Tremella aurantialba fermented lactic acid bacteria separating, screening and culturing device and separating method thereof

Abstract

The invention discloses a tremella aurantialba fermented lactic acid bacteria separating, screening and culturing device which comprises a sealing chamber, a lower isolation disc, an upper isolation disc, a heat insulation plate, a heater, a clamping sleeve and a rotating mechanism. The sealing chamber is formed by assembling a top cover and a cylindrical tank in a threaded mode, the lower isolation disc and the upper isolation disc are sequentially fixed to the inner wall of the sealing chamber from bottom to top, and the multiple heat insulation plates are arranged on the circumference and arranged between the upper isolation disc and the lower isolation disc in a sealed mode; a bottom flat cavity is formed between the lower isolation disc and the sealing chamber, the heater is arranged in the bottom flat cavity, a space between the lower isolation disc and the upper isolation disc is divided into a culture chamber and an inoculation chamber from outside to inside by a plurality of heat insulation plates, and a plurality of clamping sleeves for limiting culture dish bases are circumferentially distributed and movably arranged at the bottom of the culture chamber; a rotating mechanism for separating and screening bacterial colonies sequentially penetrates through the sealing chamber and the upper isolation disc and extends into the inoculation chamber.

Description

Technical Field

[0001] The invention relates to the technical field of lactic acid bacteria cultivation, and in particular to a device for separating, screening and cultivating Tremella fuciformis fermented lactic acid bacteria and a separation method thereof. Background Art

[0002] The isolation, screening and cultivation of Tremella fuciformis fermented lactic acid bacteria is a multi-step, sophisticated process that requires strict compliance with aseptic operation specifications and adjustment of the culture medium formula and culture conditions according to the strain characteristics and experimental requirements.

[0003] During the overall culture, the culture dishes need to be transferred manually, and separation and screening operations need to be performed on a sterile operating table. During any link, outside air will intervene, causing bacteria in the air to enter the culture dishes, resulting in a reduced culture success rate. Moreover, during the streaking operation for pure culture of colonies, the effective length and strength of the streaking are controlled by the operator, resulting in insufficient effective length of the streaking and inconsistent strength of the streaking, resulting in insufficient dispersion of the colonies and difficulty in forming individual colonies, which will also result in a reduced culture success rate.

[0004] Therefore, it is necessary to invent a device for separating, screening and culturing Tremella fuciformis fermented lactic acid bacteria to solve the above problems. Summary of the invention

[0005] In view of the above problems, the present invention provides a device for separating, screening and culturing Tremella fuciformis fermented lactic acid bacteria, which solves the problem of low culturing success rate in the lactic acid bacteria culturing process.

[0006] The technical solution adopted by the present invention is: comprising a sealed chamber, a lower isolation disk, an upper isolation disk, an insulation plate, a heater, a clamping sleeve and a rotating mechanism; the sealed chamber is formed by threaded assembly of a top cover and a cylindrical tank, the lower isolation disk and the upper isolation disk are sequentially fixed on the inner wall of the sealed chamber from bottom to top, and the insulation plate is configured with a plurality of plates on the circumference, all of which are sealed and arranged between the upper isolation disk and the lower isolation disk; Moreover, a bottom flat cavity is formed between the lower isolation disk and the sealed chamber, the heater is arranged in the bottom flat cavity, and the multiple insulation plates divide the space between the lower isolation disk and the upper isolation disk into a culture chamber and an inoculation chamber from the outside to the inside. There are multiple clamping sleeves distributed circumferentially for limiting the base of each culture dish, and they can be movably arranged at the bottom of the culture chamber. The rotating mechanism for separating and screening colonies passes through the sealed chamber and the upper isolation disk in sequence and extends into the inoculation chamber.

[0007] Further, as a preference, the rotating mechanism at least includes a connecting shaft, a knob, a disassembly and assembly assembly and a marking assembly; the connecting shaft passes through the upper isolation disk and is sealed and rotatably arranged with the upper isolation disk, the knob passes through the top cover and is connected to the extension shaft on the upper part of the connecting shaft, the disassembly and assembly assembly set for disassembling and assembling the end covers of each culture dish is fixed to one side of the bottom of the connecting shaft, and the marking assembly set for marking the culture medium is fixed to the other side of the bottom of the connecting shaft.

[0008] Further, as a preference, the marking assembly includes a multi-stage telescopic rod, a columnar base and an inoculation ring; the fixed end of the multi-stage telescopic rod is arranged on the extending end of the connecting shaft, the columnar base is fixed to the output end of the multi-stage telescopic rod and is arranged horizontally, and the multi-stage telescopic rod is connected to the external main control unit using electrical signals.

[0009] Furthermore, preferably, a spiral groove is provided at the bottom of the cylindrical base, the depth of the spiral groove gradually increases from the edge to the center, and the oblique cross-section of the spiral groove is a shape in which a circular portion and a rectangular portion are connected from top to bottom, the inoculation ring is arranged perpendicular to the cylindrical base, a ball sleeve is fixed to the upper end of the inoculation ring, a ball is rolled in the ball sleeve, the ball sleeve is slidably arranged in the circular portion of the spiral groove, the ball is in contact with the top of the circular portion, and the upper end of the inoculation ring is slidably arranged in the rectangular portion.

[0010] Further, preferably, the diameter R of the circular portion of the spiral groove is greater than the width L of the rectangular portion.

[0011] Furthermore, preferably, a heat conduction hole is opened in the center of the lower isolation plate.

[0012] Further, preferably, the lower ends of the plurality of insulation panels are configured as opening and closing panels, and the opening and closing panels have elastic deformation capability.

[0013] Furthermore, as a preference, it further comprises a lifting rod, of which a plurality is distributed around the circumference, one end of the lifting rod sealably passes through the outer wall of the sealing chamber, and the other end is sleeved and fixed in the extension portion of the clamping sleeve.

[0014] The separation method of the present invention is as follows: S1: constructing a screening medium for Lactobacillus auriculariae: MRS basic medium, primary screening medium and Lactobacillus auriculariae screening medium, and placing the completed medium in a culture room; S2: Initial screening of golden ear lactic acid bacteria: add the sample to the initial screening culture medium, culture it in the culture room at 30 ℃ under anaerobic conditions for 48 h, then re-streak the single colony with the calcium-dissolving ring through the rotating mechanism and the lifting rod, repeat the streaking culture for no less than 3 times until a single strain of pure culture is obtained, select the single colony with the calcium-dissolving ring, inoculate it in MRS liquid culture medium, move the culture dish to the culture room at 30 ℃ and culture it for 24 h, then take it out and store it at low temperature as a candidate strain of lactic acid bacteria; S3: Rescreening of Auricularia auriculariae lactic acid bacteria: The screening medium for Auricularia auriculariae lactic acid bacteria was used. The candidate strains were inoculated into the Auricularia auriculariae lactic acid bacteria screening medium and cultured at 30°C for 48 h. The operation was the same as in S2. Single colonies with calcium-dissolving circles were selected and inoculated into MRS liquid culture medium. The colonies were cultured at 30°C for 24 h and stored at low temperature as the target strains of lactic acid bacteria.

[0015] Advantages of the present invention: 1. In the present invention, the heating area, the culture area and the inoculation area are all arranged in a closed space, and the inoculation culture is completed by a rotating mechanism. Therefore, the present invention can isolate the static culture and inoculation culture processes of the Tremella fuciformis fermented lactic acid bacteria from the outside world, avoid bacterial intervention, and improve the success rate of lactic acid bacteria culture; 2. In the present invention, by using the streaking assembly, the inoculation loop can streak in a spiral shape on the surface of the culture medium, thereby increasing the effective streaking length and the dispersion area of ​​the colonies. At the same time, during the streaking process, the inoculation loop and the surface of the culture medium always maintain a constant pressure, so that the streaking force remains uniform, so that the colonies can be evenly dispersed, thereby improving the dispersion of the colonies, thereby increasing the formation rate of single colonies, and ultimately improving the success rate of lactic acid bacteria culture.

[0016] In addition to the above-described purposes, features and advantages, the present invention has other purposes, features and advantages. The present invention will be further described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 It is a schematic diagram of the overall structure of the present invention; Figure 2 is a schematic diagram of the rotating mechanism of the present invention; Figure 3 It is a schematic diagram of the columnar base structure of the present invention; Figure 4 is a partial cross-sectional view of a columnar base of the present invention; Figure 5 is a bottom-up cross-sectional view of a columnar base of the present invention; Figure 6 is a front cross-sectional view of a columnar base portion of the present invention; Figure 7 It is a motion state diagram of the columnar base and the inoculation loop of the present invention; Figure 8 It is a top view of the overall structure of the present invention.

[0019] Figure numerals: 1. Sealing chamber; 2. Lower isolation plate; 3. Upper isolation plate; 4. Insulation plate; 5. Heater; 6. Clamping sleeve; 7. Rotating mechanism; 71. Connecting shaft; 72. Knob; 73. Disassembly and assembly assembly; 74. Marking assembly; 741. Multi-stage telescopic rod; 742. Columnar base; 743. Inoculation ring; 744. Spiral groove; 7441. Circular part; 7442. Rectangular part; 745. Ball sleeve; 746. Ball; 21. Thermal hole; 8. Lifting rod. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0021] In the description of the present invention, it is necessary to understand that the terms "upper", "lower", "front", "back", "left", "right", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship are based on the orientation or position relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0022] refer to Figures 1 to 8 A device for separating, screening and cultivating golden ear fermented lactic acid bacteria, comprising a sealed chamber 1, a lower isolation plate 2, an upper isolation plate 3, an insulating plate 4, a heater 5, a clamping sleeve 6 and a rotating mechanism 7; the sealed chamber 1 is formed by threaded assembly of a top cover and a cylindrical tank, the lower isolation plate 2 and the upper isolation plate 3 are sequentially fixed on the inner wall of the sealed chamber 1 from bottom to top, and the insulating plate 4 is configured with a plurality of plates on its circumference, all of which are sealed and arranged between the upper isolation plate 3 and the lower isolation plate 2; Moreover, the lower isolation plate 2 and the sealed chamber 1 form a bottom flat cavity, the heater 5 is arranged in the bottom flat cavity, and the multiple insulation plates 4 divide the space between the lower isolation plate 2 and the upper isolation plate 3 into a culture chamber and an inoculation chamber from the outside to the inside. There are multiple clamping sleeves 6 distributed around the circumference for limiting the base of each culture dish, which can be movably arranged at the bottom of the culture chamber, and the rotating mechanism 7 for separating and screening colonies passes through the sealed chamber 1 and the upper isolation plate 3 in sequence and extends into the inoculation chamber.

[0023] For explanation, see Figure 1 The sealed chamber 1 is divided into a heating zone, a culture zone and an inoculation zone by the above structure, and the zones are all in a closed space, and the separation and screening of the colonies are completed by the rotating mechanism 7. Therefore, the present invention can isolate the static culture and inoculation culture processes of the golden ear fermented lactic acid bacteria from the outside world, avoid bacterial intervention, and improve the success rate of lactic acid bacteria culture; In addition, in the present invention, the separation and screening of lactic acid bacteria and the transfer of culture dishes can be performed through external operations of the sealed chamber 1, thereby preventing the operator's skin from being scalded and improving safety measures.

[0024] In the present invention, the rotating mechanism 7 at least includes a connecting shaft 71, a knob 72, a disassembly assembly 73 and a marking assembly 74; the connecting shaft 71 passes through the upper isolation disk 3 and is arranged to rotate in a sealed manner with the upper isolation disk 3, the knob 72 passes through the top cover and is connected to the extension shaft on the upper part of the connecting shaft 71, the disassembly assembly 73 for disassembling and assembling the end covers of each culture dish is set and fixed on one side of the bottom of the connecting shaft 71, and the marking assembly 74 for marking the culture medium is set and fixed on the other side of the bottom of the connecting shaft 71.

[0025] Among them, the disassembly and assembly component 73 is composed of a telescopic rod and a mechanical gripper. The telescopic rod and the mechanical gripper are both controlled by an external main control unit to control the telescopic amount and the clamping opening and closing angle, thereby completing the opening or closing action of the culture dish bottle cap to facilitate the marking action.

[0026] Specifically, see Figure 2 , twist the knob 72, and through the connecting shaft 71, the disassembly assembly 73 can be aligned with the culture dish below it. Similarly, the marking assembly 74 can also be aligned with the culture dish below it to complete the inoculation culture.

[0027] As a preferred embodiment, the marking assembly 74 includes a multi-stage telescopic rod 741, a columnar base 742 and an inoculation ring 743; the fixed end of the multi-stage telescopic rod 741 is arranged on the extending end of the connecting shaft 71, the columnar base 742 is fixed to the output end of the multi-stage telescopic rod 741 and is arranged horizontally, and the multi-stage telescopic rod 741 is connected to the external main control unit by electrical signals.

[0028] As a preferred embodiment, please refer to Figure 4A spiral groove 744 is provided at the bottom of the cylindrical base 742, and the depth of the spiral groove 744 gradually increases from the edge to the center, and the oblique cross-section of the spiral groove 744 is a shape in which a circular portion 7441 and a rectangular portion 7442 are connected from top to bottom. The inoculation ring 743 is arranged perpendicular to the cylindrical base 742, and a ball sleeve 745 is fixed to the upper end of the inoculation ring 743. A ball 746 is rolled in the ball sleeve 745. The ball sleeve 745 is slidably arranged in the circular portion 7441 of the spiral groove 744, and the ball 746 contacts the top of the circular portion 7441. The upper end of the inoculation ring 743 is slidably arranged in the rectangular portion 7442.

[0029] For details, please refer to Figures 2 to 6 When the streaking assembly 74 rotates to the top of the culture dish to be streaked, the multi-stage telescopic rod 741 is controlled by the external main control unit to move the multi-stage telescopic rod 741 and the column base 742 downward. When the bottom end of the inoculation loop 743 contacts the surface of the culture medium, please refer to Figure 6 A small pressure F is generated between the ball 746 and the circular portion 7441 of the spiral groove 744. When the residual force value F1 of the small pressure F reaches a certain threshold, the ball 746 and the spiral groove 744 will generate relative movement. Therefore, when the cylindrical base 742 moves downward at a uniform speed, the ball 746 will continue to roll, so that the inoculation ring 743 will form a spiral track on the surface of the culture medium, so that the effective utilization area of ​​the culture medium surface is increased, and the effective length of the streaking line is increased, thereby increasing the dispersion area of ​​the colony; Also, see Figure 7 During the uniform downward movement of the columnar base 742, the downward movement amount L is equal to the relative distance change H between the lower end of the inoculation loop 743 and the columnar base 742 during this period of time. That is to say, during the spiral streaking process, the lower end of the inoculation loop 743 always maintains a constant pressure with the surface of the culture medium, so that the streaking force remains uniform, allowing the colonies to be evenly dispersed.

[0030] Therefore, in summary, increasing the dispersion area of ​​the colonies and dispersing the colonies evenly can improve the dispersion of the colonies, thereby increasing the formation rate of single colonies and ultimately improving the success rate of lactic acid bacteria cultivation.

[0031] As a preferred embodiment, please refer to Figure 4 The diameter R of the circular portion 7441 of the spiral groove 744 is greater than the width K of the rectangular portion 7442 , and the circular portion 7441 provides a limiting and guiding function for the inoculation ring 743 , while the rectangular portion 7442 serves as an avoidance portion for the movement of the inoculation ring 743 .

[0032] As a preferred embodiment, a heat conduction hole 21 is opened in the center of the lower isolation plate 2.

[0033] It should be explained that the heater 5 generates high temperature, and the heat flows into the inoculation chamber through the heat conduction hole 21, so that the inoculation chamber is sterilized by high temperature and remains sterile during the inoculation process, thereby improving the success rate of lactic acid bacteria culture.

[0034] As a preferred embodiment, the lower ends of the plurality of insulation panels 4 are configured as open and close panels, which have elastic deformation capability.

[0035] As a preferred embodiment, it also includes a lifting rod 8, which is distributed in plurality around the circumference. One end of the lifting rod 8 is sealed and penetrates the outer wall of the sealing chamber 1, and the other end is sleeved and fixed in the extension portion of the clamping sleeve 6.

[0036] For details, please refer to Figure 1 or Figure 8 If the culture medium in two clamping sleeves 6 in the culture chamber needs to be inoculated and cultured, the culture medium is passed through the opening and closing plate into the inoculation chamber by pushing the corresponding lifting rod 8. During this process, the opening and closing plate with elastic deformation ability will overlap the lifting rod 8 to prevent the opening and closing plate from interfering with the opening and inoculation actions. After the inoculation and culture are completed under the rotating mechanism 7, the lifting rod 8 is pulled to return the clamping sleeve 6 to the culture chamber for static culture, and the opening and closing plate is restored to the initial state, that is, the culture chamber is restored to the sealed state again.

[0037] The separation method of the present invention is as follows: S1: constructing a screening medium for Lactobacillus auriculariae: MRS basic medium, primary screening medium and Lactobacillus auriculariae screening medium, and placing the completed medium in a culture room; S2: Initial screening of golden ear lactic acid bacteria: add the sample to the initial screening culture medium, culture it in the culture chamber at 30°C for 48 h under anaerobic conditions, then re-streak the single colony with the calcium-dissolving ring by the rotating mechanism 7 in cooperation with the lifting rod 8, repeat the streaking culture for no less than 3 times until a single bacterial species pure culture is obtained, select the single colony with the calcium-dissolving ring, inoculate it in the MRS liquid culture medium, move the culture dish to the culture chamber at 30°C for 24 h, and then take it out for low-temperature storage as a candidate strain of lactic acid bacteria; S3: Rescreening of Auricularia auriculariae lactic acid bacteria: The screening medium for Auricularia auriculariae lactic acid bacteria was used. The candidate strains were inoculated into the Auricularia auriculariae lactic acid bacteria screening medium and cultured at 30°C for 48 h. The operation was the same as in S2. Single colonies with calcium-dissolving circles were selected and inoculated into MRS liquid culture medium. The colonies were cultured at 30°C for 24 h and stored at low temperature as the target strains of lactic acid bacteria.

[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A device for separating, screening and cultivating Tremella fuciformis fermented lactic acid bacteria, characterized in that: The invention comprises a sealed chamber (1), a lower isolation disk (2), an upper isolation disk (3), an insulating plate (4), a heater (5), a clamping sleeve (6) and a rotating mechanism (7); the sealed chamber (1) is formed by threaded assembly of a top cover and a cylindrical tank; the lower isolation disk (2) and the upper isolation disk (3) are fixed on the inner wall of the sealed chamber (1) in sequence from bottom to top; a plurality of insulating plates (4) are arranged around the circumference and are all sealed and arranged between the upper isolation disk (3) and the lower isolation disk (2); Furthermore, a bottom flat cavity is formed between the lower isolation disk (2) and the sealed chamber (1), the heater (5) is arranged in the bottom flat cavity, and the plurality of insulation plates (4) divide the space between the lower isolation disk (2) and the upper isolation disk (3) into a culture chamber and an inoculation chamber from the outside to the inside. The clamping sleeves (6) for limiting the position of the base of each culture dish are distributed in a circle and are movably arranged at the bottom of the culture chamber. The rotating mechanism (7) for separating and screening bacterial colonies passes through the sealed chamber (1) and the upper isolation disk (3) in sequence and extends into the inoculation chamber.

2. The device for separating, screening and cultivating Tremella fuciformis fermented lactic acid bacteria according to claim 1, characterized in that: The rotating mechanism (7) at least comprises a connecting shaft (71), a knob (72), a disassembly assembly (73) and a marking assembly (74); the connecting shaft (71) passes through the upper isolation plate (3) and is arranged to rotate in a sealed manner with the upper isolation plate (3); the knob (72) passes through the top cover and is connected to the extension shaft at the top of the connecting shaft (71); the disassembly assembly (73) for disassembling the end covers of each culture dish is fixedly mounted on one side of the bottom of the connecting shaft (71); and the marking assembly (74) for marking the culture medium is fixedly mounted on the other side of the bottom of the connecting shaft (71).

3. The device for separating, screening and cultivating Tremella fuciformis fermented lactic acid bacteria according to claim 2, characterized in that: The marking assembly (74) comprises a multi-stage telescopic rod (741), a columnar base (742) and an inoculation ring (743); the fixed end of the multi-stage telescopic rod (741) is arranged on the extended end of the connecting shaft (71), the columnar base (742) is fixed to the output end of the multi-stage telescopic rod (741) and is arranged horizontally, and the multi-stage telescopic rod (741) is connected to an external main control unit using electrical signals.

4. The device for separating, screening and cultivating Tremella fuciformis fermented lactic acid bacteria according to claim 3, characterized in that: A spiral groove (744) is provided at the bottom of the columnar base (742), the depth of the spiral groove (744) gradually increases from the edge to the center, and the oblique cross-section of the spiral groove (744) is a shape in which a circular portion (7441) and a rectangular portion (7442) are connected from top to bottom. The inoculation ring (743) is arranged perpendicular to the columnar base (742), and a ball sleeve (745) is fixed to the upper end of the inoculation ring (743), and a ball (746) is rolled in the ball sleeve (745). The ball sleeve (745) is slidably arranged in the circular portion (7441) of the spiral groove (744), and the ball (746) contacts the top of the circular portion (7441), and the upper end of the inoculation ring (743) is slidably arranged in the rectangular portion (7442).

5. The device for separating, screening and cultivating Tremella fuciformis fermented lactic acid bacteria according to claim 4, characterized in that: The diameter R of the circular portion (7441) of the spiral groove (744) is greater than the width L of the rectangular portion.

6. The device for separating, screening and cultivating Tremella fuciformis fermented lactic acid bacteria according to claim 1, characterized in that: A heat conduction hole (21) is provided at the center of the lower isolation plate (2).

7. The device for separating, screening and cultivating Tremella fuciformis fermented lactic acid bacteria according to claim 1, characterized in that: The lower ends of the plurality of heat insulating plates (4) are configured as opening and closing plates, and the opening and closing plates have elastic deformation capability.

8. The device for separating, screening and cultivating Tremella fuciformis fermented lactic acid bacteria according to claim 1, characterized in that: It also includes a plurality of lifting rods (8) distributed around the circumference, one end of the lifting rod (8) sealingly passing through the outer wall of the sealing chamber (1), and the other end of the lifting rod (8) being sleeved and fixed in the extension portion of the clamping sleeve (6).

9. A method for separating, screening and culturing Tremella fuciformis fermented lactic acid bacteria is as follows: S1: constructing a screening medium for Lactobacillus auriculariae: MRS basic medium, primary screening medium and Lactobacillus auriculariae screening medium, and placing the completed medium in a culture room; S2: Initial screening of golden ear lactic acid bacteria: the sample is added to the initial screening culture medium, and cultured in the culture chamber at 30°C for 48 hours under anaerobic conditions. Subsequently, the single colony with a calcium-soluble ring is re-streak cultured by the rotating mechanism (7) in conjunction with the lifting rod (8). The streaking culture is repeated for no less than 3 times until a single bacterial species pure culture is obtained. The single colony with a calcium-soluble ring is selected and inoculated into MRS liquid culture medium. The culture dish is then moved to the culture chamber and cultured at 30°C for 24 hours. It is then taken out and stored at low temperature as a candidate strain of lactic acid bacteria; S3: Rescreening of Auricularia auriculariae lactic acid bacteria: The screening medium for Auricularia auriculariae lactic acid bacteria was used. The candidate strains were inoculated into the Auricularia auriculariae lactic acid bacteria screening medium and cultured at 30°C for 48 h. The operation was the same as in S2. Single colonies with calcium-dissolving circles were selected and inoculated into MRS liquid culture medium. The colonies were cultured at 30°C for 24 h and stored at low temperature as the target strains of lactic acid bacteria.

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