CN115011491B - Method for culturing candida utilis - Google Patents
Method for culturing candida utilis Download PDFInfo
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- CN115011491B CN115011491B CN202210846706.4A CN202210846706A CN115011491B CN 115011491 B CN115011491 B CN 115011491B CN 202210846706 A CN202210846706 A CN 202210846706A CN 115011491 B CN115011491 B CN 115011491B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/58—Reaction vessels connected in series or in parallel
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- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/18—Flow directing inserts
- C12M27/20—Baffles; Ribs; Ribbons; Auger vanes
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- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/12—Pulsatile flow
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- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/20—Degassing; Venting; Bubble traps
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- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
- C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/72—Candida
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Abstract
The invention discloses a candida utilis culture process, which comprises the following steps of S1: preparing a solid culture medium, sterilizing by high-pressure steam, and filling the upper part of a test tube on a glass rod when the solid culture medium is hot, wherein the length of the inclined surface of the culture medium is 1/2 of that of the test tube. The optimal conditions in the invention are that the culture time is 13h, the temperature is 30 ℃, the rotation speed of a shaking table is 180r/min, the density of thalli is highest when the carbon nitrogen ratio (the mass ratio of glucose to peptone) is 3:1, namely the growth effect is best, so the culture process ensures the flora activity and the spawn reproduction efficiency of candida utilis based on the parameters.
Description
Technical Field
The invention relates to the technical field of strain culture, in particular to a method for culturing candida utilis.
Background
Candida utilis (Candida utilis) is also known as torula utilis or torula utilis. The content of protein and vitamin B is higher than that of beer yeast, urea and nitric acid can be used as nitrogen sources, and the beer yeast can grow without adding any growth factors into the culture medium. It can utilize five-carbon sugar and six-carbon sugar, not only can utilize sulfurous acid waste liquor of paper industry, but also can utilize molasses and wood hydrolysate to produce human and animal edible protein.
The activity of the strain and the propagation of the flora have high requirements on the culture method, and the limitation on various parameters of the strain in the existing culture is inaccurate, so that the activity of the strain and the propagation of the flora are not optimal, and the application of the strain is affected.
Disclosure of Invention
Based on the technical problems in the background technology, the invention provides a method for culturing candida utilis.
The invention provides a method for culturing candida utilis, which comprises the following steps:
s1: preparing a solid culture medium, sterilizing by high-pressure steam, and filling the upper part of a test tube on a glass rod while the solid culture medium is hot to make the length of the inclined surface of the culture medium be 1/2 of the length of the test tube;
s2: selecting a ring of thallus from candida utilis strains, putting the thallus into a prepared liquid culture medium, shaking uniformly, and culturing at 30-32 ℃ at a rotating speed of 160-180r/min for 12-13 hours;
s3: performing plate pouring operation on the solid culture medium subjected to high-pressure steam sterilization, taking out a certain amount of bacterial liquid with a certain concentration from the liquid culture medium, injecting the bacterial liquid into the surface of the solid culture medium, and uniformly spreading the inoculated bacterial liquid on the surface of the culture medium by using a spreading rod;
s4: the culture time is 36-48 hours, and after the operation, the removed flat plate can have staggered colonies for later culture and seed taking operation.
Preferably, the solid culture medium comprises 2-5% of glucose, 2-3% of peptone, 1-2% of yeast extract powder, 2-3% of agar and distilled water in percentage by weight.
Preferably, the candida utilis is cultured in a culture medium for 13-h deg.C at 30 ℃ and at a shaking table rotation speed of 160-r/min, and the cell density is highest when the carbon nitrogen ratio (mass ratio of glucose to peptone) is about 3:1.
Preferably, in the later stage of the candida utilis culture method, a yeast culture proliferation device is also adopted for culture proliferation, the device comprises a plurality of annular pipeline type culture cavities which are connected in series, and the opening and closing of a passage between two adjacent culture cavities are controlled by a first baffle plate which can be opened and closed; the culture cavity comprises an outer wall and an inner wall sleeved in the outer wall; a first temperature regulating layer is arranged between the outer wall and the inner wall; the outer wall and the inner wall are both made of heat conducting materials; the first temperature regulating layer comprises a plurality of first semiconductor refrigerating sheets which are densely arranged; the center of the culture cavity is provided with a hollow inner temperature regulating rod along the length direction of the culture cavity; the inner temperature regulating rod is internally filled with solid-liquid phase-change materials; the internal temperature regulating rod can be communicated with the outside to circulate the solid-liquid phase material in the internal temperature regulating rod; the phase transition temperature of the solid-liquid phase change material is within the safe temperature range of saccharomycetes; the outer surface of the inner temperature regulating rod is provided with a second temperature regulating layer; the second temperature regulating layer also comprises a plurality of second semiconductor refrigerating sheets which are densely arranged; the inner temperature adjusting rod body is made of a heat conducting material; the outer wall of the culture cavity is respectively provided with an air inlet pipe which can be opened and closed respectively and is used for supplementing required sterile gas and a feed pipe which is used for supplementing required culture solution and thalli along the tangential direction of the outer wall.
The two adjacent culture cavities are communicated through a connecting pipe, and the connecting pipe is tangential to the outer walls of the two culture cavities at the same time; the first baffle is hinged at the discharge port of the previous culture cavity, the circulation passage of the previous culture cavity is kept while the connecting pipe is closed by rotation, and the circulation passage of the previous culture cavity is closed while the connecting pipe is opened.
Wherein, the feed inlet of the next culture cavity is hinged with a second baffle; the second baffle plate can keep the circulation passage of the next culture cavity while closing the connecting pipe by rotating, and can close the circulation passage of the next culture cavity while opening the connecting pipe.
Wherein the first baffle plate is of a porous structure, and a waterproof and breathable film is covered on the surface of the first baffle plate; an exhaust pipe is communicated with the connecting pipe to exhaust air outwards so as to keep negative pressure in the connecting pipe.
Wherein, the outer surface of the outer wall is uniformly provided with a plurality of annular heat conducting sheets; the annular heat conducting fin is perpendicular to the length direction of the outer wall.
Wherein, a plurality of culture cavities are distributed up and down as a whole.
Wherein, spiral rifling or smooth bulges are arranged on the inner surface of the inner wall;
the yeast culture proliferation method comprises the following steps:
(1) injecting the initial saccharomycete mixed culture solution into the culture cavity through a feed pipe at a certain speed, and simultaneously injecting compressed sterile gas which is beneficial to saccharomycete culture into the culture cavity through an air inlet pipe in a continuous or pulse mode, wherein the air inlet pipe and the feed pipe are both tangential to the culture cavity, so that the saccharomycete mixed culture solution can circularly flow in the culture cavity, and simultaneously, the spiral rifling or smooth bulge on the inner surface of the inner wall can enable the mixed culture solution to have stronger turbulence, so that the substance exchange and gas dissolution of the saccharomycete mixed culture solution are promoted, and the culture solution and gas update and the temperature uniformity in the saccharomycete mixed culture solution are facilitated; the redundant gas and waste in the culture cavity pass through the waterproof breathable film and the first baffle plate and are pumped out through the exhaust pipe, so that the air pressure stability and the air freshness in the culture cavity are ensured;
(2) the temperature of the saccharomycete mixed culture solution in the culture cavity is accurately controlled from the inner direction and the outer direction through the first semiconductor refrigerating plate and the second semiconductor refrigerating plate so as to ensure efficient culture and proliferation of saccharomycete, and the solid-liquid phase change material can rapidly remove redundant heat under the sudden situation of failure or failure of the semiconductor refrigerating plate through circulating flow and larger specific heat capacity, and can automatically balance the temperature of the saccharomycete mixed culture solution through the solid-liquid phase change characteristic of the solid-liquid phase change material so as to avoid large-scale death of saccharomycete in a period of time;
(3) after the set culture time is reached, the first baffle and the second baffle rotate in the front culture cavity and the rear culture cavity respectively, so that the flowing saccharomycete culture solution enters the rear culture cavity from the upper culture cavity through the connecting pipe under the inertia effect, after the completion, the first baffle and the second baffle are closed again, the front culture cavity is continuously filled with new saccharomycete mixed culture solution or saccharomycete mixed culture solution of the front culture cavity, and the rear culture cavity is continuously used for culturing the saccharomycete culture solution before the rear culture cavity is continuously used until the aim of culturing and proliferation is achieved after N times of culture is carried out, and then the saccharomycete culture solution is discharged.
The beneficial effects of the invention are as follows:
according to the invention, the growth influence of each factor on the saccharomycete is not obvious (p is more than 0.05) through statistical analysis, the final optimization conditions are that the culture time is 13h, the temperature is 30 ℃, the rotation speed of a shaking table is 180r/min, the density of the thalli is highest when the carbon nitrogen ratio (the mass ratio of glucose to peptone) is 3:1, namely the growth effect is the best, so that the culture method ensures the flora activity and the spawn reproduction efficiency of the candida utilis based on the parameters.
Compared with the traditional static culture proliferation by adopting a tank body, the invention can realize continuous pulsation and dynamic culture proliferation, obviously improve the production efficiency and the production consistency, reduce the residence time required by the thalli after the culture proliferation is finished to the subsequent working procedure and improve the survival rate and the culture efficiency of the thalli finished products; the culture and proliferation process of the thalli is divided into a plurality of sections to be continuously carried out, thereby realizing continuity and controllability, reducing the influence of partial thalli disqualification on the whole body and reducing the reject rate.
3. The invention adopts the semiconductor refrigerating sheets arranged inside and outside the culture cavity, not only can increase the contact area with the mixed culture solution of the saccharomycetes and improve the heat conductivity and the conduction uniformity, but also can accurately and bidirectionally control the temperature by controlling the magnitude and the direction of the current, thereby ensuring that the saccharomycetes are at the optimal culture temperature so as to ensure the efficient culture and proliferation of the saccharomycetes, and simultaneously, through the solid-liquid phase change material, the redundant heat can be rapidly removed under the sudden situation when the semiconductor refrigerating sheet fails or fails through the circulating flow and the self larger specific heat capacity, and the temperature of the mixed culture solution of the saccharomycetes can be automatically balanced through the self solid-liquid phase change characteristic, so that the saccharomycetes are prevented from dying on a large scale within a period of time.
4. The invention utilizes the characteristics that the air inlet pipe and the feeding pipe are both mutually matched with the culture cavity, not only can feed saccharomycetes and culture solution into the culture cavity and supplement gas required by saccharomycetes according to the design and calculation structure, but also can provide circulating rotation power for the mixed culture solution in the culture cavity by utilizing the impacted feed solution and air inlet, can enable the saccharomycetes mixed culture solution to circularly flow in the culture cavity, and simultaneously the spiral rifling or smooth bulge on the inner surface of the inner wall can enable the mixed culture solution to have stronger turbulence, thereby promoting the substance exchange and gas dissolution of the saccharomycetes mixed culture solution and facilitating further culture solution and gas update and temperature uniformity; and the redundant gas and waste in the culture cavity pass through the waterproof breathable film and the first baffle plate and are pumped out through the exhaust pipe, so that the air pressure stability and the air freshness in the culture cavity are ensured.
Drawings
FIG. 1 is a schematic diagram of the method of the present invention;
FIG. 2 is a schematic diagram of a yeast culture and proliferation device according to the present invention;
FIG. 3 is a schematic radial cross-sectional view of a temperature regulating rod in accordance with the present invention;
fig. 4 is a schematic cross-sectional view of a second baffle of the present invention.
In the figure: 1. a culture chamber; 11. an outer wall; 12. an inner wall; 13. a first temperature adjustment layer; 14. a first semiconductor refrigeration sheet; 15. an internal temperature regulating rod; 16. solid-liquid phase change material; 17. a second temperature adjustment layer; 18. a second semiconductor refrigeration sheet; 2. a first baffle; 21. a waterproof breathable film; 22. an exhaust pipe; 3. an air inlet pipe; 4. a feed pipe; 6. a connecting pipe; 7. and a second baffle.
Description of the embodiments
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments.
Referring to FIG. 1, a method for culturing candida utilis comprises the following steps:
s1: preparing a solid culture medium, sterilizing by high-pressure steam, and filling the upper part of a test tube on a glass rod while the solid culture medium is hot to make the length of the inclined surface of the culture medium be 1/2 of the length of the test tube;
s2: selecting a ring of thallus from candida utilis strains, putting the thallus into a prepared liquid culture medium, shaking uniformly, and culturing at 30-32 ℃ at a rotating speed of 160-180r/min for 12-13 hours;
s3: performing plate pouring operation on the solid culture medium subjected to high-pressure steam sterilization, taking out a certain amount of bacterial liquid with a certain concentration from the liquid culture medium, injecting the bacterial liquid into the surface of the solid culture medium, and uniformly spreading the inoculated bacterial liquid on the surface of the culture medium by using a spreading rod;
s4: the culture time is 36-48 hours, and after the operation, the removed flat plate can have staggered colonies for later culture and seed taking operation.
Wherein the solid culture medium comprises glucose 2-5%, peptone 2-3%, yeast extract 1-2%, agar 2-3% and distilled water.
Wherein, the cell density is highest when the candida utilis is cultured in a culture medium for 13-h at 30 ℃ and the rotation speed of a shaking table is 160-r/min and the carbon nitrogen ratio (the mass ratio of glucose to peptone) is about 3:1.
Specifically, in the invention, a yeast extract-derived glucose medium (also called YPD medium) is prepared from the following raw materials: 2-5% of glucose, 2-3% of peptone, 1-2% of yeast extract powder and distilled water; the solid culture medium is prepared by adding agar 2-3% based on liquid culture medium.
The split-charging amount is determined according to the situation, and the solid culture medium in the test tube is preferably 1/5 of the height of the tube, and the culture medium filled in the triangular flask is not preferably more than 1/2 of the volume.
Then the prepared culture medium is subjected to high-pressure steam sterilization, the articles to be sterilized are placed in a high-pressure steam sterilization pot, and the steam temperature is raised by utilizing the rising of the boiling point of water at high pressure, so that the aim of sterilizing the mixed bacteria is fulfilled by generating high temperature.
Since the medium contains saccharide, 7X 10 is generally used 4 Pa (gauge pressure) (115 ℃) for 20-30 minutes to prevent decomposition of saccharides due to high temperature.
Since autoclaving is to raise the temperature by increasing the steam pressure to kill microorganisms, the air in the pot should be exhausted as much as possible before pressurization.
After sterilization, the upper part of a test tube is padded on a glass rod when the solid culture medium which needs to be made into an inclined plane is hot, so that the inclined plane length of the culture medium is 1/2 of the length of the test tube.
Shake cultivation: picking a ring of thallus from the purchased strain, putting the thallus into the prepared liquid culture medium, and shaking uniformly.
Placing the triangular flask containing the liquid culture medium into a shaking incubator for shake cultivation, wherein the cultivation condition is 30-32 ℃, the rotation speed is 160-180r/min, and the cultivation time is 12-13 hours.
Under the culture conditions, the bacterial cells can grow rapidly and are in the logarithmic growth phase, thus providing a foundation for the subsequent culture.
Culturing under constant temperature and humidity: and (3) carrying out plate pouring operation on the solid culture medium subjected to high-pressure steam sterilization, pouring the prepared solid culture medium into the plates while the solid culture medium is hot, and then cooling the culture medium in all the plates to room temperature and solidifying.
At this time, an inoculation operation is performed, a certain amount of bacterial liquid with a certain concentration is taken out from the liquid culture medium and injected onto the surface of the solid culture medium, and then the inoculated bacterial liquid is uniformly spread on the surface of the culture medium by a spreading bar.
Finally, the flat plate is placed into a constant temperature and humidity incubator to be cultivated in a dark place for 36-48 hours, and after the operation, the dislocated colony appears on the flat plate which is taken out for later cultivation and seed taking operation application.
After the cultivation, 1mL of the bacterial liquid was taken out, diluted 5-fold with distilled water, and the absorbance was measured at 600nm with an ultraviolet-visible spectrophotometer as an initial value.
Then, the triangular flask containing the liquid culture medium is placed in a constant-temperature shaking incubator with the temperature of 30 ℃ and the rotating speed of 160r/min for shaking culture, and the absorbance value (OD value) of the bacterial liquid is measured every 1 hour by the same method, and the continuous measurement is carried out for 24 hours.
Based on the data, a growth curve (absorbance/time) of yeast was made and the log phase was found.
In the aspect of carbon source, three raw materials, namely glucose, maltose and sucrose, are selected; three raw materials, peptone, ammonium sulfate and ammonium chloride, are also selected in the aspect of nitrogen source.
Yeasts are in a sequence for the use of different carbon sources.
During intermittent fermentation, glucose and fructose are first utilized, and only when glucose and fructose are not in the culture medium, the saccharomycetes ferment maltose, and the maximum growth and propagation speed of the saccharomycetes can be ensured by using the carbon source.
And again, the nitrogen source, yeast can only utilize two forms of nitrogen: ammonia and organic nitrogen.
Among the components of the microbial medium, the most commonly used organic ammonia sources are yeast extract and various peptones obtained by enzymatic digestion of animal and vegetable proteins.
According to the optimal growth temperature of candida utilis, the culture temperature is also set to be three groups of conditions, 28 ℃, 30 ℃ and 32 ℃ respectively, according to the previous optimized conditions.
The culture of candida utilis is a facultative anaerobe and is more suitable for aerobic culture.
According to the previous determination of the influence condition on the rotation speed of the shaking table, the rotation speed of the shaking table incubator is set to be three groups of conditions of 140r/min, 160r/min and 180 r/min.
And respectively carrying out single factor and orthogonal treatment on the three factors and the three levels, and comparing and analyzing the growth condition of the saccharomycetes by each influencing factor so as to obtain the optimal condition of regrowth after mutagenesis.
The final optimization condition is that the culture time is 13h, the temperature is 30 ℃, the rotation speed of a shaking table is 160r/min, and the cell density is highest when the carbon nitrogen ratio (the mass ratio of glucose to peptone) is about 3:1, namely the growth effect is the best.
Further, referring to fig. 2 to 4, in order to allow rapid proliferation of candida utilis, a yeast culture proliferation device is designed, which comprises a plurality of annular pipeline type culture cavities 1 connected in series, wherein the opening and closing of the passages between two adjacent culture cavities 1 are controlled by a first baffle plate 2 capable of opening and closing; the culture cavity 1 comprises an outer wall 11 and an inner wall 12 sleeved in the outer wall 11; a first temperature regulating layer 13 is arranged between the outer wall 11 and the inner wall 12; the outer wall 11 and the inner wall 12 are both heat conducting materials; the first temperature regulating layer 13 includes a plurality of first semiconductor cooling fins 14 densely arranged; the center of the inside of the culture cavity 1 is provided with a hollow inner temperature regulating rod 15 along the length direction; the inner temperature regulating rod 15 is internally filled with solid-liquid phase change material 16; the internal temperature regulating rod 15 can be communicated with the outside to circulate the solid-liquid phase material 16 inside; the phase transition temperature of the solid-liquid phase change material 16 is within the safe temperature range of the yeast; the outer surface of the inner temperature regulating rod 15 is provided with a second temperature regulating layer 17; the second temperature regulating layer 17 also includes a plurality of second semiconductor cooling fins 18 densely arranged; the inner temperature regulating rod 15 is made of heat conducting material; the outer wall 11 of the culture cavity 1 is provided with an air inlet pipe 3 which can be opened and closed respectively along the tangential direction and is used for supplementing the required sterile gas and a feed pipe 4 which is used for supplementing the required culture solution and thalli.
Furthermore, two adjacent culture cavities 1 are communicated through a connecting pipe 6, and the connecting pipe 6 is tangential to the outer walls 11 of the two culture cavities 1 at the same time; the first baffle plate 2 is hinged at the discharge port of the previous culture cavity 1, and the circulation path of the previous culture cavity 1 is kept while the connecting pipe 6 is closed by rotation, and the circulation path of the previous culture cavity 1 is closed while the connecting pipe 6 is opened.
Further, a second baffle 7 is hinged at the feed inlet of the next culture cavity 1; the second shutter 7 is rotated to maintain the circulation path of the subsequent culture chamber 1 while closing the connection pipe 6, and to close the circulation path of the subsequent culture chamber 1 while opening the connection pipe 6.
Further, the first baffle plate 2 has a porous structure, and a waterproof and breathable film 21 is covered on the surface of the first baffle plate; an exhaust pipe 22 is communicated with the connecting pipe 6 to exhaust air outwards so as to keep negative pressure in the connecting pipe 6.
Further, a plurality of annular heat conducting fins are uniformly arranged on the outer surface of the outer wall 11; the annular heat conducting fin is perpendicular to the length direction of the outer wall 11.
Further, the plurality of culture chambers 1 are distributed up and down as a whole.
Further, the inner surface of the inner wall 12 is provided with spiral rifling or smooth bulges;
the yeast culture and proliferation method comprises the following steps:
(1) the initial saccharomycete mixed culture solution is injected into the culture cavity 1 through the feed pipe 4 at a certain speed, and meanwhile, compressed aseptic gas which is beneficial to saccharomycete culture is injected into the culture cavity 1 through the air inlet pipe 3 in a continuous or pulse mode, and as the air inlet pipe 3 and the feed pipe 4 are tangential to the culture cavity 1, the saccharomycete mixed culture solution can circularly flow in the culture cavity 1, and meanwhile, the spiral rifling or smooth bulge on the inner surface of the inner wall 12 can enable the mixed culture solution to have stronger turbulence, so that the substance exchange and gas dissolution of the saccharomycete mixed culture solution are promoted, and further culture solution and gas update and temperature uniformity are facilitated; the redundant gas and waste in the culture cavity 1 pass through the waterproof breathable film 21 and the first baffle plate 2 to be pumped out through the pumping pipe 22, so that the air pressure stability and the air freshness in the culture cavity 1 are ensured;
(2) the temperature of the saccharomycete mixed culture solution in the culture cavity 1 is accurately controlled from the inner direction and the outer direction through the first semiconductor refrigerating plate 14 and the second semiconductor refrigerating plate 18 so as to ensure efficient culture and proliferation of saccharomycete, and the solid-liquid phase change material 16 can rapidly remove redundant heat under the sudden situation of failure or failure of the semiconductor refrigerating plate through circulating flow and large specific heat capacity, and can automatically balance the temperature of the saccharomycete mixed culture solution through the solid-liquid phase change characteristic of the solid-liquid phase change material, so that the saccharomycete is prevented from dying in a large scale within a period of time;
(3) after the set culture time is reached, the first baffle plate 2 and the second baffle plate 7 rotate in the front culture cavity 1 and the rear culture cavity 1 respectively, so that flowing saccharomycete culture solution enters the rear culture cavity 1 from the upper culture cavity 1 through the connecting pipe 6 under the action of inertia, after the completion, the first baffle plate 2 and the second baffle plate 7 are closed again, the front culture cavity 1 is continuously filled with new saccharomycete mixed culture solution or saccharomycete mixed culture solution of the front culture cavity 1, and the rear culture cavity 1 is continuously cultured with the saccharomycete culture solution until the aim of culture proliferation is achieved after N-channel culture is carried out, and then the saccharomycete culture solution is discharged.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (9)
1. The method for culturing candida utilis is characterized by comprising the following steps of:
s1: preparing a solid culture medium, sterilizing by high-pressure steam, and filling the upper part of a test tube on a glass rod while the solid culture medium is hot to make the length of the inclined surface of the culture medium be 1/2 of the length of the test tube;
s2: selecting a ring of thallus from candida utilis strains, putting the thallus into a prepared liquid culture medium, shaking uniformly, and culturing at 30-32 ℃ at a rotating speed of 160-180r/min for 12-13 hours;
s3: performing plate pouring operation on the solid culture medium subjected to high-pressure steam sterilization, taking out a certain amount of bacterial liquid with a certain concentration from the liquid culture medium, injecting the bacterial liquid into the surface of the solid culture medium, and uniformly spreading the inoculated bacterial liquid on the surface of the culture medium by using a spreading rod;
s4: the culture time is 36-48 hours, and after the operation, the removed flat plate can have staggered colonies for later culture and seed taking operation;
s5: the yeast culture and proliferation is carried out by adopting a yeast culture and proliferation device, the device comprises a plurality of annular pipeline type culture cavities (1) which are connected in series, and the opening and closing of a passage between two adjacent culture cavities (1) are controlled by a first baffle (2) which can be opened and closed; the culture cavity (1) comprises an outer wall (11) and an inner wall (12) sleeved in the outer wall (11); a first temperature regulating layer (13) is arranged between the outer wall (11) and the inner wall (12); the outer wall (11) and the inner wall (12) are both made of heat conducting materials; the first temperature regulating layer (13) comprises a plurality of first semiconductor refrigerating sheets (14) which are densely arranged; the inner center of the culture cavity (1) is provided with a hollow inner temperature regulating rod (15) along the length direction; the inner temperature regulating rod (15) is internally filled with solid-liquid phase-change materials (16); the inner temperature regulating rod (15) can be communicated with the outside to circulate the solid-liquid phase change material (16) inside the inner temperature regulating rod; the phase transition temperature of the solid-liquid phase transition material (16) is within the safe temperature range of saccharomycetes; the outer surface of the inner temperature regulating rod (15) is provided with a second temperature regulating layer (17); the second temperature regulating layer (17) also comprises a plurality of second semiconductor refrigerating sheets (18) which are densely arranged; the rod body of the inner temperature regulating rod (15) is made of heat conducting materials; the outer wall (11) of the culture cavity (1) is respectively provided with an air inlet pipe (3) which can be opened and closed respectively along the tangential direction and is used for supplementing the required sterile gas and a feed pipe (4) which is used for supplementing the required culture solution and thalli.
2. The method for culturing candida utilis according to claim 1, wherein the solid medium comprises 2-5% of glucose, 2-3% of peptone, 1-2% of yeast extract, 2-3% of agar and distilled water.
3. The method for culturing candida utilis according to claim 1, wherein the candida utilis is cultured in a culture medium for a period of 13 to h at a temperature of 30 ℃ and a shaking table rotation speed of 160 to r/min, and the cell density is highest when the carbon nitrogen ratio is 3:1.
4. The method for culturing candida utilis according to claim 1, wherein two adjacent culture cavities (1) are communicated through a connecting pipe (6), and the connecting pipe (6) is tangential to the outer walls (11) of the two culture cavities (1) at the same time; the first baffle (2) is hinged at the discharge hole of the previous culture cavity (1), and the circulation passage of the previous culture cavity (1) is kept while the connecting pipe (6) is closed by rotation, and the circulation passage of the previous culture cavity (1) is closed while the connecting pipe (6) is opened.
5. The method for culturing candida utilis according to claim 4, wherein the feed inlet of the subsequent culture cavity (1) is hinged with a second baffle (7); the second baffle (7) can keep the circulation passage of the next culture cavity (1) while closing the connecting pipe (6) by rotating, and can close the circulation passage of the next culture cavity (1) while opening the connecting pipe (6).
6. The method for culturing candida utilis according to claim 5, wherein the first baffle plate (2) has a porous structure and is covered with a waterproof and breathable film (21); an exhaust pipe (22) is communicated with the connecting pipe (6) to exhaust air outwards so as to keep negative pressure in the connecting pipe (6).
7. The method for culturing candida utilis according to claim 6, wherein a plurality of annular heat conducting fins are uniformly arranged on the outer surface of the outer wall (11); the annular heat conducting fin is perpendicular to the length direction of the outer wall (11); the inner surface of the inner wall (12) is provided with spiral rifling or smooth bulges.
8. A method for culturing candida utilis as claimed in claim 7, wherein the plurality of culture chambers (1) are distributed up and down as a whole.
9. The method for culturing candida utilis as claimed in claim 8, wherein the method for culturing and proliferating the yeasts comprises the steps of:
(1) injecting initial saccharomycete mixed culture solution into a culture cavity (1) through a feed pipe (4) at a certain speed, and simultaneously injecting compressed sterile gas which is beneficial to saccharomycete culture into the culture cavity (1) through an air inlet pipe (3) in a continuous or pulse mode, wherein the air inlet pipe (3) and the feed pipe (4) are tangential to the culture cavity (1), so that the saccharomycete mixed culture solution can circularly flow in the culture cavity (1), and simultaneously spiral rifling or smooth bulges on the inner surface of an inner wall (12) can enable the mixed culture solution to have stronger turbulence, promote substance exchange and gas dissolution of the saccharomycete mixed culture solution, and facilitate culture solution and gas update and temperature uniformity therein; the redundant gas and waste in the culture cavity (1) pass through the waterproof breathable film (21) and the first baffle (2) to be extracted through the extraction pipe (22), so that the air pressure stability and the air freshness in the culture cavity (1) are ensured;
(2) the temperature of the saccharomycete mixed culture solution in the culture cavity (1) is accurately controlled from the inner direction and the outer direction through the first semiconductor refrigerating plate (14) and the second semiconductor refrigerating plate (18) so as to ensure efficient culture and proliferation of saccharomycetes, and the solid-liquid phase change material (16) can rapidly remove redundant heat under the sudden condition of failure or failure of the semiconductor refrigerating plate through circulating flow and larger specific heat capacity, and can automatically balance the temperature of the saccharomycete mixed culture solution through the solid-liquid phase change characteristic of the solid-liquid phase change material and avoid large-scale death of saccharomycetes in a period of time;
(3) after the set culture time is achieved, the first baffle (2) and the second baffle (7) rotate in the front culture cavity (1) and the rear culture cavity (1) respectively, so that flowing saccharomycete culture solution enters the rear culture cavity (1) from the upper culture cavity (1) through the connecting pipe (6) under the action of inertia, after the completion of the culture, the first baffle (2) and the second baffle (7) are closed again, the front culture cavity (1) is continuously filled with new saccharomycete mixed culture solution or saccharomycete mixed culture solution of the front culture cavity (1), the rear culture cavity (1) is continuously cultured with the saccharomycete culture solution before the cultivation is continuously carried out, and the saccharomycete culture solution is discharged until the goal of cultivation and proliferation is achieved after the N-channel cultivation is carried out.
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| WO2004016728A1 (en) * | 2002-08-19 | 2004-02-26 | Olympus Corporation | Incubator and culture device |
| JP2012235714A (en) * | 2011-05-10 | 2012-12-06 | Kirin Holdings Co Ltd | Method for producing useful substance by using candida utilis |
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