Relevant knowledge
The mechanism of microwave breaking cells and its application in the extraction of natural products
The mechanism of microwave breaking cells and its application in the extraction of natural products
Liu Chuanbin, Li Ning, Bai Fengwu
[Institute of Biochemical Engineering, Dalian University of Technology, Dalian] 1116012) Su Zhiguo
(State Key Laboratory of Biochemical Engineering, Institute of Chemical and Metallurgy, Chinese Academy of Sciences, Beijing.. 1000080)
Conclusion: Huibo breaking cell technology is the selectivity, instantaneity and efficiency of Huibo heating. A method of cell fragmentation through appropriate wave treatment of biomaterials shows that this technology is especially suitable for the extraction of small molecular products in cells and has broad application prospects in the extraction and separation of natural products
Key words; Natural product extraction of cell breaking mechanism
1. Introduction
The rich biological resources in nature provide tens of thousands of natural products for human beings, which are important sources of fine chemicals such as drugs, spices, food additives, etc. To develop and apply natural products, it is necessary to extract them from organisms. The traditional extraction operation is still widely used in the industry for the extraction of natural products, which is time-consuming, laborious, inefficient and backward in technology.
1z3。 On the one hand, operation
The cells were not broken before extraction, and the cell wall and cell membrane brought great extraction resistance, and the extraction time was long; In addition, the extraction operation could not inactivate the intracellular hydrolase rapidly, but the slow temperature rise promoted the degradation of the enzyme to the target product, and the precious raw material resources were not fully utilized. Therefore, long extraction time and low yield are one of the key problems that perplex the extraction of intracellular natural products. At present, people are actively engaged in the research of new extraction technologies of natural products, such as supercritical extraction, ultrasonic extraction, microwave enhanced extraction, etc.
From the perspective of kinetics, the permeability of cell wall and cell membrane is the key factor to limit the extraction rate of natural products, so the extraction of natural products will be greatly accelerated after cell breakage. The existing cell fragmentation methods such as high-speed ball milling and high-pressure homogenization can realize the rapid release of the target product, but the intracellular hydrolase is released together with the release of the target product. The released hydrolase meets the target product, resulting in the loss of the target product. Moreover, the extract contains a large amount of impurities such as protein, which increases the burden of subsequent separation and purification. Therefore, the above cell fragmentation methods are not suitable for the extraction of natural products. Therefore, it is of great significance for the research and development of natural products to study the cell fragmentation method specially suitable for the extraction of biological small molecules. In view of the good thermal stability of natural products, we applied microwave technology to cell fragmentation and established a cell fragmentation technology suitable for the extraction of small molecules in biological cells - microwave cell fragmentation.
Microwave cell fragmentation is realized by controlling the appropriate microwave conditions based on the selectivity, instantaneity and efficiency of microwave heating. Because of the reduction of extraction resistance, the dissolution of small molecular substances in cells after microwave fragmentation becomes very easy, which can shorten the extraction time. In addition, microwave treatment can also inactivate the hydrolase of the target product in the cell and avoid the degradation of the target product in the extraction process. Therefore, we put forward the concept of "microwave cell-breaking extraction", and successfully extracted trehalose in yeast cells and Rhodiola sachalinensis from callus of medicinal plant Rhodiola sachalinensis. In this paper, we first take yeast cells as an example to illustrate the mechanism of microwave breaking cells, and then illustrate the advantages of microwave breaking cell extraction technology through the comparison of examples.
2. The mechanism of the wave breaking of drunk mother cell
2.1 Features of microwave heating
Microwave is a kind of high-frequency electromagnetic wave with frequency from 3000MHz to 3000GHz. It has wave characteristics of electromagnetic wave such as reflection, transmission and interference, diffraction, polarization, and energy transmission accompanied by electromagnetic wave, and can be used for heating. Microwave obtains energy by making the rapid oscillation of polar molecules or dipoles in the microwave field produce a friction-like effect to heat the medium. Microwave heating has good selectivity. Due to the different dielectric properties of different materials, the heating characteristics in the microwave field are very different. Water and anion and cation can be heated rapidly by microwave, while glass and polytetrafluoroethylene are transparent to microwave and do not absorb microwave energy, so they cannot be heated by microwave. In addition, microwave heating is efficient and fast, and it is easy to realize continuity and automation. In view of the above characteristics of microwave technology, microwave has been widely used in food drying, sterilization, expansion and other fields.
2.22.2
Drunk yeast has a typical cell structure, which is composed of cell wall, cell membrane, nucleus, vacuole, mitochondria and cytoplasm. The protoplasm of yeast cells contains one or more vacuoles of different sizes, the diameter of which is u33m. It is especially obvious in the cells in the static growth stage, and is often a large organelle. When the cells begin to sprout in the nutrient medium, the large vacuoles are squeezed into two small vacuoles. When the buds are formed, the vacuoles are distributed to the mother cells and daughter cells. After budding, small vacuoles recombine or fuse to form large vacuoles.
According to the principle of microwave heating, polar substances such as positive and negative ions and water have strong absorption capacity for microwave, while non-polar substances hardly absorb microwave. Because yeast cells have a typical cell structure, the distribution of positive, negative ions, water and other polar substances in different parts of the cell or different organelles is uneven. Therefore, when yeast cells are placed in the microwave field for treatment, yeast cells will be locally heated.
2.3 Microwave crushing of yeast cells
Local heating of yeast cells in microwave field is the physical basis of yeast cell microwave fragmentation. Vacuole is the part rich in free water in yeast cells. When yeast cells receive microwave radiation, the free water absorbs a lot of microwave energy and is heated rapidly. The temperature reaches the boiling point and vaporizes, making the intracellular pressure rise. The internal pressure that the yeast cell wall and cell membrane can bear is limited. When the internal pressure caused by microwave heating rises to the limit that the cell can bear, the yeast cell will be expanded and broken.
2.4 Parts of yeast cells broken by microwave
Through scanning electron microscope observation of yeast before and after microwave breaking, it was found that the microwave breaking of yeast cells generally occurred at the budding site of cells first. The author explains this phenomenon as follows: For cells with buds, mother cells and daughter cells contain their own vacuoles respectively. Therefore, when yeast is treated in the microwave field, its mother cells and buds are heated to produce internal pressure, thus generating two opposite pulling forces on the cells as shown in Figure 1 (a). Make the bud break away from the mother body and the cell break at its budding site: for the cells in G, phase, as shown in Figure 1 (b), the internal pressure in all directions of the cell is equal, but the bud mark is the part where the integrity of the cell wall has been damaged, which is more likely to lead to stress concentration, so the cell breaks at the bud mark.
Under the effect of microwave field, yeast cells first break at the budding site. At this time, if the microwave radiation is continued, the cells will be heated and dehydrated and quickly dried. Accordingly, cracks appear in other parts of the cell wall. To sum up, microwave breaking cells is the result of two mechanisms. First of all, the local heating of the cell leads to the increase of internal pressure, and the cell wall and cell membrane first break at its vulnerable part; then. The cells are dehydrated and dried, and the surface is cracked. The two different stages of microwave fragmentation of yeast cells can be clearly observed from the scanning electron micrographs of yeast cells treated with different microwave times
(a) Yeast in budding stage (b) Drunken mother without buds Fig. 1. The mechanism of microwave breaking of yeast cells Fig. 3. The application of tender wave breaking cell technology in the extraction of natural products Trehalose is a non-reducing double compound formed by the combination of two glucose molecules. It is widely distributed in the body of cryptobiotic organisms, namely dehydrated animals and plants. Well endows such organisms with the ability to resist adverse environmental stresses such as high temperature, early drying, dehydration, etc. In addition. Algae enamel has the special effect of keeping the structure and function of macromolecular substances such as protein and nucleic acid from being damaged under dry conditions. Therefore, as a kind of organism
three hundred and sixty-five
As active protective agents and food additives, trehalose has broad application prospects in the production of proteins, enzymes, vaccines, vaccines, genetic engineering drugs, food and cosmetics. Saccharomyces cerevisiae, especially bakers yeast, has abundant intracellular trehalose content and is an important source of trehalose products on the market. Because trehalose hydrolase - trehalogenase and trehalose coexist in drunk mother cells. Therefore, in order to prevent the degradation of trehalose, the extraction of trehalose in drunk mother cells is mostly carried out with high concentration alcohol as solvent at boiling point. Using microwave cell-breaking extraction technology and water as solvent, only The extraction operation can be completed in 10 minutes, and the trehalose yield is% higher than the traditional extraction. In addition, compared with the traditional process, microwave cell-breaking extraction, protein and other macromolecular impurities are less dissolved, thus reducing the burden of subsequent separation of the target product "9)
As active protective agents and food additives, trehalose has broad application prospects in the production of proteins, enzymes, vaccines, vaccines, genetic engineering drugs, food and cosmetics. Saccharomyces cerevisiae, especially bakers yeast, has abundant intracellular trehalose content and is an important source of trehalose products on the market. Because trehalose hydrolase - trehalogenase and trehalose coexist in drunk mother cells. Therefore, in order to prevent the degradation of trehalose, the extraction of trehalose in drunk mother cells is mostly carried out with high concentration alcohol as solvent at boiling point. Using microwave cell-breaking extraction technology and water as solvent, only The extraction operation can be completed in 10 minutes, and the trehalose yield is% higher than the traditional extraction. In addition, compared with the traditional process, microwave cell-breaking extraction, protein and other macromolecular impurities are less dissolved, thus reducing the burden of subsequent separation of the target product "9)（
(1) 0 The above examples show that microwave fragmentation technology not only enriches the connotation of cell fragmentation in theory, but also has high practical value. This technology has certain universality for the extraction of small molecules in biological cells. Compared with traditional extraction methods, microwave cell-breaking extraction has great advantages, mainly in the aspects of short extraction time, high yield of target products, and less impurity dissolution. Therefore, microwave cell-breaking extraction is a time-saving, high-efficiency, energy-saving, clean and safe natural product extraction technology. Due to the increasing difficulty in the development of new chemical synthetic drugs, foreign famous pharmaceutical companies and research institutions have turned their eyes to nature to find new drugs from plants. Chinese herbal medicine culture is extensive and profound. It has a good knowledge accumulation and has gradually gained international recognition. We hope that microwave technology can play its due role in the modernization of Chinese traditional medicine and the research and development of natural drugs, and contribute to the development of Chinese traditional medicine in the world.
reference..
1. Gan Shijun et al., Modernization Development Strategy of Traditional Chinese Medicine, Science and Technology Press, 19
998.2 Editorial Committee of Chemical Engineering Manual, Chapter 14 of Chemical Engineering Manual: Extraction and Extraction, Chemical Industry Press, 1989
3. Xu Lianying et al., Review of the development of traditional Chinese medicine preparations, Shanghai Patent Medicine. 20
000,21 (1): 6-21.4 Display, etc., some questions about the industrialization of supercritical extraction of Chinese herbal medicine, Huyilan 19 999,30 (I): 62-65.5 Zhao Bing et al., Application of ultrasound in plant extraction, Hu Zaidong 19 999,30 (9): attached.
8. Liu Chuanbin, The accumulation law of trehalose in the cells of drunk mother and the microwave cell-breaking extraction technology, Zhang Zhugong, Zhang Zhuzhou, Shi Laozai, Zhang Ge 9
9. Liu Chuanbin et al., Microwave cell-breaking extraction of trehalose in yeast cells,
Huibo Technology in Gaoshan Country
10 Liu Chuanbin et al., Application in the extraction of effective components of Rhodiola,
The rich biological resources in nature provide tens of thousands of natural products for human beings, which are important sources of fine chemicals such as drugs, spices, food additives, etc. To develop and apply natural products, it is necessary to extract them from organisms. The traditional extraction operation is still widely used in the industry for the extraction of natural products, which is time-consuming, laborious, inefficient and backward in technology.
1z3。 On the one hand, operation
The cells were not broken before extraction, and the cell wall and cell membrane brought great extraction resistance, and the extraction time was long; In addition, the extraction operation could not inactivate the intracellular hydrolase rapidly, but the slow temperature rise promoted the degradation of the enzyme to the target product, and the precious raw material resources were not fully utilized. Therefore, long extraction time and low yield are one of the key problems that perplex the extraction of intracellular natural products. At present, people are actively engaged in the research of new extraction technologies of natural products, such as supercritical extraction, ultrasonic extraction, microwave enhanced extraction, etc.
From the perspective of kinetics, the permeability of cell wall and cell membrane is the key factor to limit the extraction rate of natural products, so the extraction of natural products will be greatly accelerated after cell breakage. The existing cell fragmentation methods such as high-speed ball milling and high-pressure homogenization can realize the rapid release of the target product, but the intracellular hydrolase is released together with the release of the target product. The released hydrolase meets the target product, resulting in the loss of the target product. Moreover, the extract contains a large amount of impurities such as protein, which increases the burden of subsequent separation and purification. Therefore, the above cell fragmentation methods are not suitable for the extraction of natural products. Therefore, it is of great significance for the research and development of natural products to study the cell fragmentation method specially suitable for the extraction of biological small molecules. In view of the good thermal stability of natural products, we applied microwave technology to cell fragmentation and established a cell fragmentation technology suitable for the extraction of small molecules in biological cells - microwave cell fragmentation.
Microwave cell fragmentation is realized by controlling the appropriate microwave conditions based on the selectivity, instantaneity and efficiency of microwave heating. Because of the reduction of extraction resistance, the dissolution of small molecular substances in cells after microwave fragmentation becomes very easy, which can shorten the extraction time. In addition, microwave treatment can also inactivate the hydrolase of the target product in the cell and avoid the degradation of the target product in the extraction process. Therefore, we put forward the concept of "microwave cell-breaking extraction", and successfully extracted trehalose in yeast cells and Rhodiola sachalinensis from callus of medicinal plant Rhodiola sachalinensis. In this paper, we first take yeast cells as an example to illustrate the mechanism of microwave breaking cells, and then illustrate the advantages of microwave breaking cell extraction technology through the comparison of examples.
2. The mechanism of the wave breaking of drunk mother cell
2.1 Features of microwave heating
Microwave is a kind of high-frequency electromagnetic wave with frequency from 3000MHz to 3000GHz. It has wave characteristics of electromagnetic wave such as reflection, transmission and interference, diffraction, polarization, and energy transmission accompanied by electromagnetic wave, and can be used for heating. Microwave obtains energy by making the rapid oscillation of polar molecules or dipoles in the microwave field produce a friction-like effect to heat the medium. Microwave heating has good selectivity. Due to the different dielectric properties of different materials, the heating characteristics in the microwave field are very different. Water and anion and cation can be heated rapidly by microwave, while glass and polytetrafluoroethylene are transparent to microwave and do not absorb microwave energy, so they cannot be heated by microwave. In addition, microwave heating is efficient and fast, and it is easy to realize continuity and automation. In view of the above characteristics of microwave technology, microwave has been widely used in food drying, sterilization, expansion and other fields.
2.22.2
Drunk yeast has a typical cell structure, which is composed of cell wall, cell membrane, nucleus, vacuole, mitochondria and cytoplasm. The protoplasm of yeast cells contains one or more vacuoles of different sizes, the diameter of which is u33m. It is especially obvious in the cells in the static growth stage, and is often a large organelle. When the cells begin to sprout in the nutrient medium, the large vacuoles are squeezed into two small vacuoles. When the buds are formed, the vacuoles are distributed to the mother cells and daughter cells. After budding, small vacuoles recombine or fuse to form large vacuoles.
According to the principle of microwave heating, polar substances such as positive and negative ions and water have strong absorption capacity for microwave, while non-polar substances hardly absorb microwave. Because yeast cells have a typical cell structure, the distribution of positive, negative ions, water and other polar substances in different parts of the cell or different organelles is uneven. Therefore, when yeast cells are placed in the microwave field for treatment, yeast cells will be locally heated.
2.3 Microwave crushing of yeast cells
Local heating of yeast cells in microwave field is the physical basis of yeast cell microwave fragmentation. Vacuole is the part rich in free water in yeast cells. When yeast cells receive microwave radiation, the free water absorbs a lot of microwave energy and is heated rapidly. The temperature reaches the boiling point and vaporizes, making the intracellular pressure rise. The internal pressure that the yeast cell wall and cell membrane can bear is limited. When the internal pressure caused by microwave heating rises to the limit that the cell can bear, the yeast cell will be expanded and broken.
2.4 Parts of yeast cells broken by microwave
Through scanning electron microscope observation of yeast before and after microwave breaking, it was found that the microwave breaking of yeast cells generally occurred at the budding site of cells first. The author explains this phenomenon as follows: For cells with buds, mother cells and daughter cells contain their own vacuoles respectively. Therefore, when yeast is treated in the microwave field, its mother cells and buds are heated to produce internal pressure, thus generating two opposite pulling forces on the cells as shown in Figure 1 (a). Make the bud break away from the mother body and the cell break at its budding site: for the cells in G, phase, as shown in Figure 1 (b), the internal pressure in all directions of the cell is equal, but the bud mark is the part where the integrity of the cell wall has been damaged, which is more likely to lead to stress concentration, so the cell breaks at the bud mark.
Under the effect of microwave field, yeast cells first break at the budding site. At this time, if the microwave radiation is continued, the cells will be heated and dehydrated and quickly dried. Accordingly, cracks appear in other parts of the cell wall. To sum up, microwave breaking cells is the result of two mechanisms. First of all, the local heating of the cell leads to the increase of internal pressure, and the cell wall and cell membrane first break at its vulnerable part; then. The cells are dehydrated and dried, and the surface is cracked. The two different stages of microwave fragmentation of yeast cells can be clearly observed from the scanning electron micrographs of yeast cells treated with different microwave times
(a) Yeast in budding stage (b) Drunken mother without buds Fig. 1. The mechanism of microwave breaking of yeast cells Fig. 3. The application of tender wave breaking cell technology in the extraction of natural products Trehalose is a non-reducing double compound formed by the combination of two glucose molecules. It is widely distributed in the body of cryptobiotic organisms, namely dehydrated animals and plants. Well endows such organisms with the ability to resist adverse environmental stresses such as high temperature, early drying, dehydration, etc. In addition. Algae enamel has the special effect of keeping the structure and function of macromolecular substances such as protein and nucleic acid from being damaged under dry conditions. Therefore, as a kind of organism
three hundred and sixty-five
As active protective agents and food additives, trehalose has broad application prospects in the production of proteins, enzymes, vaccines, vaccines, genetic engineering drugs, food and cosmetics. Saccharomyces cerevisiae, especially bakers yeast, has abundant intracellular trehalose content and is an important source of trehalose products on the market. Because trehalose hydrolase - trehalogenase and trehalose coexist in drunk mother cells. Therefore, in order to prevent the degradation of trehalose, the extraction of trehalose in drunk mother cells is mostly carried out with high concentration alcohol as solvent at boiling point. Using microwave cell-breaking extraction technology and water as solvent, only The extraction operation can be completed in 10 minutes, and the trehalose yield is% higher than the traditional extraction. In addition, compared with the traditional process, microwave cell-breaking extraction, protein and other macromolecular impurities are less dissolved, thus reducing the burden of subsequent separation of the target product "9)
As active protective agents and food additives, trehalose has broad application prospects in the production of proteins, enzymes, vaccines, vaccines, genetic engineering drugs, food and cosmetics. Saccharomyces cerevisiae, especially bakers yeast, has abundant intracellular trehalose content and is an important source of trehalose products on the market. Because trehalose hydrolase - trehalogenase and trehalose coexist in drunk mother cells. Therefore, in order to prevent the degradation of trehalose, the extraction of trehalose in drunk mother cells is mostly carried out with high concentration alcohol as solvent at boiling point. Using microwave cell-breaking extraction technology and water as solvent, only The extraction operation can be completed in 10 minutes, and the trehalose yield is% higher than the traditional extraction. In addition, compared with the traditional process, microwave cell-breaking extraction, protein and other macromolecular impurities are less dissolved, thus reducing the burden of subsequent separation of the target product "9)（
(1) 0 The above examples show that microwave fragmentation technology not only enriches the connotation of cell fragmentation in theory, but also has high practical value. This technology has certain universality for the extraction of small molecules in biological cells. Compared with traditional extraction methods, microwave cell-breaking extraction has great advantages, mainly in the aspects of short extraction time, high yield of target products, and less impurity dissolution. Therefore, microwave cell-breaking extraction is a time-saving, high-efficiency, energy-saving, clean and safe natural product extraction technology. Due to the increasing difficulty in the development of new chemical synthetic drugs, foreign famous pharmaceutical companies and research institutions have turned their eyes to nature to find new drugs from plants. Chinese herbal medicine culture is extensive and profound. It has a good knowledge accumulation and has gradually gained international recognition. We hope that microwave technology can play its due role in the modernization of Chinese traditional medicine and the research and development of natural drugs, and contribute to the development of Chinese traditional medicine in the world.
reference..
1. Gan Shijun et al., Modernization Development Strategy of Traditional Chinese Medicine, Science and Technology Press, 19
998.2 Editorial Committee of Chemical Engineering Manual, Chapter 14 of Chemical Engineering Manual: Extraction and Extraction, Chemical Industry Press, 1989
3. Xu Lianying et al., Review of the development of traditional Chinese medicine preparations, Shanghai Patent Medicine. 20
000,21 (1): 6-21.4 Display, etc., some questions about the industrialization of supercritical extraction of Chinese herbal medicine, Huyilan 19 999,30 (I): 62-65.5 Zhao Bing et al., Application of ultrasound in plant extraction, Hu Zaidong 19 999,30 (9): attached.
8. Liu Chuanbin, The accumulation law of trehalose in the cells of drunk mother and the microwave cell-breaking extraction technology, Zhang Zhugong, Zhang Zhuzhou, Shi Laozai, Zhang Ge 9
9. Liu Chuanbin et al., Microwave cell-breaking extraction of trehalose in yeast cells,
Huibo Technology in Gaoshan Country
10 Liu Chuanbin et al., Application in the extraction of effective components of Rhodiola,