于蛋黄卵磷脂提取的技术特点,因此开展此方面研究对促进蛋品的深加工和加快蛋黄卵磷脂的工业化生产进程具有重要的意义。
本文以蛋黄粉为实验原料,以获得具有高效、快速、节能特点的蛋黄卵磷脂生产技术为主要研究目的,在建立蛋黄卵磷脂检测方法和预处理的基础上,采用线性回归正交组合设计、均匀试验设计和响应面设计与分析,创新性地开展了蛋黄卵磷脂的高压脉冲电场提取、柱层析精制条件、脱色和干燥以及增强记忆功能评价等方面的研究工作,构建出蛋黄卵磷脂生产关键技术的回归模型,确定了最佳技术参数,建立了一套完整的PEF技术提取蛋黄卵磷脂的新工艺和新方法,并对PEF技术的作用机理进行了初步探讨。结果显示:蛋黄卵磷脂提取率达到22.8%,PC含量为97.53%,提取效率显著提高,并且对改善小鼠的记忆功能具有功效。
研究结果证明:PEF技术提取蛋黄卵磷脂具有高效、快速、节能等特点,可为PEF技术的提取研究提供理论依据,也可为促进我国蛋黄卵磷脂的产业化生产提供新的思路。
This paper is attached to the important project of Department of Agriculture of Jilin Province which called Developing and Researching of Neotype Functional Food– extracting active compound of high added value from eggs and developing of its functional food(20050202-3). And it is also attached to the Start Fund of Jilin Province called Research on Egg Yolk Lecithin Extracted by PEF(2006018).It has been identified to scientific research achievement of Jilin Province(2008317). The research of this paper takes egg yellow powder as experimental material. Its purpose is to manufacture a kind of technology that consisted characteristic of rapid, high performance and save energy to producing egg yolk lecithin The main achievement of this paper is listed below in 6 points.
1.Research on the detection methods of egg yolk lecithin
It established UV method and HPLC method to detecting egg yolk lecithin. Some conclusion are listed below:
(1) The regression equation of standard curve is A=0.92917C+0.14811 with correlation coefficient of 0.99952 in UV method which had been verificated by precision experiment, repeatability experiment, stability experiment, recovery after application of sample experiment and so on. This proved that UV method fit to detecting egg yolk lecithin.
(2)The chromatographic condition in HPLC method in detecting egg yolk lecithin is listed below. chromatographic column: Lichrosorb Si60(4.6mm×150mm,5μm); chromatographic column: methanol/ acetonitrile/water=60/30/10; column temperature: 40℃; wave length: 206nm. The standard curve is Y=7.512941×10-4X-27.06823(R2=0.99998)between content of egg yolk lecithin and peak area. The precision experiment, repeatability experiment, stability experiment, and recovery after application of sample experiment show the HPLC method is a kind of feasible method for detecting egg yolk lecithin. But it also has some shortage such as costing much too expensive solutions and wasting materials and its high cost price.
2.Research on raw material pretreatment of egg yolk lecithin:
In the foundation of detecting the content of protein, fat, total phospholipids, phospholipids and water of egg yolk powder, the regression model of conditioning of egg yolk powder to obtain insoluble substance in acetone on project was obtained by multiple linear regression orthogonal combination design. The flowing is the equation : =92.708-0.687Z1-0.085Z2-0.823Z3 . In the equation,independent variables are liquid-solid ratio(Z1),extcition time (Z2)and drying temperature(Z3) . By Sylvester inequality extremums can be obtained directly, so by working out differentials of Regression model of conditioning of egg yolk powder to obtain insoluble substance in acetone , ratio, diffusion time and diffusion can be optimized. They wre 8.8:1,39.6min and 28.5℃. the yield of insoluble substance in acetone was above 59.84% on average.
3.Research on extraction of lecithin from egg yolk by PEF
(1)Regression equation about diffusion egg white lecithin by ethanol in homogeneous design. Y=3.79+0.402X1+ 0.141X2+0.300X3-8.34×10-2X4. From the regression equation , the order of contribution of factors to extraction yield solid-liquid ratio ,concentration of alcohol extraction temperature and extraction time. Optimizations of experimental condition of obtaining lecithin from egg yolk by alcohol are ratio of alcohol to obtain insoluble substance in acetone is 11:1,and concentration of alcohol is 95%,and extraction temperature is 35℃,and extraction time is 2h. Extraction yield of lectin can be to reach 19.72%.
(2)By response surface design three rounds quadratic regression equation was Y=-72.55+2.79X1+1.655X2+2.425X3-0.031X1X2+0.018X1X3-0.0017X2X3-0.031X12-0.0085X22-0.0903X32, Independent variables are pulse numbers(X1), electrical field strength(X2), concentration of accessory ingredient(X3). Optimization of parameter of extraction lecithin from egg yolk by PEF are pulse number is 35, and electrical field strength is 30kv, concentration of accessory ingredient is 15mg/L,and extraction yield is 22.8%. The extraction effectiveness raised significantly. I designed a kind of method by PEF that consisted characteristic of rapid, high performance and save energy to producing egg yolk lecithin. This makes a precedent in extraction and extraction egg yolk lecithin.
(3)By designhomogeneous design, regression equation of extraction lectin from egg yolk by ultrasonic wave was obtained. Y=58.697+0.02333 X1+1.087 X2+0.84 X3,independent variables are ultrasonic frequency (X1), ultrasonic time (X2), solid-liquid ratio (X3).Optimization of parameter of extraction lectin from egg yolk by ultrasonic wave are ultrasonic frequency is 600w, and ultrasonic time is 11min, solid-liquid ratio is10mL/g,and extraction yield of lectin is 19.38%.
(4)Compared the efficiency of extraction lectin from egg yolk ,PEF had the biggest efficiency.at the same time .By HPLC PC content of lectin obtaind by PEF was known,and residual volume of acetone was detected by GC.The results were purity coefficient of lectin obtained was 72.12%,and the concentration of residual volume of acetone was 0.00071%.
4.Screening condition of column chromatography in purificating egg yolk lectin
Compared to rate of eluent, alcohol concentration in eluent, the percentage of sample injection in column volume which was utility,which showed that it was not completely same that effect of peak width, peak height, half band width, retention time andpeak area on items in chromatogram.
Expression of main constituent was obtained by investigating there fators which have effect on column chromatography depurattion by principal component analysis method.
The principal constituent expression of flow rate of eluent was
F=-0.25437ZX1-0.00881ZX2+0.408034ZX3+0.3906ZX4+0.439036ZX5
The principal constituent expression of Alcohol concentration was F=-0.2559ZX1+0.433251ZX2+0.365095ZX3+0.434916ZX4+0.218003ZX5
The principal constituent expression of the percentage of sample injection in column volume which was utility was
F=0.233169ZX1+0.448146ZX2+0.177647ZX3+0.292246ZX4+0.388087ZX5 and ZX1,ZX2,ZX3,ZX4,ZX5 represented standard data.
The optimization conditions of purificating lectin from egg yolk by column chromatography were the rate of eluent was 0.1mL/min,the concentration of ethanol was 95%,the percentage of sample was 5%,concentration of lectin in sample was 97.53% by HPLC.
5.Research on decolorization and drying of egg yolk.lectin
(1)The results showed: Regression model of physical decolorization obtained by homogeneous design and regression analysis was Y1=51.076+0.468 X1+4.365 X2+0.08478 X3;The effect of three independent variables to dependent variable(Y1) were different, and the contribution of factors to Y1 is decolorization temperature(X 1) which is the most important ,amount of sample(X2 )less important, decolorization time(X 3) was the least important .
(2)Regression model of chemical decolorization was Y1’=-2.297+0.112 X1’+1.525X3’-0.00261 X4’,and correlation coefficient was 0.872897;and the contribution of factors to Y1 is concentration (X 1) which is the most important ,amount of sample(X2 )less important, decolorization time and decolorization temperature was the least important .
(3)Physical decolorization had more advantages than chemical decolorization by homogeneous design and analysis.Results showed peroxide value was 10meg/kg,and ratio of decolorization reached 88%,and conditions of physical decolorization were decolorization temperature(X1)was 45℃,and amount of sample(X2)was 3.0%,and decolorization time(X 3)70min,and
(4)Conditions of chemical decolorization were concentration(X1’)was 70%, decolorization temperature(X2’)was 30℃,and amount of sample(X3’)was 3.0%,and decolorization time(X4’)70min,under which decolorization ratio reached to 10% and peroxide value was 10meg/kg, larger than government standard level.
(5)Five dying methods of lectin had been estimated on time,cost,and peroxide value.the order of optimization was vacuo heating dying, far infrared dying, centre atomization dying and hot air drying.Drying lectin by vacuo heating was the best method among the five methods.
6.Effect of Egg Yolk Lecithin to the memory of Mice
The effect of lectin extracted by PEF under the optimization conditions from egg yolk has been studied . By incubation period in water maze experiment, lightning stroke times , autonomic activities times, times in hole and incubation period and mistakes times in o avoiding dark experiment and by Appraisement procedure and measurement of Health food, feed with egg yolk lecithin can improve the memory function in the mice.,Which provid new evidence for serial products of lectin from egg yolk.
To sum up, we have explored a new way to preparation of egg yolk lecithin through the study of this paper. The method of PEF has the advantages of non-thermal, fast responsive, efficient, easy to operate, low cost, which will greatly reduce the cost of extraction of lecithin, reduce environmental pollution and saving energy too. With the development of science and technology and mature of high-voltage pulsed electric field technology gradually, this technology will be used in a wide range of applications in the industrial production, and it will make a greater contribution to society consequentially.
蛋黄卵磷脂是一种制作保健品和药用辅料的优良原料,由于目前其提取方法普遍存在效率低、产品稳定性差等问题,还不能实现大规模工业化生产,满足不了社会的需求。我国作为产蛋大国,其深加工水平较低的现状也亟需改善。PEF技术作为一种非热加工高新技术,具有适合于蛋黄卵磷脂提取的技术特点,因此开展此方面研究对促进蛋品的深加工和加快蛋黄卵磷脂的工业化生产进程具有重要的意义。
本文以蛋黄粉为实验原料,以获得具有高效、快速、节能特点的蛋黄卵磷脂生产技术为主要研究目的,在建立蛋黄卵磷脂检测方法和预处理的基础上,采用线性回归正交组合设计、均匀试验设计和响应面设计与分析,创新性地开展了蛋黄卵磷脂的高压脉冲电场提取、柱层析精制条件、脱色和干燥以及增强记忆功能评价等方面的研究工作,构建出蛋黄卵磷脂生产关键技术的回归模型,确定了最佳技术参数,建立了一套完整的PEF技术提取蛋黄卵磷脂的新工艺和新方法,并对PEF技术的作用机理进行了初步探讨。结果显示:蛋黄卵磷脂提取率达到22.8%,PC含量为97.53%,提取效率显著提高,并且对改善小鼠的记忆功能具有功效。
研究结果证明:PEF技术提取蛋黄卵磷脂具有高效、快速、节能等特点,可为PEF技术的提取研究提供理论依据,也可为促进我国蛋黄卵磷脂的产业化生产提供新的思路。
This paper is attached to the important project of Department of Agriculture of Jilin Province which called Developing and Researching of Neotype Functional Food– extracting active compound of high added value from eggs and developing of its functional food(20050202-3). And it is also attached to the Start Fund of Jilin Province called Research on Egg Yolk Lecithin Extracted by PEF(2006018).It has been identified to scientific research achievement of Jilin Province(2008317). The research of this paper takes egg yellow powder as experimental material. Its purpose is to manufacture a kind of technology that consisted characteristic of rapid, high performance and save energy to producing egg yolk lecithin The main achievement of this paper is listed below in 6 points.
1.Research on the detection methods of egg yolk lecithin
It established UV method and HPLC method to detecting egg yolk lecithin. Some conclusion are listed below:
(1) The regression equation of standard curve is A=0.92917C+0.14811 with correlation coefficient of 0.99952 in UV method which had been verificated by precision experiment, repeatability experiment, stability experiment, recovery after application of sample experiment and so on. This proved that UV method fit to detecting egg yolk lecithin.
(2)The chromatographic condition in HPLC method in detecting egg yolk lecithin is listed below. chromatographic column: Lichrosorb Si60(4.6mm×150mm,5μm); chromatographic column: methanol/ acetonitrile/water=60/30/10; column temperature: 40℃; wave length: 206nm. The standard curve is Y=7.512941×10-4X-27.06823(R2=0.99998)between content of egg yolk lecithin and peak area. The precision experiment, repeatability experiment, stability experiment, and recovery after application of sample experiment show the HPLC method is a kind of feasible method for detecting egg yolk lecithin. But it also has some shortage such as costing much too expensive solutions and wasting materials and its high cost price.
2.Research on raw material pretreatment of egg yolk lecithin:
In the foundation of detecting the content of protein, fat, total phospholipids, phospholipids and water of egg yolk powder, the regression model of conditioning of egg yolk powder to obtain insoluble substance in acetone on project was obtained by multiple linear regression orthogonal combination design. The flowing is the equation : =92.708-0.687Z1-0.085Z2-0.823Z3 . In the equation,independent variables are liquid-solid ratio(Z1),extcition time (Z2)and drying temperature(Z3) . By Sylvester inequality extremums can be obtained directly, so by working out differentials of Regression model of conditioning of egg yolk powder to obtain insoluble substance in acetone , ratio, diffusion time and diffusion can be optimized. They wre 8.8:1,39.6min and 28.5℃. the yield of insoluble substance in acetone was above 59.84% on average.
3.Research on extraction of lecithin from egg yolk by PEF
(1)Regression equation about diffusion egg white lecithin by ethanol in homogeneous design. Y=3.79+0.402X1+ 0.141X2+0.300X3-8.34×10-2X4. From the regression equation , the order of contribution of factors to extraction yield solid-liquid ratio ,concentration of alcohol extraction temperature and extraction time. Optimizations of experimental condition of obtaining lecithin from egg yolk by alcohol are ratio of alcohol to obtain insoluble substance in acetone is 11:1,and concentration of alcohol is 95%,and extraction temperature is 35℃,and extraction time is 2h. Extraction yield of lectin can be to reach 19.72%.
(2)By response surface design three rounds quadratic regression equation was Y=-72.55+2.79X1+1.655X2+2.425X3-0.031X1X2+0.018X1X3-0.0017X2X3-0.031X12-0.0085X22-0.0903X32, Independent variables are pulse numbers(X1), electrical field strength(X2), concentration of accessory ingredient(X3). Optimization of parameter of extraction lecithin from egg yolk by PEF are pulse number is 35, and electrical field strength is 30kv, concentration of accessory ingredient is 15mg/L,and extraction yield is 22.8%. The extraction effectiveness raised significantly. I designed a kind of method by PEF that consisted characteristic of rapid, high performance and save energy to producing egg yolk lecithin. This makes a precedent in extraction and extraction egg yolk lecithin.
(3)By designhomogeneous design, regression equation of extraction lectin from egg yolk by ultrasonic wave was obtained. Y=58.697+0.02333 X1+1.087 X2+0.84 X3,independent variables are ultrasonic frequency (X1), ultrasonic time (X2), solid-liquid ratio (X3).Optimization of parameter of extraction lectin from egg yolk by ultrasonic wave are ultrasonic frequency is 600w, and ultrasonic time is 11min, solid-liquid ratio is10mL/g,and extraction yield of lectin is 19.38%.
(4)Compared the efficiency of extraction lectin from egg yolk ,PEF had the biggest efficiency.at the same time .By HPLC PC content of lectin obtaind by PEF was known,and residual volume of acetone was detected by GC.The results were purity coefficient of lectin obtained was 72.12%,and the concentration of residual volume of acetone was 0.00071%.
4.Screening condition of column chromatography in purificating egg yolk lectin
Compared to rate of eluent, alcohol concentration in eluent, the percentage of sample injection in column volume which was utility,which showed that it was not completely same that effect of peak width, peak height, half band width, retention time andpeak area on items in chromatogram.
Expression of main constituent was obtained by investigating there fators which have effect on column chromatography depurattion by principal component analysis method.
The principal constituent expression of flow rate of eluent was
F=-0.25437ZX1-0.00881ZX2+0.408034ZX3+0.3906ZX4+0.439036ZX5
The principal constituent expression of Alcohol concentration was F=-0.2559ZX1+0.433251ZX2+0.365095ZX3+0.434916ZX4+0.218003ZX5
The principal constituent expression of the percentage of sample injection in column volume which was utility was
F=0.233169ZX1+0.448146ZX2+0.177647ZX3+0.292246ZX4+0.388087ZX5 and ZX1,ZX2,ZX3,ZX4,ZX5 represented standard data.
The optimization conditions of purificating lectin from egg yolk by column chromatography were the rate of eluent was 0.1mL/min,the concentration of ethanol was 95%,the percentage of sample was 5%,concentration of lectin in sample was 97.53% by HPLC.
5.Research on decolorization and drying of egg yolk.lectin
(1)The results showed: Regression model of physical decolorization obtained by homogeneous design and regression analysis was Y1=51.076+0.468 X1+4.365 X2+0.08478 X3;The effect of three independent variables to dependent variable(Y1) were different, and the contribution of factors to Y1 is decolorization temperature(X 1) which is the most important ,amount of sample(X2 )less important, decolorization time(X 3) was the least important .
(2)Regression model of chemical decolorization was Y1’=-2.297+0.112 X1’+1.525X3’-0.00261 X4’,and correlation coefficient was 0.872897;and the contribution of factors to Y1 is concentration (X 1) which is the most important ,amount of sample(X2 )less important, decolorization time and decolorization temperature was the least important .
(3)Physical decolorization had more advantages than chemical decolorization by homogeneous design and analysis.Results showed peroxide value was 10meg/kg,and ratio of decolorization reached 88%,and conditions of physical decolorization were decolorization temperature(X1)was 45℃,and amount of sample(X2)was 3.0%,and decolorization time(X 3)70min,and
(4)Conditions of chemical decolorization were concentration(X1’)was 70%, decolorization temperature(X2’)was 30℃,and amount of sample(X3’)was 3.0%,and decolorization time(X4’)70min,under which decolorization ratio reached to 10% and peroxide value was 10meg/kg, larger than government standard level.
(5)Five dying methods of lectin had been estimated on time,cost,and peroxide value.the order of optimization was vacuo heating dying, far infrared dying, centre atomization dying and hot air drying.Drying lectin by vacuo heating was the best method among the five methods.
6.Effect of Egg Yolk Lecithin to the memory of Mice
The effect of lectin extracted by PEF under the optimization conditions from egg yolk has been studied . By incubation period in water maze experiment, lightning stroke times , autonomic activities times, times in hole and incubation period and mistakes times in o avoiding dark experiment and by Appraisement procedure and measurement of Health food, feed with egg yolk lecithin can improve the memory function in the mice.,Which provid new evidence for serial products of lectin from egg yolk.
To sum up, we have explored a new way to preparation of egg yolk lecithin through the study of this paper. The method of PEF has the advantages of non-thermal, fast responsive, efficient, easy to operate, low cost, which will greatly reduce the cost of extraction of lecithin, reduce environmental pollution and saving energy too. With the development of science and technology and mature of high-voltage pulsed electric field technology gradually, this technology will be used in a wide range of applications in the industrial production, and it will make a greater contribution to society consequentially.
引文
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