Device for producing cordycepin at high yield through liquid fermentation

Abstract

The invention discloses a device for high yield of cordycepin through liquid fermentation, and relates to the technical field of microbial fermentation, the device comprises a closed box body, a magnetic stirrer is arranged at the bottom in the closed box body, and the magnetic stirrer is used for stirring a culture medium in a conical flask; a lamp is arranged on the inner side wall of the closed box body; fans are arranged on the two sides of the closed box body; a temperature sensor and an observation window are arranged on the closed box body; and a controller and an ultraviolet sterilizing lamp are arranged at the top in the closed box body. According to the device for producing cordycepin at high yield through liquid fermentation, the magnetic stirrer is used for replacing an oscillating incubator, the LED lamp tubes are used for replacing common fluorescent lamp tubes, a totally-closed lightproof culture space is provided, and the ultraviolet sterilizing lamp is additionally arranged to sterilize the space before culture; in the culture period, sterile air is introduced into the space by using a filter fan, and the illumination time, the stirring time, the ventilation time and the ultraviolet sterilization time can be respectively and independently controlled, so that the optimal conditions for culturing the cordyceps militaris liquid are provided.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, in particular to a device for high-yield cordycepin by liquid fermentation. Background Art

[0002] Cordyceps militaris is a rare edible and medicinal fungus with nutritional and tonic effects. It can synthesize and secrete active substances with a variety of health-care effects, mainly including immunomodulatory, antiviral, anti-inflammatory, hypolipidemic, anti-oxidant, anti-inflammatory, anti-aging and other effects. Therefore, the content of active substances in Cordyceps militaris is the main evaluation index of its quality. Among them, cordycepin is one of the core high-value-added secondary metabolites produced by Cordyceps fungi. At present, it is mainly focused on molecular breeding of high-yield cordycepin and research on cordycepin metabolism regulation; one of the patents reported is to increase the cordycepin content in Cordyceps militaris fruiting bodies, and the other is to develop a medium formula in liquid mycelium fermentation combined with shake flask fermentation or liquid deep fermentation, but the yield of cordycepin is limited. In recent years, research on light quality (fluorescent light, blue light, red light) and light intensity in the cultivation of Cordyceps militaris fruiting bodies has significantly increased the cordycepin content in the fruiting bodies; suitable hypoxia stress can increase the cordycepin content. Based on this theory, the present invention avoids the defects that shake flask fermentation is easily contaminated with bacteria and deep fermentation cannot be evenly illuminated. Firstly, the present invention uses magnetic stirring and box sealing to pass sterile air to avoid contamination by foreign bacteria; secondly, by adjusting the switching time of the fan and the magnetic stirrer, the low oxygen stress of the fermentation culture is achieved; finally, by adding lamps with different light qualities and light intensities and maintaining a distance from the fermentation bottle, multiple combinations of light quality and light intensity are achieved; finally, the high yield of cordycepin in liquid fermentation is promoted, and equipment is provided for the industrial production of cordycepin. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a liquid fermentation device for high-yield cordycepin to provide the most suitable conditions for liquid culture of Cordyceps militaris.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The invention provides a device for high-yield cordycepin by liquid fermentation, comprising a closed box, wherein a magnetic stirrer is arranged at the bottom of the closed box, and the magnetic stirrer is used to stir the culture medium in a conical flask; a lamp is arranged on the inner wall of the closed box; fans are arranged on both sides of the closed box; a temperature sensor and an observation window are arranged on the closed box; an ultraviolet sterilization lamp is arranged at the top of the closed box; a controller is arranged on the upper left outer side of the front of the closed box, and the controller is electrically connected with the magnetic stirrer, the lamp, the fan, the temperature sensor and the ultraviolet sterilization lamp.

[0006] Optionally, a box door is provided on the top of the sealed box body, and a sealing strip is provided around the box door.

[0007] Optionally, the sealed box is a light-proof box, and the sealed box is made of stainless steel or aluminum alloy or wood or opaque plastic.

[0008] Optionally, 8-10 magnetic stirrers are arranged in the closed box, and the 8-10 magnetic stirrers are placed in double rows; the magnetic stirrer is digitally displayed, constant temperature and speed adjustable, with a temperature range of room temperature-200°C, a rotation speed of 0-2000r / min, and a stirring capacity range of 50-3000mL.

[0009] Optionally, the lamp adopts an LED lamp, and the lamp includes a white light LED lamp, a blue light LED lamp and a red light LED lamp; 1 to 2 of the white light LED lamps, the blue light LED lamps and the red light LED lamps are respectively arranged on the front and the back of the closed box.

[0010] Optionally, the white light LED lamp has a color temperature of 3000-6500K and an illumination of 1000-4000lux; the blue light LED lamp has a wavelength of 460nm and an illumination of 200-2000lux; and the red light LED lamp has a wavelength of 660nm and an illumination of 200-2000lux.

[0011] Optionally, the sealed box has openings on both sides, one opening is inlaid with an air inlet fan, and the other opening is inlaid with an air outlet fan, and the air volume of the air inlet fan and the air outlet fan is 1-50m 3 / min, the speed is 2000-3000r / min.

[0012] Optionally, a dust removal and sterilization filter is provided on the air inlet side outside the box of the air inlet fan; and a dust removal and sterilization filter is provided on the air outlet side of the box of the air outlet fan.

[0013] Optionally, the measurable temperature range of the temperature sensor is -20°C-200°C.

[0014] Optionally, the power range of the ultraviolet sterilization lamp is 15W-40W.

[0015] Compared with the prior art, the present invention has achieved the following technical effects:

[0016] The liquid fermentation high-yield cordycepin device of the present invention uses a magnetic stirrer to replace an oscillating culture box, uses LED lamps with different light qualities to replace ordinary fluorescent lamps, provides a fully enclosed light-proof culture space, installs an ultraviolet sterilization lamp to sterilize the space before culture, and uses a dust removal, sterilization and filtering fan to introduce sterile air into the space during the culture period. The stirring time, different light qualities, light intensity combinations and times, ventilation time and ultraviolet sterilization time can be separately controlled, so as to provide the most suitable conditions for the liquid culture of Cordyceps militaris. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is a schematic diagram of the structure of the device for high-yield cordycepin by liquid fermentation of the present invention;

[0019] Figure 2 This is a schematic diagram of the lateral structure of the liquid fermentation high-yield cordycepin device of the present invention;

[0020] Figure 3 Comparison of cordycepin production between white light LED illumination culture and traditional shaking flask fermentation in a shaking incubator in the dark;

[0021] Figure 4 Comparison of cordycepin yields between cultivation under mixed illumination of white, blue and red LEDs and cultivation under single white LED illumination;

[0022] Figure 5 The cordycepin production was compared between continuous illumination culture with a mixture of white, blue and red LED light and intermittent illumination, intermittent ventilation and intermittent magnetic stirring culture with a mixture of white, blue and red LED light.

[0023] Explanation of the accompanying reference numerals: 1. Sealed box; 2. Magnetic stirrer; 3. Conical flask; 4. LED lamp; 5. Fan; 6. Temperature sensor; 7. Observation window; 8. Ultraviolet sterilization lamp; 9. Controller. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] like Figure 1 and 2 As shown, this embodiment provides a device for high-yield cordycepin by liquid fermentation, including a closed box 1, a magnetic stirrer 2 is arranged at the bottom of the closed box 1, and the magnetic stirrer 2 is used to stir the culture medium in the conical flask 3; a lamp 4 is arranged on the inner wall of the closed box 1; fans 5 are arranged on both sides of the closed box 1; a temperature sensor 6 and an observation window 7 are arranged on the closed box 1; an ultraviolet sterilization lamp 8 is arranged on the top of the closed box 1; a controller 9 is arranged on the upper left outer side of the front of the closed box 1; the controller 9 is electrically connected to the magnetic stirrer 2, the lamp 4, the fan 5, the temperature sensor 6 and the ultraviolet sterilization lamp 8.

[0026] In this specific embodiment, a box door is arranged on the top of the sealed box body 1, and a sealing strip is arranged around the box door.

[0027] The sealed box 1 is a light-proof box. In this specific embodiment, the sealed box 1 is made of stainless steel.

[0028] The magnetic stirrer 2 is placed on the bottom plate of the box, and 8-10 magnetic stirrers are placed according to the size of the box, and are evenly distributed in the box to ensure that the conical flask 3 is evenly illuminated. Preferably, a 1000mL conical flask 3 is filled with 500-600mL Cordyceps militaris liquid culture medium and a magnetic stirrer, which is placed on the panel of the magnetic stirrer 2, and all magnetic stirrers 2 are connected to a magnetic stirring timing controller 9.

[0029] Two white LED tubes with a color temperature of 6500K and an illumination of 1000 lux, one blue LED tube with a color temperature of 460nm and an illumination of 300 lux, and one red LED tube with a color temperature of 660nm and an illumination of 300 lux are installed on the front side of the inner wall of the box and its opposite side. The tubes of different colors are connected in series to the lighting timing controller 9.

[0030] The fan 5 is installed in the two sides of the box body, the air inlet opening is located at about one-third of the lower part, and the air outlet opening is located at about one-third of the upper part, so that a diagonal air duct with air inlet at the bottom and air outlet at the top is formed inside the box body. Filter cotton is installed on the outside of the two fans 5 to fill the device with clean air. The two fans 5 are connected in series to the fan timing controller 9.

[0031] The temperature sensor 6 probe is placed at the center of the box to measure the temperature inside the box, and the temperature sensor display is placed at the controller 9.

[0032] A hole of suitable size is opened on the upper front part of the box body as an observation window 7, a transparent acrylic plate is sealed and pasted on the inside, and a light shielding plate consistent with the box body is used as a baffle on the outside. When observation is needed, the baffle can be removed to observe the culture situation inside.

[0033] An ultraviolet sterilization lamp 8 is installed on the inner wall of the top surface of the box, preferably 30W, 90cm each, and connected to an ultraviolet sterilization timing controller 9.

[0034] A circle of silicone sealing strip is pasted around the edge of the inner wall of the top surface of the box body so that the top cover can achieve sealing performance when the device is running.

[0035] All timing controllers and temperature sensor displays are installed on the upper left outer side of the front of the box, and all controllers 9 and sensor display panels are exposed. The gap between the panel and the box is sealed with silicone sealant to prevent air leakage.

[0036] The controller 9 includes a magnetic stirrer timing controller, a lighting timing and combination controller, a fan timing controller, a temperature sensor display and an ultraviolet sterilization timing controller, which can control the timing start and shut down of each accessory respectively.

[0037] Embodiment 1:

[0038] The present embodiment specifically discloses a device application method for high-yield cordycepin in liquid fermentation, a white light LED illumination culture and a traditional shaking incubator shake flask fermentation light-proof culture cordycepin yield comparison experiment, using a 1000mL conical flask to hold 500mL liquid fermentation medium and place a magnetic stirrer, and inoculate the activated Cordyceps militaris strain into the liquid fermentation medium under aseptic conditions. After the internal part of the device is sterilized using an ultraviolet sterilization lamp, the conical flask containing the strain and the culture medium is placed on the magnetic stirrer platform in the device, and the box door is closed to make the device reach a closed state.

[0039] When the inside of the device is sealed, turn on the LED white light, inlet and outlet fans, and magnetic stirrer timing controller respectively, so that the LED white light reaches a color temperature of 6500K and an illumination of 2000lux, and the inlet and outlet fans reach a speed of 2500r / min and an air volume of 1.0m 3 / min, and the magnetic stirrer reaches 500r / min. After the liquid fermented Cordyceps militaris is cultured under continuous illumination and magnetic stirring for 10 days, samples are taken to detect the cordycepin content in the fermentation liquid.

[0040] like Figure 3 As shown, compared with the traditional shake flask fermentation in a shaking incubator in the dark, the cordycepin content of the shake flask fermentation in the shaking incubator in the dark was 109.82±5.49μg / mL. The cordycepin production of the device with continuous white light and magnetic stirring for 10 days was 550.64±27.53μg / mL, which was 5.014 times that of the traditional shake flask fermentation in a shaking incubator in the dark.

[0041] Embodiment 2:

[0042] The present embodiment specifically discloses a laboratory application method of a device for high yield of cordycepin by liquid fermentation, a comparison experiment of cordycepin yield by mixed illumination culture of white, blue and red LED and single illumination culture of white LED, a 1000mL conical flask is used to hold 500mL of liquid fermentation medium and a magnetic stirrer is placed, and the activated Cordyceps militaris strain is inoculated into the liquid fermentation medium under aseptic conditions. After the inside of the device is sterilized using an ultraviolet sterilization lamp, the conical flask containing the strain and the culture medium is placed on the magnetic stirrer platform in the device, and the door of the box is closed to make the device reach a closed state.

[0043] When the inside of the device is sealed, turn on the LED white light, LED blue light, LED red light, inlet and outlet fans, and magnetic stirrer timing controller respectively, so that the LED white light reaches a color temperature of 6500K and an illumination of 2000lux, the LED blue light reaches 460nm and an illumination of 300lux, the LED red light reaches 660nm and an illumination of 300lux, and the inlet and outlet fans reach a speed of 2500r / min and an air volume of 1.0m 3 / min, and the magnetic stirrer reached 500r / min. After the liquid fermentation Cordyceps militaris was cultured for 10 days with continuous mixing, illumination and magnetic stirring, samples were taken to detect the cordycepin content in the fermentation liquid. Figure 4 As shown, the cordycepin content of the culture under white light LED illumination alone is 553.26±27.62μg / mL, and the cordycepin content of the culture under white, blue and red LED mixed illumination is 1080.32±54.01μg / mL. Using this device, the cordycepin content of the culture under white, blue and red LED mixed illumination is about 1.95 times that of the culture under white light LED illumination alone.

[0044] Embodiment three:

[0045] The present embodiment specifically discloses a laboratory application method of a device for high yield of cordycepin by liquid fermentation, a comparison experiment of cordycepin yield by mixed continuous illumination culture of white, blue and red LED and mixed intermittent illumination, intermittent ventilation and intermittent magnetic stirring culture of white, blue and red LED, a 1000mL conical flask is used to hold 500mL of liquid fermentation medium and a magnetic stirrer is placed, and the activated Cordyceps militaris strain is inoculated into the liquid fermentation medium under sterile conditions. After the inside of the device is sterilized using an ultraviolet sterilization lamp, the conical flask containing the culture medium inoculated with the strain is placed on the magnetic stirrer platform in the device, and the door is closed to make the device reach a closed state.

[0046] When the inside of the device is sealed, turn on the LED white light, LED blue light, LED red light, inlet and outlet fans, and magnetic stirrer timing controller respectively, so that the LED white light reaches a color temperature of 6500K and an illumination of 2000lux, the LED blue light reaches 460nm and an illumination of 300lux, and the LED red light reaches 660nm and an illumination of 300lux, and the inlet and outlet fans reach a speed of 2500r / min and an air volume of 1.0m 3 / min, the magnetic stirrer reaches 500r / min. Different from continuous illumination, all controllers are adjusted to time switches, turning on for 3h and then off for 1h, for intermittent illumination. When the illumination is stopped, the operation of the magnetic stirrer is stopped, and appropriate hypoxic stress is applied to the Cordyceps, which is conducive to further increase of cordycepin content. After 10 days of intermittent illumination and magnetic stirring cultivation of liquid fermentation Cordyceps militaris, samples are taken to detect the cordycepin content in the fermentation liquid. Figure 5 The cordycepin content of the mixed continuous illumination culture of white, blue and red LEDs is 1045.26±52.26μg / mL, and the cordycepin content of the mixed intermittent illumination, intermittent ventilation and intermittent magnetic stirring culture of white, blue and red LEDs is 1982.34±99.12μg / mL. Fermentation using this device can increase the cordycepin yield by about 1 times.

[0047] It should be noted that it is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention, and any figure mark in the claims should not be regarded as limiting the claims involved.

[0048] The present specification uses specific examples to illustrate the principles and implementation methods of the present invention. The above examples are only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A liquid fermentation device for high yield of cordycepin, It is characterized in that It includes a closed box, a magnetic stirrer is arranged at the bottom of the closed box, and the magnetic stirrer is used to stir the culture medium in the conical flask; a lamp is arranged on the inner wall of the closed box; fans are arranged on both sides of the closed box; a temperature sensor and an observation window are arranged on the closed box; an ultraviolet sterilization lamp is arranged on the top of the closed box; a controller is arranged on the upper left outer side of the front of the closed box; the controller is electrically connected to the magnetic stirrer, the lamp, the fan, the temperature sensor and the ultraviolet sterilization lamp.

2. The liquid fermentation high-yield cordycepin device according to claim 1, It is characterized in that A box door is arranged on the top of the sealed box body, and a sealing strip is arranged around the box door.

3. The liquid fermentation high-yield cordycepin device according to claim 1, It is characterized in that The sealed box is a light-proof box, and is made of stainless steel, aluminum alloy, wood board, or opaque plastic.

4. The liquid fermentation high-yield cordycepin device according to claim 1, It is characterized in that 8-10 magnetic stirrers are arranged in the closed box, and the 8-10 magnetic stirrers are placed in double rows; the magnetic stirrer is digitally displayed, constant temperature and speed adjustable, with a temperature range of room temperature-200°C, a rotation speed of 0-2000r / min, and a stirring capacity range of 50-3000mL.

5. The liquid fermentation high-yield cordycepin device according to claim 1, It is characterized in that The lamp adopts an LED lamp, which includes a white light LED lamp, a blue light LED lamp and a red light LED lamp; 1 to 2 white light LED lamps, blue light LED lamps and red light LED lamps are respectively arranged on the front and the back of the closed box.

6. The liquid fermentation high-yield cordycepin device according to claim 5, It is characterized in that The white light LED lamp has a color temperature of 3000-6500K and an illumination of 1000-4000lux; The blue light LED lamp has a wavelength of 460nm and an illumination of 200-2000lux; The red light LED lamp has a wavelength of 660nm and an illumination of 200-2000lux.

7. The liquid fermentation high-yield cordycepin device according to claim 1, It is characterized in that The sealed box has openings on both sides, one side of the opening is inlaid with an air inlet fan, and the other side of the opening is inlaid with an air outlet fan. The air volume of the air inlet fan and the air outlet fan is 1-50m 3 / min, the speed is 2000-3000r / min.

8. The liquid fermentation high-yield cordycepin device according to claim 7, It is characterized in that The air inlet side of the air inlet fan is provided with a dust removal and sterilization filter; the air outlet side of the air outlet fan is provided with a dust removal and sterilization filter.

9. The liquid fermentation high-yield cordycepin device according to claim 1, It is characterized in that The measurable temperature range of the temperature sensor is -20°C to 200°C.

10. The liquid fermentation high-yield cordycepin device according to claim 1, It is characterized in that The power range of the ultraviolet sterilization lamp is 15W-40W.

Images