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Study on Process and Mechanism of Pulsed Electric Field on Several Components Extraction from Animal and Plant

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Tutor: YinYongGuang
School: Jilin University
Course: Agricultural Mechanization Engineering
Keywords: Pulsed Electric Fields,Extract,Green tea polysaccharide,Green tea polyphenols,Gr
CLC: Q50
Type: PhD thesis
Year:  2007
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Pulsed electric field (PEF) non-thermal pasteurization technique is known as the one of the most interesting and advanced technology internationally. It is a revolution of traditional pasteurization technique. It is unassailable that it is innovative, scientific and advanced. PEF technology is noticeable in being a crossed branch of electronics, chemistry, microbiology, physics and engineering technique. Its mechanism research to organism¡¯s inactivation will provide a new method to resolve life sciences question, such as cell components extraction, cell fusion et al. But there is no any effective research in this way. So it is necessary to develop the PEF technology to meet the market¡¯s need for low cost and high effective new technique.The extraction, separation and purification of components within cells are the key basic technologies for modernization of biological pharmacy. Nowadays, the traditional extraction methods give priority to the modernization of biological pharmacy, which are low effect, require long time and have low extraction rate. So it is in dire need of a new method to quicken the biological pharmacy¡¯s step to the world.We come to a conclusion on the PEF as follows:extracting at normal temperature, the essential components without loss and denaturalization, time of extraction being short, the achievable yield being high, purification and separation being easy, economizing on energy and without contamination. PEF is better than the traditional technologies. The PEF technique was firstly used to extract Green tea leaves¡¯s Polysaccharides(TPS)¡¢Polyphenols(TP)¡¢Caffeine(Caf), Deoxyribonucleic acid(DNA) and Bovine spleen¡¯s DNA in this paper. Several key technical problems of the technology were studied as follows:1. PEF technique was used to extract Green tea leaves¡¯s TPS¡¢TP¡¢Caf and DNA for the first time. This is a new exploration and probing to extract the important components from Green tea leaves. Factors affecting PEF treatment were analyzed in detail with the extraction of Green tea leaves¡¯s TPS¡¢TP¡¢Caf and DNA. The basic parameters of PEF treatment for extraction the Green tea leaves¡¯sTPS¡¢TP¡¢Caf and DNA are as follows:TPS:pH=9.5,2N=10,E=25kV/cm;TP :pH=9.5,2N=12,E=25kV/cm;Caf :pH=4.0,2N=10,E=25kV/cm;DNA: pH=8.5,2N=14,E=25kV/cm; (E=electric field strength (kV/cm),N=pulse number ) These results provide not only experimental and theoretical basis for extraction of Green tea leaves¡¯s TPS¡¢TP¡¢Caf and DNA, but also good experience for extracting the other different kinds of plants.2. We developed a high throughput DNA extraction method from bovine spleen with PEF, thus effectively decreasing the time, cost and handling error of the DNA extraction stage. The effectiveness of PEF treatment depended on the electrical conditions such as electric field strengths, total number of pulses (based on treatment time, pulse frequency). A maximum isolation was achieved when materials suspended in buffer (pH 5.5) was treated for 2¦Ìs pulse width at 30 kV/cm, 140 Hz and n = 8, flow velocity = 2 ml/min, diluted time = 4¡Á, 65¡æheating treatment in bipolar mode. Finally, PEF processing was two times more effective than untreated in extraction. This work clearly demonstrated that the PEF treatment at high-salted, low-acidic conditions was an effective method to achieve a high throughput DNA extraction.The extraction time of PEF is ultra short. PEF can be operated at normal temperature, without heating. As proved by the animal test, the final products of PEF are as active as those of compound extraction method. It shows that PEF is not only feasible and has a good development prospect, but also has the characteristics of operating simple, safe and keeping ahead in the world. In addition, the economic and social benefits are better than that of the other extracting ways.3. It is very important to resolve the PEF extraction mechanism of the biological useful components. High electric field strengths can cause some structural changes, such as cell deformation, cell membrane damage, protein denaturalization and so on in biological materials. On the PEF treatment, the electric field strengths between the interior and the exterior of cell membranes is so large that it leads to electroporation and rapid permeation. The higher the electric field strengths, the more solvent can enter into the cell and the more components can permeate the cell membrane and flow out from a cell interior to exterior. Consequently, the concentration of cell¡¯s components between the cell interior and the cell exterior can reach equilibrium in an ultra short time. An electric field strengths of at least 30kV/cm and more than 4¡«8 pulse numbers can damage cell membrane non-reversibly(for example, bovine spleen). The electric field strengths is a key factor to damage cell membrane, and the pulse number decides the existent duration of structural changes on cell membrane. But it is improper to increase the electric field intensity and pulse number overly.The principle of PEF not only explains the phenomena of achievable yield being high and time being short, but also provides some valuable references for the studies of the other extracting way. 4. The higher electric field strengths and pulse number (treatment time,¦Ìs) were, the more the extraction rate increased, but when PEF treatment exceeds the limitation, the extraction rate decrease reversely, because of denaturalization and conformation of the biological components in the molecular level. Hence the extraction rate of biological components decrease. The molecular mass of TPS is very big and its structure is complex, too. There are many glycoside bond and different groups in TPS structure and different methods of separation and purification has effect on TPS character. The experiment proved that TPS of green tea leaves have got absorbance apices in 3392cm-1 (O-H , N-H)¡¢2927cm-1 (C-H), 1604cm-1 (C=O), 1051~1145cm-1 (C-O), 927cm-1, 875cm-1, 829cm-1, 788cm-1, 767cm-1, 713cm-1, 619cm-1 and 529cm-1, and so TPS consisted of mannose and galactose.When TPS was treated with PEF, the position of absorbance apices was changed in 3400cm-1 and 1600cm-1;3392 cm-1 (0kV/cm), 3410 cm-1 (15kV/cm), 3421 cm-1(25kV/cm), 3430 cm-1 (30kV/cm);1600 cm-1 (0 kV/cm), 1616 cm-1 (15 kV/cm), 1623 cm-1 (25 kV/cm), 1652 cm-1 (30 kV/cm). The reason considered was that changing of atom angle between O-H and C=O or changing of distance between atoms led to improvement of the shaking-kinetic energy and the absorbance energy. The experiment proved that this model could reflect accurately the experiment result for the electric field intensity range of 0 to 30kV/cm. The dynamic model of PEF has a wide application, which can provide a theoretical method and application value for important component¡¯s extraction. Generally, a new way, which is feasible to extract the essential components of the biological material without heating, is found out and the optimum process parameters are also decided. PEF technology has the following good characteristics : the achievable yield is high and the time of extraction is short. It can be operated at normal temperature without the disadvantage of denaturalization of heating. The process parameters can be controlled and adjusted easily for the PEF equipment; a set of equipment can be used for different raw materials, and can carry out the extraction of different essential components. PEF technology is simply and economical. So PEF method is a novel and promising method to extract the essential and important components.
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