CN111298477A

CN111298477A - Microorganism broken wall extraction system

Info

Publication number: CN111298477A
Application number: CN202010166291.7A
Authority: CN
Inventors: 卢婷婷; 李翠; 赵丽; 王文佳; 吕慧芳; 李存法; 连瑞丽; 王瑞宁
Original assignee: Henan University of Animal Husbandry and Economy
Current assignee: Henan University of Animal Husbandry and Economy
Priority date: 2020-03-11
Filing date: 2020-03-11
Publication date: 2020-06-19
Anticipated expiration: 2040-03-11
Also published as: CN111298477B

Abstract

The invention discloses a microorganism wall breaking extraction system which comprises a permeation impact tank, a permeation liquid tank, a cleaning tank, a freezing tank, a refrigeration system, a heat exchange system, an ice-containing crusher and a pumping mechanism, wherein the permeation impact tank comprises a permeation tank body, a conical sealing cover is fixed at the center of the bottom in the permeation tank body, the conical sealing cover and the inner wall of the tank body enclose a ring cavity, a filter layer is fixed at the upper end of the conical sealing cover, outlets are respectively arranged at two sides of the bottom of the ring cavity, and the two sides of the bottom of the ring cavity are respectively communicated with the permeation liquid tank and the cleaning tank through; the invention firstly reduces the strength of microbial cell walls through the penetration impact tank, and the process does not need direct wall breaking, so the invention is a milder crushing pretreatment process, water in cells seeps outwards under the action of repeated osmotic pressure of the penetration impact tank, the cells shrink, and after the balance is reached, the medium is quickly diluted, or the cells are transferred into water or buffer solution, so the quantity of peptide bonds and the crosslinking degree are reduced, and further, the reticular structure of peptidoglycan in the cell walls is reduced.

Description

Microorganism broken wall extraction system

Technical Field

The invention belongs to the field of microbial cell disruption and plasmid extraction equipment, and particularly relates to a mechanical and non-mechanical combined microbial wall breaking extraction technology for continuous production.

Background

The cell disruption technique is a technique for releasing cell contents including target product components by destroying cell membranes and cell walls by external force, and it is necessary to disrupt tissues and cells by an appropriate method. The difficulty of cell disruption varies among organisms or tissues in different parts of the same organism, and the method used varies. The current cell disruption methods mainly comprise: mechanical crushing and non-mechanical crushing. The mechanical crushing method also comprises the methods of high-pressure homogenizing, bead grinding, impact crushing, ultrasonic crushing and the like, and is characterized by large treatment capacity, high crushing efficiency, high speed and mainly depending on the homogenizing action and the grinding action; during the treatment, the cell wall cannot be broken by strong mechanical shear forces, resulting in cytosolic efflux. The above wall-breaking methods are mainly applied to the wall-breaking of plant cells such as traditional Chinese medicine materials, etc., and the non-mechanical disruption method is a chemical or biochemical infiltration method and a physical infiltration method, and the chemical and biochemical infiltration methods mainly include: acid-base treatment, chemical reagent treatment, enzymatic lysis and the like, a small amount of cell wall breaking experiments are usually performed under experimental conditions (many lysis methods are needed for stubborn positive bacteria). Compared with the mechanical crushing method, the chemical infiltration method has low processing speed and poor crushing efficiency, and the addition of chemical or biochemical reagents can cause new pollution and bring troubles for further separation and purification. The method often causes social problems of waste of a large amount of raw materials, increase of byproducts, environmental pollution caused by a large amount of chemical reagents and the like, and is not suitable for large-scale industrial use.

In addition, the cell membrane of animal organs is fragile and easily broken by common cell breaking equipment such as mechanical breaking method, but the cell wall breaking difficulty of microorganisms is large because of complex cell wall (the cell wall of gram-positive bacteria can endure 20kg/cm2 pressure). For example, yeast has a specific chemical component of cell wall, mainly composed of "yeast cellulose", which is similar to a sandwich structure, with an outer layer of mannan, an inner layer of glucan, a middle layer of protein molecules, and a small amount of lipids and chitin on the cell wall. The main component of the bacterial cell wall is peptidoglycan. Peptidoglycan from gram-positive bacteria accounts for more than 50% of the dry weight of the cell wall. It is a slightly soluble glycan chain, and is surrounded around cells by a network structure formed by crosslinking short peptides, so that the cells have certain shape and strength. The main resistance to break-up bacteria is from the network of peptidoglycans, whose compactness and strength depend on the number of peptide bonds present on the glycan chain and the extent of their cross-linking, if large. The heating phenomenon of crushing can be caused by adopting the existing mechanical crushing method, which is a common problem of the existing mechanical crushing equipment, and the local high temperature generated by friction and impact in the working process of the equipment can cause the deterioration of substances required in cytoplasm. In the prior art, the protein is denatured by simple repeated freezing and thawing, thereby influencing the recovery rate of the active protein.

Therefore, it is an urgent technical problem to be solved by those skilled in the art to provide a cell disruption method or cell disruptor which is suitable for large-scale industrial use, simple, efficient, energy-saving and environment-friendly.

Disclosure of Invention

Aiming at the defects and problems of the prior cell disruption device in the bacterial cell disruption process, the invention provides a microorganism wall-breaking extraction system combining a mechanical mode and a non-mechanical mode. The method realizes mechanical crushing under low temperature, prevents the problem that the local high temperature generated by friction and impact can cause the deterioration of the substances required in cytoplasm, ensures that the activity of protein is not influenced, and improves the recovery rate.

The technical scheme adopted by the invention for solving the technical problems is as follows: a microorganism wall breaking extraction system comprises a penetration impact tank, a penetration liquid tank, a cleaning tank, a freezing tank, a refrigerating system, a heat exchange system, an ice-containing crusher and a pumping mechanism, wherein the penetration impact tank comprises a penetration tank body, a conical sealing cover is fixed at the center of the bottom in the penetration tank body, the conical sealing cover and the inner wall of the tank body enclose a ring cavity, a filtering layer is fixed at the upper end of the conical sealing cover, outlets are respectively arranged at two sides of the bottom of the ring cavity, and the two sides of the bottom of the ring cavity are respectively communicated with the penetration liquid tank and the cleaning tank through corresponding pipelines; an outlet is arranged in the center of the filter layer, a discharge electromagnetic ball valve and a discharge pipe are arranged on the filter layer, the discharge pipe is communicated with an inlet at the upper end of the freezing tank through the pumping mechanism, and the lower end of the freezing tank is communicated with an inlet at the upper end of the ice-containing crusher; the permeate liquid tank comprises a permeate tank body, the bottom of the permeate tank body is provided with an inlet and is communicated with an outlet at the bottom of the annular cavity through a permeate communicating pipe, a permeate closing valve is installed on the permeate communicating pipe, and the top of the permeate tank body is provided with an air suction port and is hermetically provided with a negative pressure branch pipe I; the cleaning tank comprises a clean water tank body, the bottom of the clean water tank body is provided with an inlet and is communicated with the other outlet at the bottom of the annular cavity through a clean water discharge main pipe and a clean water discharge main pipe, and a clean water sealing valve is arranged on the clean water discharge main pipe; an air exhaust hole is formed in the top of the clean water tank body, and a negative pressure branch pipe II is hermetically installed on the top of the clean water tank body; the first negative pressure branch pipe and the second negative pressure branch pipe are connected with an air suction port of the vacuum pump through a three-way pipe, and a first air path electromagnetic valve and a second air path electromagnetic valve are respectively arranged on the first negative pressure branch pipe and the second negative pressure branch pipe; the freezing tank comprises a cooling tank main body, a top cover and a bottom plate, wherein a liquid feeding hopper is arranged on the top cover, an ice discharging port is arranged on the bottom plate, a central rotating shaft is arranged at the centers of the top cover and the bottom plate through a bearing, one end of the central rotating shaft is in transmission connection with a driving mechanism, a plurality of layers of rotating disks are uniformly distributed and arranged on the part of the central rotating shaft in the tank body, the outer edge and the inner edge of each layer of rotating disk are respectively provided with a flange so as to form an annular accommodating area, each layer of rotating disk is provided with a material leaking hole, an ice shoveling plate is arranged on the upper side of each layer of rotating disk, the outer end of each ice shoveling plate is fixed on the inner wall of the tank body, the lower side of each ice shoveling plate is provided with a shoveling surface and is in matched contact; the positions of the material leaking holes of the upper and lower adjacent turntables are distributed in a staggered way in sequence; the ice crusher comprises a cylindrical section, a conical section and a cover plate, wherein an ice adding opening is arranged on the cover plate, a filter screen bracket and a lower shaft bracket are fixed at the bottom of the cylindrical section for a wall breaking outlet at the center of the conical section, the outer ends of the conical section and the conical section are respectively fixed on the inner walls of the cylindrical section and the conical section, the inner ends of the conical section and the cylindrical section are jointed and fixed together, a fine filter screen is fixed on the upper side of the filter screen bracket, an upper bearing is arranged in the center of the cover plate, meanwhile, a lower bearing is arranged in the center of the lower shaft support, a crushing rotating shaft is sleeved in the upper bearing and the lower bearing, the crushing rotating shaft is in transmission connection with a corresponding driving mechanism, a crushing hammer, a crushing knife and a ball poking rod are fixed on the crushing rotating shaft along the radial direction, the ball pulling rod is positioned at the lowest layer and is close to the fine filter screen, abrasive is laid on the upper side of the fine filter screen, and the ball pulling rod can contact the abrasive and drive the abrasive to roll along the surface of the fine filter screen.

Still include mixed liquid collection box, the clear water discharges the lower extreme of house steward and communicates in mixed liquid collection box through mixed liquid closed valve, the bottom of wasing the jar communicates to mixed liquid collection box in through the evacuation pipe, installs the evacuation closed valve on the evacuation pipe simultaneously.

The penetration impact tank comprises a penetration tank body, wherein a material cleaning stirring motor is fixed in the middle of an upper end socket of the penetration tank body, the upper end of a motor rotating shaft of the material cleaning stirring motor is arranged in the center of the upper end socket of the tank body through an upper bearing and an upper bearing seat, the lower end of the motor rotating shaft of the material cleaning stirring motor is arranged on a lower bearing support through a lower bearing and a lower bearing seat, and the lower bearing support is fixed in the center of the middle of the tank; and meanwhile, a hinge seat is fixed on the motor rotating shaft, a hinge rod is hinged to the side face of the hinge seat through a hinge pin, a scraper is hinged to the tail end of the hinge rod through a hinge pin, the lower surface of each scraper is matched and attached to the upper surface of the filter layer, and the adjacent scrapers are distributed in a staggered mode along the radial direction.

The microbial wall-breaking extraction system of claim, wherein the peripheral edge of the filter layer is fixed on the inner wall of the tank body through a mesh end support, mesh lower support ribs are uniformly distributed and fixed below the filter layer, the outer ends of the mesh lower support ribs are respectively fixed on the mesh end support, and the inner ends of the mesh lower support ribs are respectively fixed on the peripheral edge of the lower bearing support.

And a penetrating fluid replenishing pipe is also arranged at the top of the penetrating fluid tank, and a sealing valve is arranged.

The adopted penetrating fluid is a mixed solution of high-concentration glycerol or sucrose or sodium chloride.

The driving mechanism of the ice crusher comprises a crushing motor and a crushing gearbox, the crushing motor and the crushing gearbox are fixed on the cover plate and are in transmission connection, and an output shaft of the crushing gearbox is in transmission connection with the crushing rotating shaft through a coupler.

The top of the ice crusher is provided with a water replenishing pipe, a plurality of nozzles are distributed and fixed in the cover plate or the inner wall of the tank body, and the nozzles are respectively communicated with the water replenishing pipe.

The grinding agent is ceramic balls, quartz sand or alumina which are mixed together in a ratio of 4:2:1, and the mixture is rapidly stirred to break cells.

The driving mechanism of the freezing tank comprises a cold tank rotating motor and a freezing gearbox, the cold tank rotating motor and the freezing gearbox are fixed on the top cover and are in transmission connection, and a rotating shaft of the freezing gearbox is in transmission connection with the central rotating shaft through a coupler.

And one side of the cover plate positioned at the upper end of the cylindrical section is hinged at one side edge of the cylindrical section through a pin shaft, and the other side of the cover plate is fixed at the other side of the cylindrical section through a fastener or a bolt.

The invention has the beneficial effects that: the invention firstly reduces the strength of the microbial cell wall through the osmotic shock tank, the process does not need direct wall breaking, so the process is a milder crushing pretreatment process, the water in the cell is exuded outwards under the action of the repeated osmotic pressure of the osmotic shock tank, the cell shrinks, after the balance is reached, the medium is quickly diluted, or the cell is transferred into water or buffer solution, and the extracellular water quickly infiltrates into the cell due to the sudden change of the osmotic pressure to cause the quick expansion of the cell, thereby reducing the number of peptide bonds and the crosslinking degree, and further reducing the network structure of peptidoglycan in the hard cell wall.

After osmotic pressure impact treatment is carried out by the osmotic impact tank, microbial cells are rapidly frozen by the freezing tank and are matched with scraping to form a frozen body with a smaller volume, intracellular water is crystallized to form ice crystal grains, and the cells are expanded and broken. This process only involves one freezing and one thawing and thawing process, so that the desired substance in the cytoplasm is not substantially deteriorated. The invention is mechanically crushed in a frozen state, a crusher containing a blade and a hammer head is used for crushing a frozen body into smaller volume, and then an abrasive and frozen cells are quickly stirred and ground together to crush the cells. The problem of deterioration of substances required in cytoplasm can not be caused by local high temperature generated by friction and impact in the process, the activity of protein is not influenced, the recovery rate is improved, frozen cells on a grinding surface can be rapidly melted by high-speed grinding in a frozen state, the hydrophobic bond structure of a cell membrane can be broken, and therefore the hydrophilic performance of the cell is improved. The grinding in a frozen state can realize high wall-breaking rate of bacterial cells, and the wall-breaking rate is usually over 95-98%.

Drawings

FIG. 1 is a schematic diagram of the system connection of the present invention.

Fig. 2 is a schematic sectional view of the osmotic shock tank of fig. 1.

Fig. 3 is a schematic view of the internal structure of the freezing cylinder of fig. 1.

Fig. 4 is a schematic cross-sectional view of the ice crusher of fig. 1.

Fig. 5 is a control section block diagram.

Reference numbers in the figures: a penetration impact tank 1, a penetration liquid tank 2, a cleaning tank 3, a mixed liquid recovery tank 4, a freezing tank 5, a refrigerating system 6, a heat exchange system 7, an ice-containing crusher 8, a pumping mechanism 9, a charging port 10, a penetration tank 11, a cleaning stirring motor 12, a motor rotating shaft 13, an upper bearing seat 131, a lower bearing seat 132, a lower bearing support 133, a conical sealing cover 14, an annular cavity 15, a filter layer 16, a net end support 161, a net lower support rib 162, a hinge seat 17, a hinge rod 171, a scraper 172, a discharge electromagnetic ball valve 18, a discharge pipe 19, a water replenishing pipe 20, a penetration tank 21, a penetrating fluid 22, a negative pressure branch pipe 23, a penetrating fluid closing valve 24, a penetrating fluid replenishing pipe 25, a clear water tank 31, a clear water discharge main pipe 32, a clear water discharge branch pipe 33, a discharge pipe 34, a negative pressure branch pipe 35, a clear water closing valve 36, a mixed liquid closing valve 37, the ice making machine comprises a cold tank main body 51, a top cover 52, a bottom plate 53, a central rotating shaft 54, a cold tank rotating motor 55, a freezing gearbox 56, a rotary disc 57, a material leakage hole 571, an ice shoveling plate 572, a liquid feeding hopper 58, an ice discharging hole 59, a cylindrical section 81, a conical section 82, a cover plate 83, a crushing motor 84, a crushing gearbox 841, a crushing rotating shaft 842, a crushing hammer 843, a crushing cutter 844, a ball stirring rod 845, a lower shaft seat 846, a lower shaft support 847, an ice adding hole 85, an outlet 86, a water supplementing pipe 87, a spray head 871, a ceramic ball 88, a fine filter screen 89 and a filter screen support 891.

Detailed Description

The following example 1 is applied to a way for obtaining a large amount of beneficial products aiming at yeast cell wall breaking, is a microbial wall breaking and extracting process combining mechanical and non-mechanical modes, avoids the problem of deterioration of substances required in cytoplasm due to local high temperature generated by friction and impact through a mechanical breaking mode under a low temperature condition, ensures that protein activity is not influenced, the recovery rate is improved.

Example 1: the main parts of the microbial wall breaking and extracting system are shown in figure 1, and the main parts comprise a permeation impact tank 1, a permeation liquid tank 2, a cleaning tank 3, a mixed liquid recycling tank 4, a freezing tank 5, a refrigerating system 6, a heat exchange system 7, an ice-containing crusher 8, a pumping mechanism 9 and the like.

Wherein, the internal structure of the osmotic shock tank 1 is shown in fig. 2, one side of the upper end socket of the osmotic shock tank 1 is provided with a feed inlet for introducing cell suspension prepared by yeast (the bacterial mass ratio pbs volume when the slow release liquid is proportioned is 10-30%) into the osmotic tank body 11, the other side of the upper end socket of the osmotic shock tank 1 is also provided with a liquid supplementing pipe and a corresponding liquid supplementing motor M2, and a breathing pipeline and a breathing valve. The middle part of the upper end socket of the infiltration tank body 11 is fixed with a material cleaning stirring motor 12, the upper end of a motor rotating shaft 13 of the material cleaning stirring motor 12 is arranged in the center of the upper end socket of the tank body through an upper bearing and an upper bearing seat 131, and the lower end is arranged on a lower bearing bracket 133 through a lower bearing and a lower bearing seat 132. The lower bearing support 133 is fixed at the center of the middle part of the tank body and is positioned at the upper side of the filter layer 16, the aperture of the filter layer 16 is designed based on the permeation of water, the retention of cells and suspended impurities, and part of culture medium, and sundries which cannot permeate the net are regularly cleaned, and the filter layer 16 comprises an upper net layer, a lower net layer and a semi-permeable film layer in the middle, and is formed by fixing and compounding the edges. Before discharging, firstly the penetration impact tank 1 is acted by a penetration liquid tank 2 and a cleaning tank 3, so that the penetration impact tank reduces the strength of microbial cell walls, and the process does not need direct wall breaking.

The motor rotating shaft 13 is fixed with a hinge seat 17, the side surface of the hinge seat 17 is hinged with a hinge rod 171 through a pin shaft, the tail end of the hinge rod 171 is hinged with a scraper 172 through a pin shaft, the lower surface of each scraper 172 is matched and attached with the upper surface of the filter layer 16, the adjacent scrapers 172 are distributed in a staggered mode along the radial direction, the scrapers are ensured to cover the whole or most of filter screens, and when the scrapers rotate along with the motor rotating shaft, the surfaces of the filter screens can be cleaned to ensure the permeability of the filter screens.

A conical sealing cover 14 is fixed at the center of the bottom in the infiltration tank body 11, and the conical sealing cover 14 and the inner wall of the tank body form an annular cavity 15. The upper end of the conical sealing cover 14 is fixedly connected with the central bottom of the filter layer 16, and the peripheral edge of the filter layer 16 is fixed on the inner wall of the tank body through a net end support 161. Mesh lower support ribs 162 are uniformly distributed and fixed below the filter layer 16 for the purpose of improving the strength of the filter layer, the outer ends of the mesh lower support ribs 162 are respectively fixed on the mesh end supports 161, and the inner ends of the mesh lower support ribs are respectively fixed on the peripheral edge of the lower bearing support 133.

As can be seen in fig. 2, outlets are respectively arranged at two sides of the bottom of the annular cavity 15 and are respectively communicated with the permeate liquid tank 2 (the upper end of which contains a first breathing pipe and a control valve Q31) and the cleaning tank 3 (the upper end of which contains a second breathing pipe and a control valve Q32) through corresponding pipelines. An outlet is arranged at the center of the filter layer 16, a discharge electromagnetic ball valve 18 and a discharge pipe 19 are arranged, the discharge pipe 19 is communicated with the inlet at the upper end of the freezing tank 5 through the pumping mechanism 9, and the lower end of the freezing tank 5 is communicated with the inlet at the upper end of the ice-containing crusher 8.

As shown in fig. 1 and 2, the bottom of the permeate tank body 21 of the permeate tank 2 is provided with an inlet and is communicated with an outlet at the bottom of the annular cavity 15 through a permeate communicating pipe 22. A penetrating fluid closing valve 24 is arranged on the penetrating fluid communicating pipe 22, and a suction opening is arranged at the top of the penetrating tank body 21 and a negative pressure branch pipe I23 is arranged in a sealing way.

A permeate make-up pipe 25 is also installed on top of the permeate tank 2 and a sealing valve is provided. The adopted penetrating fluid is a mixed solution of high-concentration glycerol or sucrose or sodium chloride.

The cleaning tank 3 comprises a clean water tank 31, the bottom of the clean water tank 31 is provided with an inlet and is communicated with another outlet at the bottom of the annular cavity 15 through a clean water discharge main pipe 32 and a clean water discharge main pipe 32, and a clean water closing valve 36 is arranged on the clean water discharge main pipe 32. An air exhaust hole is formed in the top of the clean water tank body 31, and a second negative pressure branch pipe 35 is hermetically installed on the top of the clean water tank body; the first negative pressure branch pipe 23 and the second negative pressure branch pipe 35 are connected with an air suction opening of the vacuum pump through a three-way pipe, and meanwhile, a first air path electromagnetic valve and a second air path electromagnetic valve are respectively installed on the first negative pressure branch pipe 23 and the second negative pressure branch pipe 35.

Under the action of the M1, the permeate liquid tank 2 and the cleaning tank 3 work alternately under the control of electromagnetic valves Q1 and Q2. As shown in fig. 5, the control part adopts a siemens PLC controller to respectively receive signals of a plurality of sensors T1, S1, BL1 and BL2, respectively controls a relay group KM1-KM6 according to program design so as to control a plurality of motors M1-M6 to respectively work, and controls valve groups Q1-Q3, Q11 and Q21-Q23 to function. Wherein, T1 is a temperature sensor, S1 is a liquid level sensor, BL1 and BL2 are penetrant concentration sensors. M1 is a vacuum pump of a negative pressure system, M2 is a liquid supplementing motor, M3 is a feeding pump, M4 is a stirring motor, M5 is a first rotating motor, and M6 is a second rotating motor. The specific control process is that after cell suspension containing yeast is supplemented into the osmotic shock tank 1, the stirring motor M4 continuously rotates, (1) Q1 is closed, Q2 is opened, Q11 is closed, Q21 is opened, Q22 and Q23 are closed, and M1 is started, so that liquid in the osmotic shock tank 1 is pumped to one side of the cleaning tank 3, and the liquid part in the osmotic shock tank 1 is emptied; (2) q1 is closed, Q2 is closed, Q11 is opened, Q21 is closed, Q22 or Q23 is opened, so that the penetrating fluid enters the penetration and impact tank 1 from the penetrating fluid tank 2; (3) q1 is opened, Q2 is closed, Q11 is opened, M1 is started, and the penetrating fluid is enabled to flow back into the penetrating fluid tank 2 from the penetrating shock tank 1; (4) q11 is closed and Q21 is opened to allow clean water to enter the osmotic shock tank 1. And circulating the steps. The osmotic shock tank reduces the strength of microbial cell walls, and the process does not need direct wall breaking and is a milder crushing pretreatment process. Through the effect of the repeated osmotic pressure of the osmotic shock tank, the water in the cells seeps outwards, the cells shrink, after the balance is reached, the medium is quickly diluted, or the cells are transferred into water or buffer solution, and the water outside the cells quickly seeps into the cells due to the sudden change of the osmotic pressure, so that the cells are quickly expanded, the crosslinking degree of cell walls is reduced, and the hardness is further reduced.

The treated cells are transferred to the charge tank 58 of the freezing cylinder 5 through the discharge electromagnetic ball valve 18 and the discharge pipe 19 and the pumping mechanism 9. The freezing tank 5 shown in fig. 3 includes a freezing tank main body 51, a top cover 52 and a bottom plate 53, wherein a liquid adding hopper 58 is provided on the top cover 52, and an ice discharge port 59 is provided on the bottom plate 53. A central rotating shaft 54 is arranged at the centers of the top cover 52 and the bottom plate 53 through bearings, one end of the central rotating shaft 54 is in transmission connection with a driving mechanism, a plurality of layers of turntables 57 are uniformly arranged at the part of the central rotating shaft 54 positioned in the tank body, the outer edge and the inner edge of each layer of turntables 57 are respectively provided with a flange so as to form an annular accommodating area, each layer of turntables 57 is provided with a material leakage hole 571, meanwhile, the upper side of each layer of turntables 57 is respectively provided with an ice shoveling plate 572, the outer end of each ice shoveling plate 572 is fixed on the inner wall of the tank body, the lower side of each ice shoveling plate 572 is provided with a shoveling surface which is in matching contact with the bottom surface of the annular accommodating area of each layer of turn; the positions of the material leaking holes 571 of the upper and lower adjacent turntables 57 are sequentially distributed in a staggered manner; when the motor M5 works, the central rotating shaft 54 is driven to rotate to drive the turntables 57 of each layer to rotate synchronously, and the ice shoveling plate 572 on the upper side of the ice discharge port 59 of the turntables 57 of each layer can shovel ice in the turntables of the upper layer to the lower layer, but the ice shoveling plate is not synchronous when the upper layer and the lower layer are transferred, so that the freezing time is prolonged. The ice shoveling plate 572 can also prevent the ice from being excessively hardened and being unable to be separated from the turntable, so that the ice is always in a smaller particle size. The driving mechanism of the freezing tank 5 comprises a cold tank rotating motor 55 and a freezing gearbox 56 which are fixed on the top cover 52 and are in transmission connection, and a rotating shaft of the freezing gearbox 56 is in transmission connection with the central rotating shaft 54 through a coupler.

The ice crusher 8 as in fig. 4 comprises a cylindrical section 81 and a conical section 82 and a cover plate 83. One side of the cover plate 83 positioned at the upper end of the cylindrical section 81 is hinged at one side of the cylindrical section 81 through a pin shaft, and the other side is fixed at the other side of the cylindrical section 81 through a fastener or a bolt. An ice adding opening 85 is arranged on the cover plate 83, the center of the conical section 82 is a wall breaking outlet 86, a filter screen bracket 891 and a lower shaft bracket 847 are fixed at the bottom of the cylindrical section 81, the outer ends of the two are respectively fixed on the inner walls of the cylindrical section 81 and the conical section 82, the inner ends of the two are converged and fixed together, a fine filter screen 89 is fixed on the upper side of the filter screen bracket 891, an upper bearing is installed at the center of the cover plate, meanwhile, a lower shaft seat 846 is arranged at the center of the lower shaft support 847, a lower bearing is arranged in the lower shaft seat 846, a crushing rotating shaft 842 is sleeved in the upper bearing and the lower bearing, the crushing rotating shaft 842 is in transmission connection with a corresponding driving mechanism, a crushing hammer 843, a crushing knife 844 and a cue 845 are fixed on the crushing rotating shaft 842 along the radial direction, wherein the ball pulling rod 845 is positioned at the lowest layer and is close to the fine filter screen 89, the upper side of the fine filter screen 89 is paved with grinding agents, the paddle 845 is capable of contacting the abrasives and driving the abrasives to roll along the surface of the fine screen 89. The grinding agent is ceramic balls 88, quartz sand or alumina which are mixed together in a ratio of 4:2:1, and the mixture is rapidly stirred to break cells.

The driving mechanism of the ice crusher 8 comprises a crushing motor 84 and a crushing gearbox 841, the crushing motor 84 and the crushing gearbox 841 are fixed on the cover plate 83 and are in transmission connection, and an output shaft of the crushing gearbox 841 is in transmission connection with the crushing rotating shaft 842 through a coupler.

A water replenishing pipe 87 is arranged at the top of the ice crusher 8, a plurality of nozzles 871 are distributed and fixed in the cover plate 83 or the inner wall of the tank body, and each nozzle 871 is respectively communicated with the water replenishing pipe 87.

After osmotic pressure impact treatment is carried out through the osmotic impact tank, microbial cells are rapidly frozen through the freezing tank and are matched with scraping to form a small-volume frozen body, intracellular water is crystallized to form ice crystal grains, and the cells are expanded and broken. This process only involves one freezing and one thawing and thawing process, so that the desired substance in the cytoplasm is not substantially deteriorated. The invention is mechanically crushed in a frozen state, a crusher containing a blade and a hammer head is used for crushing a frozen body into smaller volume, and then an abrasive and frozen cells are quickly stirred and ground together to crush the cells. The process can not cause the problem of deterioration of substances required in cytoplasm due to local high temperature generated by friction and impact, ensures that the activity of protein is not influenced, improves the recovery rate, and can also cause frozen cells on a grinding surface to be quickly melted by high-speed grinding in a frozen state, and can break the hydrophobic bond structure of cell membranes, thereby increasing the hydrophilic performance of the cells, wherein the whole treatment process is shown in figure 5. The grinding in a frozen state can realize high wall-breaking rate of bacterial cells, and the wall-breaking rate is usually over 95-98%.

Example 2: on the basis of embodiment 1, the cleaning device further comprises a mixed liquid recycling tank 4, the lower end of the clean water discharge header pipe 32 is communicated with the inside of the mixed liquid recycling tank 4 through a mixed liquid closing valve 37, the bottom of the cleaning tank 3 is communicated with the inside of the mixed liquid recycling tank 4 through an emptying pipe 34, and an emptying closing valve 38 is installed on the emptying pipe 34. The Siemens PLC controller is adopted to respectively receive signals of a plurality of sensors T1, S1, BL1 and BL2, respectively control a relay group KM1-KM6 according to program design so as to control a plurality of motors M1-M6 to respectively work, and control valve groups Q1-Q3, Q11, Q21-Q23, Q31 and Q32 to act. Wherein, T1 is a temperature sensor, S1 is a liquid level sensor, BL1 and BL2 are penetrant concentration sensors. M1 is a vacuum pump of a negative pressure system, M2 is a liquid supplementing motor, M3 is a feeding pump, M4 is a stirring motor, M5 is a first rotating motor, and M6 is a second rotating motor. The specific control process is that after the cell suspension containing the yeast is supplemented into the osmotic shock tank 1, the stirring motor M4 continuously rotates, Q1 is closed, Q2 is opened, Q11 is closed, Q32 is closed, Q21 is opened, Q22 and Q23 are closed, M1 is started, liquid in the osmotic shock tank 1 is pumped to one side of the cleaning tank 3, and the liquid part in the osmotic shock tank 1 is emptied. Q1 is then closed, Q2 closed, Q11 open, Q31 open, Q32 open, Q21 closed, Q22 or Q23 open, allowing permeate from permeate tank 2 to enter permeate surge tank 1. Next Q1 is opened, Q2 is closed, Q11 is opened, Q31 is closed, and M1 is activated to return permeate from the permeate surge tank 1 to the permeate tank 2. Q11 is closed again, Q21 is closed, and M2 is started to make clear water enter the infiltration surge tank 1. And circulating the steps.

Example 3: the process of breaking the walls of specific bacteria was performed using a technique similar to the mechanical and non-mechanical combination described in example 1. The bacteria are widely distributed in nature, large in quantity and multiple in types, can benefit mankind, and can also become a cause of diseases. In the field of biotechnology, beneficial bacteria are widely used, and most of the beneficial bacteria can be cultured by an artificial method, namely, the bacteria are inoculated on a culture medium to grow and reproduce, and the cultured bacteria are used for research, identification and application, cell disruption protein extraction, medicine application and the like. The cell wall is positioned at the outermost layer of the bacterial cell and wraps the periphery of the cell membrane, the composition is complex and is different with different bacteria, the cell walls of almost all bacteria are net structures of insoluble peptidoglycan, the net structures have large cross-linking degree, the quantity of peptide bonds causes high compactness, the wall breaking difficulty is very large, the existing filtering device for breaking and extracting the bacterial cells has complex structure and complicated operation process, and cannot achieve good extraction and filtration effects of object cells, thereby affecting the working quality, and is not suitable for large-scale industrial use, the following method is used for breaking the wall of the streptomyces and extracting crude enzyme liquid (the sub-cells after the bacteria are split are separated to form single thalli, and some sub-cells are not separated to form a certain arrangement mode, such as streptococcus, streptomyces and the like), the bacteria and the extraction process which combine mechanical mode with non-mechanical mode are broken by the mechanical breaking mode under the low temperature condition, avoid the problem that local high temperature generated by friction and impact can cause the deterioration of substances required in cytoplasm, ensure that the activity of protein is not influenced, and improve the recovery rate.

The apparatus and treatment method used in this example are substantially the same as those used in example 1, and the same points are not repeated, except that the ratio of the amount of bacteria to the volume of pbs is 1: 3. Through permeate liquid jar 2 and washing jar 3 to 1 effect of osmotic shock jar, make the osmotic shock jar reduce microorganism cell wall intensity, this process need not direct broken wall, so be a milder broken pretreatment process, effect through osmotic shock jar's repeated osmotic pressure, intracellular moisture just outwards oozes, the cell takes place the shrink, after reaching balance, dilute the medium fast, or with in the cell changes into water or buffer solution, because the abrupt change of osmotic pressure, extracellular water infiltrates the cell rapidly, arouse the cell and expand fast, thereby reduce the quantity and the crosslinking degree of peptide bond, and then reduce and the network structure of peptidoglycan in the hard its cell wall.

Claims

1. A microorganism wall breaking extraction system is characterized by comprising a penetration impact tank (1), a penetration liquid tank (2), a cleaning tank (3), a freezing tank (5), a refrigerating system (6), a heat exchange system (7), an ice-containing crusher (8) and a pumping mechanism (9), wherein the penetration impact tank (1) comprises a penetration tank body (11), a conical sealing cover (14) is fixed at the center of the bottom in the penetration tank body (11), the conical sealing cover (14) and the inner wall of the tank body enclose a ring cavity ((15)), a filter layer (16) is fixed at the upper end of the conical sealing cover (14), outlets are respectively arranged at two sides of the bottom of the ring cavity (15), and the outlets are respectively communicated with the penetration liquid tank (2) and the cleaning tank (3) through corresponding pipelines; an outlet is arranged at the center of the filter layer (16), a discharge electromagnetic ball valve (18) and a discharge pipe (19) are installed on the filter layer, the discharge pipe (19) is communicated with an inlet at the upper end of the freezing tank (5) through the pumping mechanism (9), and the lower end of the freezing tank (5) is communicated with an inlet at the upper end of the ice-containing crusher (8); the permeate liquid tank (2) comprises a permeate tank body (21), the bottom of the permeate tank body (21) is provided with an inlet and is communicated with an outlet at the bottom of the annular cavity (15) through a permeate communicating pipe (22), a permeate closing valve (24) is installed on the permeate communicating pipe (22), the top of the permeate tank body (21) is provided with an air suction port, and a negative pressure branch pipe I (23) is installed in a sealing mode; the cleaning tank (3) comprises a clear water tank body (31), the bottom of the clear water tank body (31) is provided with an inlet and is communicated with the other outlet at the bottom of the annular cavity (15) through a clear water discharge main pipe (32) and a clear water discharge main pipe (32), and a clear water closing valve (36) is arranged on the clear water discharge main pipe (32); an air suction hole is formed in the top of the clear water tank body (31) and a second negative pressure branch pipe (35) is hermetically installed on the top of the clear water tank body; the first negative pressure branch pipe (23) and the second negative pressure branch pipe (35) are connected with an air suction port of the vacuum pump through a three-way pipe, and a first air path electromagnetic valve and a second air path electromagnetic valve are respectively arranged on the first negative pressure branch pipe (23) and the second negative pressure branch pipe (35); the freezing tank (5) comprises a cooling tank main body (51), a top cover (52) and a bottom plate (53), a liquid feeding hopper (58) is arranged on the top cover (52), an ice discharging port (59) is arranged on the bottom plate (53), a central rotating shaft (54) is arranged at the centers of the top cover (52) and the bottom plate (53) through a bearing, one end of the central rotating shaft (54) is in transmission connection with a driving mechanism, a plurality of layers of turntables (57) are uniformly arranged at the part of the central rotating shaft (54) in the tank body, the outer edge and the inner edge of each layer of turntables (57) are respectively provided with a flange so as to form an annular accommodating area, each layer of turntables (57) is respectively provided with a material leaking hole (571), meanwhile, an ice shoveling plate (572) is respectively arranged at the upper side of each layer of turntables (57), the outer end of each ice shoveling plate (572) is fixed on the inner wall of the tank body, a shoveling surface is arranged at the lower side, the length of each ice shoveling plate (572) is slightly larger than that of the corresponding material leakage hole (571); the positions of the material leaking holes (571) of the upper and lower adjacent turntables (57) are distributed in a staggered way in sequence; the ice-containing crusher (8) comprises a cylindrical section (81), a conical section (82) and a cover plate (83), wherein an ice adding opening (85) is formed in the cover plate (83), the center of the conical section (82) is a wall breaking outlet (86), a filter screen support (891) and a lower shaft support (847) are fixed at the bottom of the cylindrical section (81), the outer ends of the two are respectively fixed on the inner walls of the cylindrical section (81) and the conical section (82), the inner ends of the two are crossed and fixed together, a fine filter screen (89) is fixed on the upper side of the filter screen support (891), an upper bearing is installed at the center of the cover plate, a lower bearing is installed at the center of the lower shaft support (847), a crushing rotating shaft (842) is sleeved in the upper bearing and the lower bearing, the crushing rotating shaft (842) is in transmission connection with a corresponding driving mechanism, a crushing hammer (843), a crushing cutter (844) and a ball stirring rod (845) are radially fixed on the, wherein, the ball pulling rod (845) is positioned at the lowest layer and is close to the fine filter screen (89), the grinding agent is paved on the upper side of the fine filter screen (89), and the ball pulling rod (845) can contact the grinding agent and drive the grinding agent to roll along the surface of the fine filter screen (89).

2. A microbial wall breaking extraction system according to claim 1, further comprising a mixed liquor recovery tank (4), wherein the lower end of the clean water discharge header pipe (32) is communicated with the mixed liquor recovery tank (4) through a mixed liquor closing valve (37), the bottom of the cleaning tank (3) is communicated with the mixed liquor recovery tank (4) through an emptying pipe (34), and an emptying closing valve (38) is installed on the emptying pipe (34).

3. A microbial wall breaking extraction system according to claim 1, wherein the penetration impact tank (1) comprises a penetration tank body (11), a material cleaning stirring motor (12) is fixed in the middle of an upper end socket of the penetration tank body, the upper end of a motor rotating shaft (13) of the material cleaning stirring motor (12) is installed in the center of the upper end socket of the tank body through an upper bearing and an upper bearing seat (131), the lower end of the motor rotating shaft is installed in a lower bearing support (133) through a lower bearing and a lower bearing seat (132), and the lower bearing support (133) is fixed in the center of the middle of the tank body and is positioned on the upper side of the filter layer (16); simultaneously be fixed with articulated seat (17) on motor shaft (13), the side of articulated seat (17) articulates through the round pin axle has articulated pole (171), and the end of articulated pole (171) articulates through the round pin axle has scraper blade (172), the lower surface of each scraper blade (172) with the upper surface of filter layer (16) matches the laminating, and adjacent each scraper blade (172) are along radial crisscross distribution.

4. A microbial wall breaking extraction system according to claim 1, wherein the peripheral edge of the filter layer (16) is fixed on the inner wall of the tank body through a mesh end support (161), mesh lower support ribs (162) are uniformly distributed and fixed below the filter layer (16), the outer ends of the mesh lower support ribs (162) are respectively fixed on the mesh end support (161), and the inner ends of the mesh lower support ribs are respectively fixed on the peripheral edge of the lower bearing support (133).

5. A broken microbial wall extraction system according to claim 1, wherein a permeate replenishing pipe (25) is further installed on the top of the permeate tank (2), and a sealing valve is arranged.

6. A microbial wall-breaking extraction system according to claim 1 or 5, wherein the adopted penetrating fluid is a high-concentration glycerol or sucrose or sodium chloride mixed solution.

7. A microbial wall breaking extraction system according to claim 1, wherein the driving mechanism of the ice crusher (8) comprises a crushing motor (84) and a crushing gearbox (841), the two are fixed on the cover plate (83) and are in transmission connection, and an output shaft of the crushing gearbox (841) is in transmission connection with the crushing rotating shaft (842) through a coupling.

8. The microbial wall-breaking extraction system of claim 1, wherein a water replenishing pipe (87) is arranged at the top of the ice crusher (8), a plurality of nozzles (871) are distributed and fixed in the cover plate (83) or the inner wall of the tank body, and each nozzle (871) is respectively communicated with the water replenishing pipe (87).

9. A microbial wall breaking extraction system according to claim 1, wherein the grinding agent is ceramic balls (88), quartz sand or alumina mixed together at a ratio of 4:2: 1.

10. A microbial wall breaking extraction system according to claim 1, wherein the cover plate (83) is arranged at the upper end of the cylindrical section (81), one side of the cover plate is hinged to one side of the cylindrical section (81) through a pin shaft, and the other side of the cover plate is fixed to the other side of the cylindrical section (81) through a fastener or a bolt.