CN112625927A - Kluyveromyces marxianus strain culture medium and culture method thereof - Google Patents
Kluyveromyces marxianus strain culture medium and culture method thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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Abstract
The invention belongs to the technical field of microbial culture, and particularly relates to a kluyveromyces marxianus strain culture medium and a culture method thereof, wherein the kluyveromyces marxianus strain culture medium is a massive solid in an initial state and slowly expands in an environment with the temperature of more than 24 ℃; the kluyveromyces marxianus strain culture medium is composed of the following raw materials: 15-20 parts of vegetable sponge, 5-8 parts of potato starch, 30-35 parts of deionized water, 1-1.5 parts of peptone, 1-1.5 parts of agar and 0.5-1.5 parts of gelatin; according to the invention, the culture medium volume is slowly increased, so that the bacterial strains inoculated on the surface layer gradually permeate into the expanded culture medium along with the time, the contact area of the culture medium and air is effectively increased, meanwhile, the metabolic products gradually drip towards the bottom along with the loofah fiber under the action of gravity, and further, the bacterial strains mixed in the metabolic products are accelerated to permeate into the culture medium, and the culture rate of the bacterial strains is effectively accelerated.
Description
Technical Field
The invention belongs to the technical field of microbial culture, and particularly relates to a kluyveromyces marxianus strain culture medium and a culture method thereof.
Background
In the prior art, most of kluyveromyces marxianus is cultured by adopting a solid medium or a liquid medium, a solid medium culture method enables bacterial colonies to grow and breed on the surface of the medium in the culture process, the solid medium has limited surface area, the solid medium has low space utilization rate in an incubator in the process of culturing a large number of strains, the propagation rate of the strains is greatly slowed down after the strains are spread on the surface of the solid medium, the culture rate of the strains is slow, the liquid medium can grow and breed in a liquid environment because the kluyveromyces marxianus is facultative anaerobe in the culture process, the utilization degree of space is high, but the kluyveromyces marxianus can generate carbon dioxide and acid products in the metabolic process, so the pH value in the liquid medium is gradually reduced along with the time, and alcohol in the kluyveromyces marxianus product in the anaerobic environment has toxicity to the kluyveromyces marxianus, so that the growth and the breeding of the kluyveromyces marxianus can be inhibited in the liquid environment in the liquid culture medium.
Kluyveromyces marxianus strain for milk beer production, a special culture medium and application thereof, which are issued by Chinese patents, have the following application numbers: 201711120989X, the bacterial strain provided by the proposal and the prepared liquid bacterial strain can contain more than 1.0 multiplied by 1011 viable bacteria/mL when stored for 4 days at 0-4 ℃, the raw material source is sufficient, the price is low, the ingredients are convenient, the preparation method of the high-density liquid leavening agent is adopted, the culture volume is reduced, the equipment investment and the production time are reduced, and the purchase cost of the bacterial strain is also saved.
In view of the above, the invention develops a kluyveromyces marxianus strain culture medium and a culture method thereof, which are used for solving the technical problems.
Disclosure of Invention
In order to make up for the defects of the prior art and solve the problems that in the prior art, the pH value in the liquid culture medium is gradually reduced along with the time due to the fact that carbon dioxide and acid products are generated in the metabolism process of the kluyveromyces marxianus, and the alcohol in the kluyveromyces marxianus product in the anaerobic environment has toxicity to the kluyveromyces marxianus and further causes the liquid environment in the liquid culture medium to play a role in inhibiting the growth and the breeding of the kluyveromyces marxianus.
The technical scheme adopted by the invention for solving the technical problems is as follows: the kluyveromyces marxianus strain culture medium is a massive solid in an initial state and slowly expands in an environment with the temperature of more than 24 ℃; the kluyveromyces marxianus strain culture medium is composed of the following raw materials:
15-20 parts of vegetable sponge, 5-8 parts of potato starch, 30-35 parts of deionized water, 1-1.5 parts of peptone, 1-1.5 parts of agar and 0.5-1.5 parts of gelatin;
the preparation method of the kluyveromyces marxianus strain culture medium comprises the following steps:
s1: selecting naturally aged loofah pulp, removing surface loofah skin, cutting into rectangular sheet bodies of 2 x 1 x 0.8cm, continuously drying the sheet bodies at 45-50 ℃ for 15-18 min, introducing into an ultraviolet sterilizer, and performing conventional disinfection and sterilization treatment;
s2: placing agar into 3/4 deionized water heated to 100-105 ℃, carrying out heat preservation and heating for 5-8 min, carrying out continuous stirring in the heat preservation and heating process, introducing boiling water into a filter screen after heating, controlling the mesh number of the filter screen to be 160-200 meshes, and filtering to obtain solidified water;
s3: adding potato starch and peptone into the coagulating water, continuously stirring for 5-10 min, naturally cooling the solution to 55-60 ℃, soaking the vegetable sponge sheet body subjected to sterilization treatment in S1 for 10-12 min, taking out the vegetable sponge sheet body after soaking, rapidly ventilating and cooling to 16-24 ℃, pressing the cooled vegetable sponge by using a pressing plate, and compressing the thickness of the vegetable sponge to 0.35-0.45cm for later use;
s4: dissolving gelatin in 1/4 deionized water heated to 80-90 ℃, continuously stirring for 2-3 min, cooling the solution to 30-32 ℃, pouring the gelatin solution on the surface of the compressed loofah sponge, and sending the loofah sponge into a quick freezing box to reduce the temperature of the loofah sponge to 15-20 ℃ to prepare a compressed tablet;
s5: stacking the compressed sheets in a vertically staggered manner, placing the stacked compressed sheets in a temperature circulating box, controlling the temperature in the temperature circulating box to be 18-24 ℃ and circulating at a constant speed, controlling the circulating interval to be 5-6 min, and taking out the compressed sheets after circulating treatment for 10-12 min to obtain the kluyveromyces marxianus strain culture medium;
in the prior art, most of kluyveromyces marxianus is cultured by adopting a solid medium or a liquid medium, a solid medium culture method enables bacterial colonies to grow and breed on the surface of the medium in the culture process, the solid medium has limited surface area, the solid medium has low space utilization rate in an incubator in the process of culturing a large number of strains, the propagation rate of the strains is greatly slowed down after the strains are spread on the surface of the solid medium, the culture rate of the strains is slow, the liquid medium can grow and breed in a liquid environment because the kluyveromyces marxianus is facultative anaerobe in the culture process, the utilization degree of space is high, but the kluyveromyces marxianus can generate carbon dioxide and acid products in the metabolic process, so the pH value in the liquid medium is gradually reduced along with the time, in addition, alcohol in the kluyveromyces marxianus product in an anaerobic environment has toxicity to the kluyveromyces marxianus, so that the growth and the breeding of the kluyveromyces marxianus can be inhibited in a liquid environment in a liquid culture medium;
when the invention works, potato starch is used as a sugar source, peptone is used as a nitrogen source, the potato starch is dissolved in water to form a culture solution, agar is dissolved in the culture solution, the culture solution is converted into a gel state after the culture solution is cooled, because the reticular fibrous layer structure of the vegetable sponge has excellent conductivity, the peeled vegetable sponge is cut into a sheet body with a standard size, the sheet body is soaked in the uncooled culture solution, and then the vegetable sponge is fished out and rapidly cooled, so that the culture solution is uniformly condensed on fibers in the vegetable sponge, the whole vegetable sponge is converted into a solid culture medium, meanwhile, because the reticular structure of the vegetable sponge has stronger deformation capability, the vegetable sponge is compressed, a solution prepared from gelatin is poured around the vegetable sponge sheet body, and when the gelatin solution is cooled to 15-20 ℃, the gelatin solution is solidified, further fixing the compressed luffa pith to reduce the volume of a solid culture medium made of luffa pith, overlapping a plurality of luffa culture mediums in a staggered manner, placing the luffa culture mediums in a temperature circulation box, melting and solidifying gelatin on the surface layer of the luffa pith in the process of temperature circulation, further bonding the plurality of layers of luffa pith solid culture mediums, wherein the reduction of the volume enables the prepared culture medium to be convenient to store and transport, meanwhile, in the process of strain culture, slowly raising the temperature to enable the volume of the culture medium to be slowly increased, further enabling the strain inoculated on the surface layer to gradually permeate into the expanded culture medium along with the passage of time, further effectively increasing the contact area of the culture medium and air, and the kluyveromyces marxianus has a higher breeding speed in an aerobic environment, can effectively promote the breeding of kluyveromyces marxianus, and simultaneously in the process of the metabolism of the kluyveromyces marxianus, the metabolite gradually drips to the bottom under the effect of gravity along the loofah fiber under the effect of gravity, and then the bacterial strain that mixes in the metabolite is accelerated to the inside infiltration of culture medium, and then the effectual cultivation rate that accelerates the bacterial strain, simultaneously because the culture medium slowly expands, and then make the inside negative pressure that forms of culture medium, and then make the culture medium inside form the extraction to the outside air, the flow of the in-process air current of air extraction plays the assistance to the bacterial strain to the inside diffusion of culture medium, and then the speed of bacterial strain to the inside diffusion of culture medium is accelerated to the flow effect of effectual cooperation resultant, the loofah itself can be decomposed by the bacterial strain simultaneously, also can regard as fermented material to use in follow-up fermentation process, make the utilization degree of loofah higher.
Preferably, the watermelon pulp in the S1 is soaked in alkaline water for 40-60 min before being dried, and then is sent into a refrigeration house at-8 to-5 ℃ for freezing treatment, wherein the freezing time is controlled to be 10-15 min; the alkaline water is a 5-7% sodium hydroxide solution;
during operation, because fibre itself is comparatively thin in the vegetable sponge, make the culture solution comparatively difficult at the in-process that the wall built-up solidifies to the vegetable sponge fibre, through soaking the vegetable sponge in sodium hydroxide solution before drying, utilize the swelling effect of sodium hydroxide solution to the cellulose, effectual get rid of the inside tiny fibre of vegetable sponge, still make the cellulose in the cellosilk dissolve simultaneously, after soaking the completion, carry out the refrigeration treatment with the vegetable sponge simultaneously, utilize the moisture to freeze, the principle of volume expansion, make the texture of cellosilk itself softer, make the culture solution can permeate to the inside of vegetable sponge, make the culture solution condense more smoothly on the inside fibre of vegetable sponge.
Preferably, the watermelon pulp in the S3 and the S4 are continuously turned in the process of rapid cooling, and the turning rate is controlled to be 35-40 r/min;
during operation, because the liquid possesses gravity, through quick upset at the in-process that the cooling condenses, and then make culture solution evenly distributed in the vegetable sponge, it is inhomogeneous to avoid the culture solution to condense in the vegetable sponge is inside, leads to even that the culture solution blocks up the inside space of vegetable sponge in the in-process that condenses, forms the hindrance to the invasion of bacterial strain.
Preferably, the raw materials also comprise sputtering microbeads; the content of the sputtering microbeads accounts for 20-25% of the weight of the loofah pulp; the sputtering micro-beads are in a granular structure coated with compressed carbon dioxide; the surface of the sputtering micro-bead is composed of white granulated sugar and propolis;
during operation, by adding the sputtering micro-beads, in the culture process of the strains, water, carbon dioxide and alcohol are generated in the metabolic process of the kluyveromyces marxianus strains, water drops are dripped on the surface layers of the sputtering micro-beads under the action of gravity, so that sugar shells on the surface layers of the sputtering micro-beads are dissolved, carbon dioxide bubbles in the sputtering micro-beads are broken, the carbon dioxide is sprayed outwards, impact is further formed on the water drops on the surfaces of the sputtering micro-beads, the water drops are transversely sputtered inside the vegetable sponge along with the carbon dioxide, and the kluyveromyces marxianus strains are contained in the water drops.
Preferably, the method for manufacturing the sputtering microbead comprises the following steps:
i: mixing white granulated sugar and propolis according to a ratio of 5:1, introducing into a heating dish, controlling the temperature in the heating dish to be maintained at 95-100 ℃, continuously preserving heat and dissolving, and performing circle-drawing stirring in the process of preserving heat and dissolving, wherein the stirring speed is controlled to be 35-40 r/min;
II: pouring the syrup which is kept warm and dissolved for 10-12 min onto a marble table, filling carbon dioxide gas into the syrup, repeatedly folding and compressing the syrup until the syrup is cooled and solidified, and preparing syrup solid containing uniform and dense compressed bubbles;
III: feeding the syrup solid in the step II into a shearing machine, controlling the rotating speed of the shearing machine to be 60-80 r/min, shearing and crushing, feeding a crushed product into a double-filtering sieve, intercepting the crushed product with the particle size of 2-3 mm by using the double-filtering sieve to obtain sputtering microbeads, and directly and uniformly spraying the sputtering microbeads into the loofah before ventilation and cooling in S3 when the sputtering microbeads are used;
the during operation, through folding the compression repeatedly, with carbon dioxide bubble even breakage in the syrup, dispersion, and then make the carbon dioxide compression bubble distribution in the syrup that solidifies more even, intensive degree is great, uses dual filtration simultaneously, gets rid of broken less piece of bubble that contains and the broken thing of large granule, obtains the comparatively suitable broken microballon of particle size, and then makes broken microballon convenient, the sputtering effect is better in what the inside dispersion of luffa flesh.
Preferably, the carbon dioxide gas filled in the syrup in II is mixed with sodium bicarbonate solution; the carbon dioxide is carbon dioxide gas heated to 65-70 ℃; the carbon dioxide gas is contacted with the sodium bicarbonate solution in a flowing process; the during operation, because kluyveromyces marxianus all can make culture medium pH value to acid conversion at metabolic process and the in-process that carbon dioxide dissolved in aqueous, through flowing through the sodium bicarbonate solution with carbon dioxide to the inside in-process flow of syrup, and then make sodium bicarbonate solution atomize and flow to syrup inside along with carbon dioxide, and then utilize the alkalescence of sodium bicarbonate solution to carry out the neutralization to acidic material, and then the inside pH value of effectual maintenance culture medium, make the inside pH value of culture medium be partial to neutral, the growth of kluyveromyces marxianus of being convenient for, breed.
A culture method of a Kluyveromyces marxianus strain comprises the following steps:
a1: filling the prepared kluyveromyces marxianus strain culture medium into an incubator, inoculating the kluyveromyces marxianus strain on the surface of the culture medium in the incubator by using a scribing method, and placing the incubator in a thermostat for culture;
a2: controlling the temperature in the incubator to slowly rise to 20-23 ℃, carrying out constant-temperature culture for 2-3H, then controlling the temperature in the incubator to rise to 30-35 ℃, continuously carrying out heat preservation culture for 14-16H, and continuously supplementing fresh sterile air to the incubator in the culture process;
a3: and (3) using the residual culture medium and the bacterial colony in the incubator with the culture time of 16-19H for inoculating the production starter, and cooling to 0-4 ℃ after fermentation is completed to obtain the liquid starter containing a large amount of Kluyveromyces marxianus strains.
The invention has the following beneficial effects:
1. the invention relates to a Kluyveromyces marxianus strain culture medium and a culture method thereof, which can slowly increase the volume of a culture medium by increasing the temperature in the process of strain culture, further enable a strain inoculated on a surface layer to gradually permeate into an expanded culture medium along with the passage of time, further effectively increase the contact area of the culture medium and air, enable the Kluyveromyces marxianus to have a higher breeding rate in an aerobic environment, effectively promote the breeding of the Kluyveromyces marxianus, simultaneously enable metabolites to gradually drip to the bottom under the action of gravity along with vegetable sponge fibers in the process of metabolism of the Kluyveromyces marxianus, further enable strains mixed in the metabolites to accelerate the permeation into the culture medium, further effectively accelerate the culture rate of the strains, and simultaneously enable the culture medium to slowly expand, and then negative pressure is formed in the culture medium, so that the culture medium is internally used for extracting the outside air, the flowing of the air flow assists the strain to diffuse into the culture medium in the air extraction process, the speed of the strain diffusing into the culture medium is accelerated by effectively matching the flowing effect of the resultant, meanwhile, the vegetable sponge can be decomposed by the strain and can also be used as a fermentation material in the subsequent fermentation process, and the utilization degree of the vegetable sponge is higher.
2. The kluyveromyces marxianus strain culture medium and the culture method thereof have the advantages that sputtering microbeads are added, in the culture process of the strain, along with the production of water, carbon dioxide and alcohol in the metabolic process of the kluyveromyces marxianus strain, dripping water drops on the surface layer of the sputtering micro-bead under the action of gravity, further dissolving the sugar shell on the surface layer of the sputtering micro-bead, breaking the carbon dioxide bubbles in the sputtering micro-bead, spraying the carbon dioxide outwards, further forming impact on water drops sputtered on the surfaces of the microbeads, so that the water drops are transversely sputtered in the loofah sponge along with carbon dioxide, and because the water drops contain Kluyveromyces marxianus strains, in the process of water drop sputtering, the Kluyveromyces marxianus strain diffuses transversely into the vegetable sponge, and the diffusion efficiency of the Kluyveromyces marxianus strain in the vegetable sponge is effectively enhanced by the vertical diffusion of the water drops under the action of gravity.
Drawings
The invention will be further explained with reference to the drawings.
FIG. 1 is a flow chart of a method for preparing a culture medium of Kluyveromyces marxianus strain;
FIG. 2 is a method flow diagram of a sputtering bead fabrication method;
FIG. 3 is a flow chart of a method for culturing Kluyveromyces marxianus strain;
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
As shown in fig. 1 to 3, the kluyveromyces marxianus strain culture medium of the present invention is a massive solid in an initial state and slowly swells in an environment of 24 ℃ or higher; the kluyveromyces marxianus strain culture medium is composed of the following raw materials:
15-20 parts of vegetable sponge, 5-8 parts of potato starch, 30-35 parts of deionized water, 1-1.5 parts of peptone, 1-1.5 parts of agar and 0.5-1.5 parts of gelatin;
the preparation method of the kluyveromyces marxianus strain culture medium comprises the following steps:
s1: selecting naturally aged loofah pulp, removing surface loofah skin, cutting into rectangular sheet bodies of 2 x 1 x 0.8cm, continuously drying the sheet bodies at 45-50 ℃ for 15-18 min, introducing into an ultraviolet sterilizer, and performing conventional disinfection and sterilization treatment;
s2: placing agar into 3/4 deionized water heated to 100-105 ℃, carrying out heat preservation and heating for 5-8 min, carrying out continuous stirring in the heat preservation and heating process, introducing boiling water into a filter screen after heating, controlling the mesh number of the filter screen to be 160-200 meshes, and filtering to obtain solidified water;
s3: adding potato starch and peptone into the coagulating water, continuously stirring for 5-10 min, naturally cooling the solution to 55-60 ℃, soaking the vegetable sponge sheet body subjected to sterilization treatment in S1 for 10-12 min, taking out the vegetable sponge sheet body after soaking, rapidly ventilating and cooling to 16-24 ℃, pressing the cooled vegetable sponge by using a pressing plate, and compressing the thickness of the vegetable sponge to 0.35-0.45cm for later use;
s4: dissolving gelatin in 1/4 deionized water heated to 80-90 ℃, continuously stirring for 2-3 min, cooling the solution to 30-32 ℃, pouring the gelatin solution on the surface of the compressed loofah sponge, and sending the loofah sponge into a quick freezing box to reduce the temperature of the loofah sponge to 15-20 ℃ to prepare a compressed tablet;
s5: stacking the compressed sheets in a vertically staggered manner, placing the stacked compressed sheets in a temperature circulating box, controlling the temperature in the temperature circulating box to be 18-24 ℃ and circulating at a constant speed, controlling the circulating interval to be 5-6 min, and taking out the compressed sheets after circulating treatment for 10-12 min to obtain the kluyveromyces marxianus strain culture medium;
in the prior art, most of kluyveromyces marxianus is cultured by adopting a solid medium or a liquid medium, a solid medium culture method enables bacterial colonies to grow and breed on the surface of the medium in the culture process, the solid medium has limited surface area, the solid medium has low space utilization rate in an incubator in the process of culturing a large number of strains, the propagation rate of the strains is greatly slowed down after the strains are spread on the surface of the solid medium, the culture rate of the strains is slow, the liquid medium can grow and breed in a liquid environment because the kluyveromyces marxianus is facultative anaerobe in the culture process, the utilization degree of space is high, but the kluyveromyces marxianus can generate carbon dioxide and acid products in the metabolic process, so the pH value in the liquid medium is gradually reduced along with the time, in addition, alcohol in the kluyveromyces marxianus product in an anaerobic environment has toxicity to the kluyveromyces marxianus, so that the growth and the breeding of the kluyveromyces marxianus can be inhibited in a liquid environment in a liquid culture medium;
when the invention works, potato starch is used as a sugar source, peptone is used as a nitrogen source, the potato starch is dissolved in water to form a culture solution, agar is dissolved in the culture solution, the culture solution is converted into a gel state after the culture solution is cooled, because the reticular fibrous layer structure of the vegetable sponge has excellent conductivity, the peeled vegetable sponge is cut into a sheet body with a standard size, the sheet body is soaked in the uncooled culture solution, and then the vegetable sponge is fished out and rapidly cooled, so that the culture solution is uniformly condensed on fibers in the vegetable sponge, the whole vegetable sponge is converted into a solid culture medium, meanwhile, because the reticular structure of the vegetable sponge has stronger deformation capability, the vegetable sponge is compressed, a solution prepared from gelatin is poured around the vegetable sponge sheet body, and when the gelatin solution is cooled to 15-20 ℃, the gelatin solution is solidified, further fixing the compressed luffa pith to reduce the volume of a solid culture medium made of luffa pith, overlapping a plurality of luffa culture mediums in a staggered manner, placing the luffa culture mediums in a temperature circulation box, melting and solidifying gelatin on the surface layer of the luffa pith in the process of temperature circulation, further bonding the plurality of layers of luffa pith solid culture mediums, wherein the reduction of the volume enables the prepared culture medium to be convenient to store and transport, meanwhile, in the process of strain culture, slowly raising the temperature to enable the volume of the culture medium to be slowly increased, further enabling the strain inoculated on the surface layer to gradually permeate into the expanded culture medium along with the passage of time, further effectively increasing the contact area of the culture medium and air, and the kluyveromyces marxianus has a higher breeding speed in an aerobic environment, can effectively promote the breeding of kluyveromyces marxianus, and simultaneously in the process of the metabolism of the kluyveromyces marxianus, the metabolite gradually drips to the bottom under the effect of gravity along the loofah fiber under the effect of gravity, and then the bacterial strain that mixes in the metabolite is accelerated to the inside infiltration of culture medium, and then the effectual cultivation rate that accelerates the bacterial strain, simultaneously because the culture medium slowly expands, and then make the inside negative pressure that forms of culture medium, and then make the culture medium inside form the extraction to the outside air, the flow of the in-process air current of air extraction plays the assistance to the bacterial strain to the inside diffusion of culture medium, and then the speed of bacterial strain to the inside diffusion of culture medium is accelerated to the flow effect of effectual cooperation resultant, the loofah itself can be decomposed by the bacterial strain simultaneously, also can regard as fermented material to use in follow-up fermentation process, make the utilization degree of loofah higher.
The preparation method is an embodiment of the invention, wherein the watermelon pulp in S1 is soaked in alkaline water for 40-60 min before being dried, and then is sent into a refrigeration house with the temperature of-8 to-5 ℃ for freezing treatment after soaking, and the freezing time is controlled to be 10-15 min; the alkaline water is a 5-7% sodium hydroxide solution;
during operation, because fibre itself is comparatively thin in the vegetable sponge, make the culture solution comparatively difficult at the in-process that the wall built-up solidifies to the vegetable sponge fibre, through soaking the vegetable sponge in sodium hydroxide solution before drying, utilize the swelling effect of sodium hydroxide solution to the cellulose, effectual get rid of the inside tiny fibre of vegetable sponge, still make the cellulose in the cellosilk dissolve simultaneously, after soaking the completion, carry out the refrigeration treatment with the vegetable sponge simultaneously, utilize the moisture to freeze, the principle of volume expansion, make the texture of cellosilk itself softer, make the culture solution can permeate to the inside of vegetable sponge, make the culture solution condense more smoothly on the inside fibre of vegetable sponge.
The method is an embodiment of the invention, wherein the watermelon pulp in S3 and S4 is continuously turned in the process of rapid cooling, and the turning rate is controlled to be 35-40 r/min;
during operation, because the liquid possesses gravity, through quick upset at the in-process that the cooling condenses, and then make culture solution evenly distributed in the vegetable sponge, it is inhomogeneous to avoid the culture solution to condense in the vegetable sponge is inside, leads to even that the culture solution blocks up the inside space of vegetable sponge in the in-process that condenses, forms the hindrance to the invasion of bacterial strain.
As an embodiment of the present invention, wherein the raw material further comprises sputtered microbeads; the content of the sputtering microbeads accounts for 20-25% of the weight of the loofah pulp; the sputtering micro-beads are in a granular structure coated with compressed carbon dioxide; the surface of the sputtering micro-bead is composed of white granulated sugar and propolis;
during operation, by adding the sputtering micro-beads, in the culture process of the strains, water, carbon dioxide and alcohol are generated in the metabolic process of the kluyveromyces marxianus strains, water drops are dripped on the surface layers of the sputtering micro-beads under the action of gravity, so that sugar shells on the surface layers of the sputtering micro-beads are dissolved, carbon dioxide bubbles in the sputtering micro-beads are broken, the carbon dioxide is sprayed outwards, impact is further formed on the water drops on the surfaces of the sputtering micro-beads, the water drops are transversely sputtered inside the vegetable sponge along with the carbon dioxide, and the kluyveromyces marxianus strains are contained in the water drops.
As an embodiment of the present invention, the method for manufacturing sputtering micro beads includes the steps of:
i: mixing white granulated sugar and propolis according to a ratio of 5:1, introducing into a heating dish, controlling the temperature in the heating dish to be maintained at 95-100 ℃, continuously preserving heat and dissolving, and performing circle-drawing stirring in the process of preserving heat and dissolving, wherein the stirring speed is controlled to be 35-40 r/min;
II: pouring the syrup which is kept warm and dissolved for 10-12 min onto a marble table, filling carbon dioxide gas into the syrup, repeatedly folding and compressing the syrup until the syrup is cooled and solidified, and preparing syrup solid containing uniform and dense compressed bubbles;
III: feeding the syrup solid in the step II into a shearing machine, controlling the rotating speed of the shearing machine to be 60-80 r/min, shearing and crushing, feeding a crushed product into a double-filtering sieve, intercepting the crushed product with the particle size of 2-3 mm by using the double-filtering sieve to obtain sputtering microbeads, and directly and uniformly spraying the sputtering microbeads into the loofah before ventilation and cooling in S3 when the sputtering microbeads are used;
the during operation, through folding the compression repeatedly, with carbon dioxide bubble even breakage in the syrup, dispersion, and then make the carbon dioxide compression bubble distribution in the syrup that solidifies more even, intensive degree is great, uses dual filtration simultaneously, gets rid of broken less piece of bubble that contains and the broken thing of large granule, obtains the comparatively suitable broken microballon of particle size, and then makes broken microballon convenient, the sputtering effect is better in what the inside dispersion of luffa flesh.
In one embodiment of the present invention, the carbon dioxide gas filled in the syrup is mixed with a sodium bicarbonate solution; the carbon dioxide is carbon dioxide gas heated to 65-70 ℃; the carbon dioxide gas is contacted with the sodium bicarbonate solution in a flowing process; the during operation, because kluyveromyces marxianus all can make culture medium pH value to acid conversion at metabolic process and the in-process that carbon dioxide dissolved in aqueous, through flowing through the sodium bicarbonate solution with carbon dioxide to the inside in-process flow of syrup, and then make sodium bicarbonate solution atomize and flow to syrup inside along with carbon dioxide, and then utilize the alkalescence of sodium bicarbonate solution to carry out the neutralization to acidic material, and then the inside pH value of effectual maintenance culture medium, make the inside pH value of culture medium be partial to neutral, the growth of kluyveromyces marxianus of being convenient for, breed.
A culture method of a Kluyveromyces marxianus strain comprises the following steps:
a1: filling the prepared kluyveromyces marxianus strain culture medium into an incubator, inoculating the kluyveromyces marxianus strain on the surface of the culture medium in the incubator by using a scribing method, and placing the incubator in a thermostat for culture;
a2: controlling the temperature in the incubator to slowly rise to 20-23 ℃, carrying out constant-temperature culture for 2-3H, then controlling the temperature in the incubator to rise to 30-35 ℃, continuously carrying out heat preservation culture for 14-16H, and continuously supplementing fresh sterile air to the incubator in the culture process;
a3: and (3) using the residual culture medium and the bacterial colony in the incubator with the culture time of 16-19H for inoculating the production starter, and cooling to 0-4 ℃ after fermentation is completed to obtain the liquid starter containing a large amount of Kluyveromyces marxianus strains.
The specific implementation flow is as follows:
when the culture medium is in work, potato starch is used as a sugar source, peptone is used as a nitrogen source, the potato starch is dissolved in water to form a culture solution, agar is dissolved in the culture solution, the culture solution is converted into a gel state after the culture solution is cooled, the vegetable sponge is cut into a sheet-shaped body with a standard size due to the excellent conductivity of the reticular fiber layer structure of the vegetable sponge, the sheet-shaped body is soaked in the uncooled culture solution, then the vegetable sponge is taken out and rapidly cooled, the culture solution can be uniformly condensed on fibers in the vegetable sponge, the vegetable sponge is integrally converted into a solid culture medium, meanwhile, the vegetable sponge is compressed due to the strong deformation capability of the reticular structure of the vegetable sponge, a solution prepared from gelatin is poured around the vegetable sponge, and the gelatin solution is solidified after the gelatin solution is cooled to 15-20 ℃, and then fixing the compressed loofah pulp to reduce the volume of a solid culture medium prepared from the loofah pulp, overlapping a plurality of loofah culture mediums in a staggered manner, placing the loofah culture mediums in a temperature circulation box, melting and solidifying gelatin on the surface layer of the loofah pulp in the temperature circulation process, and further adhering the plurality of layers of loofah pulp solid culture mediums, wherein the prepared culture medium is convenient to store in transportation due to the reduction of the volume.
In order to verify the using effect of the kluyveromyces marxianus strain culture medium prepared by the invention, the following groups of experiments are specially set up for verifying the promotion effect of the kluyveromyces marxianus strain culture medium prepared by the invention in the culture process of the kluyveromyces marxianus strain;
example 1
The kluyveromyces marxianus strain culture medium is composed of the following raw materials:
5-8 parts of potato starch, 30-35 parts of deionized water, 1-1.5 parts of peptone and 1-1.5 parts of agar;
placing agar in 3/4 deionized water heated to 100-105 ℃, carrying out heat preservation and heating for 5-8 min, carrying out continuous stirring in the heat preservation and heating process, introducing boiling water into a filter sieve after heating, controlling the mesh number of the filter sieve to be 160-200 meshes, filtering to obtain solidified water, adding potato starch and peptone into the solidified water, carrying out continuous stirring for 5-10 min, naturally cooling the solution to normal temperature to obtain a Kluyveromyces marxianus strain culture medium, filling the prepared Kluyveromyces marxianus strain culture medium into 3 culture boxes in equal parts, inoculating 100 Kluyveromyces marxianus strains on the surface of the culture medium in the culture boxes by using a scribing method, placing the culture boxes in a constant temperature box, controlling the temperature in the constant temperature box to rise to 30-35 ℃, and carrying out heat preservation and culture for 4H, 12H and 19H in 3 culture boxes continuously, after the constant-temperature cultivation is finished, completely dissolving the culture medium in 1L of deionized water, carrying out centrifugal stirring treatment, immediately extracting 5 groups of 1ml of centrifugate after the centrifugal treatment is finished, and carrying out strain quantity detection by using a chromosome fluorescent staining technique;
example 2
The kluyveromyces marxianus strain culture medium is a massive solid in an initial state and slowly expands in an environment above 24 ℃; the kluyveromyces marxianus strain culture medium is composed of the following raw materials:
15-20 parts of vegetable sponge, 5-8 parts of potato starch, 30-35 parts of deionized water, 1-1.5 parts of peptone, 1-1.5 parts of agar and 0.5-1.5 parts of gelatin;
selecting naturally aged loofah pulp, removing surface loofah peel, cutting the loofah pulp into rectangular sheet bodies with the length of 2 x 1 x 0.8cm, continuously drying the sheet bodies at 45-50 ℃ for 15-18 min, introducing the sheet bodies into an ultraviolet sterilizer, carrying out conventional disinfection and sterilization treatment, synchronously placing agar into 3/4 deionized water heated to 100-105 ℃, carrying out heat preservation and heating for 5-8 min, carrying out continuous stirring in the heat preservation and heating process, introducing boiling water into a filter screen after heating, controlling the mesh number of the filter screen to be 160-200 meshes, filtering to obtain solidified water, adding potato starch and peptone into the solidified water, continuously stirring for 5-10 min, naturally cooling the solution to 55-60 ℃, soaking the loofah pulp sheet bodies subjected to disinfection treatment in S1 for 10-12 min, taking out the loofah pulp sheet bodies after soaking, rapidly ventilating and cooling to 16-24 ℃, pressing the cooled loofah pulp by using a pressing plate, compressing the thickness of the luffa pith to 0.35-0.45cm for later use, dissolving gelatin in 1/4 deionized water heated to 80-90 ℃, continuously stirring for 2-3 min, cooling the solution to 30-32 ℃, pouring the gelatin solution on the surface of the luffa pith in a compressed state, sending the luffa pith into an emergency freezer to reduce the temperature of the luffa pith to 15-20 ℃ to prepare compressed sheets, stacking the compressed sheets in a vertically staggered manner, placing the compressed sheets in a temperature circulating box, controlling the temperature in the temperature circulating box to be 18-24 ℃ for uniform circulation, controlling the circulation interval to be 5-6 min, carrying out circulation treatment for 10-12 min, taking out the sheets to prepare a kluyveromyces marxianus strain culture medium, filling the prepared kluyveromyces marxianus strain culture medium into 3 culture boxes in equal intervals, inoculating 100 kluyveromyces marxianus strains on the surface of the culture medium in the culture boxes by a scribing method, placing the incubator in a thermostat, controlling the temperature in the thermostat to slowly rise to 20-23 ℃, carrying out constant temperature culture for 3H, then controlling the temperature in the thermostat to rise to 30-35 ℃, controlling the three incubators to continuously culture 1H, 9H and 16H in a heat preservation manner, continuously supplementing fresh sterile air into the incubators in the culture process, completely dissolving a culture medium in 1L of deionized water after the constant temperature culture is finished, carrying out centrifugal stirring treatment, immediately extracting 5 groups of 1ml of centrifugate after the centrifugal treatment is finished, and carrying out strain quantity detection by using a chromosome fluorescent staining technique;
example 3
The kluyveromyces marxianus strain culture medium is a massive solid in an initial state and slowly expands in an environment above 24 ℃; the kluyveromyces marxianus strain culture medium is composed of the following raw materials:
15-20 parts of vegetable sponge, 5-8 parts of potato starch, 30-35 parts of deionized water, 1-1.5 parts of peptone, 1-1.5 parts of agar and 0.5-1.5 parts of gelatin;
selecting naturally aged loofah sponge, removing surface loofah peel, cutting the loofah sponge into rectangular sheets with the length of 2 x 1 x 0.8cm, continuously drying the sheets at 45-50 ℃ for 15-18 min, introducing the sheets into an ultraviolet sterilizer, carrying out conventional disinfection and sterilization treatment, synchronously placing agar into 3/4 deionized water heated to 100-105 ℃, carrying out heat preservation and heating for 5-8 min, carrying out continuous stirring in the heat preservation and heating process, introducing boiling water into a filter screen after heating is finished, controlling the mesh number of the filter screen to be 160-200 meshes, filtering to obtain solidified water, adding potato starch and peptone into the solidified water, continuously stirring for 5-10 min, naturally cooling the solution to 55-60 ℃, soaking the disinfected loofah sponge sheets for 10-12 min, taking out the loofah sponge sheets after soaking, rapidly ventilating and cooling to 16-24 ℃, and uniformly scattering sputtered microbeads with the content of 20-25% of the weight of the loofah sponge into the interior of the loofah sponge, pressing the cooled loofah pulp by using a pressing plate, compressing the thickness of the loofah pulp to 0.35-0.45cm for later use, dissolving gelatin in 1/4 deionized water heated to 80-90 ℃, continuously stirring for 2-3 min, cooling the solution to 30-32 ℃, pouring the gelatin solution on the surface of the compressed loofah pulp, sending the loofah pulp into an emergency freezer, reducing the temperature of the loofah pulp to 15-20 ℃ to obtain a compressed sheet, vertically stacking the compressed sheet in a staggered manner, placing the stacked sheets in a temperature circulating box, controlling the temperature in the temperature circulating box to 18-24 ℃ and circulating at a constant speed, controlling the circulation interval to be 5-6 min, carrying out circulating treatment for 10-12 min, then taking out the sheets to obtain the kluyveromyces marxianus strain culture medium, equally filling the prepared kluyveromyces marxianus strain culture medium into 3 culture boxes, inoculating 100 kluyveromyces marxianus strains on the surface of the culture medium by using a scribing method, placing the incubator in a thermostat, controlling the temperature in the thermostat to slowly rise to 20-23 ℃, carrying out constant temperature culture for 3H, then controlling the temperature in the thermostat to rise to 30-35 ℃, controlling the three incubators to continuously culture 1H, 9H and 16H in a heat preservation manner, continuously supplementing fresh sterile air into the incubators in the culture process, completely dissolving a culture medium in 1L of deionized water after the constant temperature culture is finished, carrying out centrifugal stirring treatment, immediately extracting 5 groups of 1ml of centrifugate after the centrifugal treatment is finished, and carrying out strain quantity detection by using a chromosome fluorescent staining technique;
TABLE 1 (Strain content/ml)
According to the experimental data of the three groups of examples, the kluyveromyces marxianus strain culture medium prepared by the method and the formula has the advantages that the growth of the kluyveromyces marxianus strain and the continuous growth of the kluyveromyces marxianus strain are not hindered in the breeding process along with the time in the process of culturing the kluyveromyces marxianus strain, and in the examples without the method and the method, the breeding of the kluyveromyces marxianus strain is inhibited by space and products respectively along with the time, so that the growth and breeding efficiency of the kluyveromyces marxianus strain in the kluyveromyces marxianus strain culture medium prepared by the method is higher.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. A Kluyveromyces marxianus strain culture medium is characterized in that: the kluyveromyces marxianus strain culture medium is a massive solid in an initial state and slowly expands in an environment above 24 ℃; the kluyveromyces marxianus strain culture medium is composed of the following raw materials:
15-20 parts of vegetable sponge, 5-8 parts of potato starch, 30-35 parts of deionized water, 1-1.5 parts of peptone, 1-1.5 parts of agar and 0.5-1.5 parts of gelatin;
the preparation method of the kluyveromyces marxianus strain culture medium comprises the following steps:
s1: selecting naturally aged loofah pulp, removing surface loofah skin, cutting into rectangular sheet bodies of 2 x 1 x 0.8cm, continuously drying the sheet bodies at 45-50 ℃ for 15-18 min, introducing into an ultraviolet sterilizer, and performing conventional disinfection and sterilization treatment;
s2: placing agar into 3/4 deionized water heated to 100-105 ℃, carrying out heat preservation and heating for 5-8 min, carrying out continuous stirring in the heat preservation and heating process, introducing boiling water into a filter screen after heating, controlling the mesh number of the filter screen to be 160-200 meshes, and filtering to obtain solidified water;
s3: adding potato starch and peptone into the coagulating water, continuously stirring for 5-10 min, naturally cooling the solution to 55-60 ℃, soaking the vegetable sponge sheet body subjected to sterilization treatment in S1 for 10-12 min, taking out the vegetable sponge sheet body after soaking, rapidly ventilating and cooling to 16-24 ℃, pressing the cooled vegetable sponge by using a pressing plate, and compressing the thickness of the vegetable sponge to 0.35-0.45cm for later use;
s4: dissolving gelatin in 1/4 deionized water heated to 80-90 ℃, continuously stirring for 2-3 min, cooling the solution to 30-32 ℃, pouring the gelatin solution on the surface of the compressed loofah sponge, and sending the loofah sponge into a quick freezing box to reduce the temperature of the loofah sponge to 15-20 ℃ to prepare a compressed tablet;
s5: stacking the compressed sheets in a staggered manner from top to bottom, placing the stacked compressed sheets in a temperature circulating box, controlling the temperature in the temperature circulating box to be 18-24 ℃ and circulating at a constant speed, controlling the circulating interval to be 5-6 min, and taking out the compressed sheets after circulating treatment for 10-12 min to obtain the kluyveromyces marxianus strain culture medium.
2. The kluyveromyces marxianus strain culture medium as claimed in claim 1, wherein: the watermelon pulp in the S1 is soaked in alkaline water for 40-60 min before being dried, and then is sent into a refrigeration house with the temperature of-8 to-5 ℃ for freezing treatment, wherein the freezing time is controlled to be 10-15 min; the alkaline water is 5-7% of sodium hydroxide solution.
3. The kluyveromyces marxianus strain culture medium as claimed in claim 1, wherein: the watermelon pulp in the S3 and the S4 are continuously turned in the process of rapid cooling, and the turning speed is controlled to be 35-40 r/min.
4. The kluyveromyces marxianus strain culture medium as claimed in claim 1, wherein: wherein the raw materials also comprise sputtering microbeads; the content of the sputtering microbeads accounts for 20-25% of the weight of the loofah pulp; the sputtering micro-beads are in a granular structure coated with compressed carbon dioxide; the surface of the sputtering micro-bead is composed of white granulated sugar and propolis.
5. The Kluyveromyces marxianus strain culture medium as claimed in claim 4, wherein: the manufacturing method of the sputtering micro-bead comprises the following steps:
i: mixing white granulated sugar and propolis according to a ratio of 5:1, introducing into a heating dish, controlling the temperature in the heating dish to be maintained at 95-100 ℃, continuously preserving heat and dissolving, and performing circle-drawing stirring in the process of preserving heat and dissolving, wherein the stirring speed is controlled to be 35-40 r/min;
II: pouring the syrup which is kept warm and dissolved for 10-12 min onto a marble table, filling carbon dioxide gas into the syrup, repeatedly folding and compressing the syrup until the syrup is cooled and solidified, and preparing syrup solid containing uniform and dense compressed bubbles;
III: and (3) introducing the syrup solid in the step (II) into a shearing machine, controlling the rotating speed of the shearing machine to be 60-80 r/min, shearing and crushing, introducing a crushed product into a double-filtering sieve, intercepting the crushed product with the particle size of 2-3 mm by using the double-filtering sieve to obtain sputtering microbeads, and directly and uniformly spraying the sputtering microbeads into the loofah before ventilation and cooling in S3 when the sputtering micro-beads are used.
6. The kluyveromyces marxianus strain culture medium as claimed in claim 5, wherein: wherein the carbon dioxide gas filled in the syrup is mixed with sodium bicarbonate solution; the carbon dioxide is carbon dioxide gas heated to 65-70 ℃; the carbon dioxide gas is contacted with the sodium bicarbonate solution during the flow.
7. A culture method of Kluyveromyces marxianus strain is characterized in that: the method for culturing the kluyveromyces marxianus strain is suitable for the culture medium of the kluyveromyces marxianus strain of any one of the claims 1-6; the culture method of the Kluyveromyces marxianus strain comprises the following steps:
a1: filling the prepared kluyveromyces marxianus strain culture medium into an incubator, inoculating the kluyveromyces marxianus strain on the surface of the culture medium in the incubator by using a scribing method, and placing the incubator in a thermostat for culture;
a2: controlling the temperature in the incubator to slowly rise to 20-23 ℃, carrying out constant-temperature culture for 2-3H, then controlling the temperature in the incubator to rise to 30-35 ℃, continuously carrying out heat preservation culture for 14-16H, and continuously supplementing fresh sterile air to the incubator in the culture process;
a3: and (3) using the residual culture medium and the bacterial colony in the incubator with the culture time of 16-19H for inoculating the production starter, and cooling to 0-4 ℃ after fermentation is completed to obtain the liquid starter containing a large amount of Kluyveromyces marxianus strains.
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| CN101911977A (en) * | 2008-10-24 | 2010-12-15 | 卡夫食品环球品牌有限责任公司 | Gas-effusing compositions and methods of making and using same |
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