CN110643520A - Antarctic fungus Geomyyces sp - Google Patents
Antarctic fungus Geomyyces sp Download PDFInfo
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Abstract
The invention provides a high-yield Antarctic fungus Geomycosis sp mutant strain of Antarctic purpurin and application thereof. The inventor screens a mutant strain with greatly improved purplish red pigment yield from a large number of mutant strains of Geomyces sp, and the mutant strain is named as Geomyes sp.M 210. Compared with the original strain, the strain greatly provides the yield of the purple red pigment, shortens the fermentation period to about 2/3 of the original strain, has strong temperature adaptability, and can ferment at room temperature and generate relatively high yield of the purple red pigment. The Geomyyces sp.M210 strain and the culture, metabolite, culture supernatant or lysate thereof have good industrial application prospects.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a high-yield Antarctic bacteria sp mutant strain of Antarctic bacteria of Antarctic purple red pigment and application thereof.
Background
The pigment endows food with rich and gorgeous color, not only can greatly increase the appetite of people, but also can promote the desire of people to buy, thereby being widely used in the industry. The edible pigment is obtained by natural extraction, artificial synthesis or biological production.
The naturally extracted haematochrome/purple haematochrome has rich sources, but the haematochrome is still in a state of short supply and short demand in China; and because of its complex structure, the natural edible red pigment/purple red pigment has poor stability compared with the artificial synthetic pigment, but the artificial synthetic pigment can cause great harm to human health when being improperly used. In view of the potential hazard of artificially synthesized pigments, the production of natural haematochrome/purple haematochrome with high safety by utilizing biotechnology is a more ideal choice, and under the condition of having ideal strains and ideal fermentation conditions, the natural haematochrome/purple haematochrome can be produced in a larger scale and meet the market requirements. However, the bottleneck in such techniques is that microorganisms with high production efficiency are not available, and fermentation process optimization is sometimes a challenging aspect.
The Antarctic fungus Geomyyces sp.WNF-15A is a Geotrichum fungus with high yield of the purple red pigment obtained from Antarctic soil, and compared with the physicochemical properties of other natural edible purple red pigments such as red yeast purple red pigment and the like, the purple red pigment generated by the Antarctic fungus has the huge potential of becoming a natural edible purple red pigment which is safer, low in cost and easy to obtain, and the capability of the strain for secreting the pigment is far higher than that of the same type of strain. However, although the strain is considered to be an ideal industrial production strain at present, the strain per se and the production process of the purplish red pigment still have a space for further improvement so as to meet the requirement of more efficient industrial or large-scale production; on the other hand, the Antarctic fungi is from Antarctic, so the requirement on the temperature environment is severe, the fermentation process is required to be controlled at a lower temperature such as 14 ℃, and the yield is greatly reduced at normal temperature, namely the production at the normal temperature cannot meet the industrial requirement.
In view of the above, research and development of a high-yield rhodochrous pigment strain which can be stably inherited, can tolerate a room temperature environment and has a performance of efficiently producing a rhodochrous pigment are urgently needed in the field, so that an industrial process of producing the rhodochrous pigment by an Antarctic fungus is greatly promoted.
Disclosure of Invention
The invention aims to provide a high-yield Antarctic fungus Geomyyces sp.
In the first aspect of the invention, an isolated rhodochrous pigment producing strain is provided, and the strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2019507.
In a preferred embodiment, the purplish red pigment producing strain is a strain of the genus Geomycosis sp.
In another preferred example, the purplish red pigment producing strain is obtained by mutation of a strain of Geomycosis sp; more preferably, it is obtained by mutation of Geomycosis sp.WNF-15A strain of Geomycosis. The mutation is normal pressure room temperature plasma (ARTP) mutation.
In another preferred example, the purple red pigment is a south pole purple red pigment.
In another aspect of the present invention, there is provided a cell culture, a cell metabolite, a cell culture supernatant or a cell lysate of the purplish red pigment producing strain.
In a preferred embodiment, the cell culture, cell metabolite, cell culture supernatant or cell lysate of said magenta pigment-producing strain contains or is capable of producing a magenta pigment.
In another aspect of the present invention, there is provided a use of the purplish red pigment producing strain for: (1) producing the purplish red pigment; or (2) preparing a cell culture, cell metabolite, cell culture supernatant, or cell lysate.
In another aspect of the present invention, there is provided the use of said cell culture, cell metabolite, cell culture supernatant or cell lysate for the preparation of a pigment (pigment exhibiting red color) as a food additive or an industrial additive.
In another aspect of the present invention, there is provided a composition for producing or isolating a magenta pigment, comprising an ingredient selected from the group consisting of: a purplish red pigment producing strain according to any one of the preceding claims; or a cell culture, cell metabolite, cell culture supernatant or cell lysate of any of the foregoing.
In a preferred embodiment, the composition further comprises: an industrially acceptable carrier or a microbiologically acceptable carrier.
In another aspect of the present invention, there is provided a method for producing a magenta pigment using any one of the magenta pigment-producing strains described above, comprising: the purplish red pigment producing strain was cultured under the following conditions: the temperature is 10 to 25 ℃, preferably 12 to 22 ℃, more preferably 14 to 20 ℃; a rotation speed of 50 to 300rpm, preferably 100 to 250rpm, more preferably 100 to 150 rpm; and/or, the cultivation time is 6-12 days, preferably 7-10 days, more preferably 7-9 days.
In a preferred embodiment, the method uses soluble starch, glucose, mannose or maltose as a carbon source, preferably uses soluble starch as a carbon source; preferably, the concentration of the soluble starch is 15-35 g/L; more preferably 20 to 30 g/L.
In another preferred example, in the method, peptone is used as a nitrogen source; preferably, the concentration of peptone is 1-2.5 g/L, preferably 1.5-2 g/L.
In another preferred example, the method further comprises: and (4) purifying or separating the purplish red pigment from the culture product (fermentation liquor).
In another aspect of the present invention, there is provided a kit for producing or isolating a magenta pigment, comprising: a container, and a purplish red pigment producing strain of any one of the preceding claims or a cell culture, cell metabolite, cell culture supernatant or cell lysate of any one of the preceding claims in the container.
In another preferred example, the kit further comprises instructions for use, which describe the method for culturing the rhodochrous pigment-producing strain, the method for producing a cell culture, a cell metabolite, a cell culture supernatant or a cell lysate thereof, and the like.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
FIG. 1 antagonism experiments with the Antarctic fungus Geomyyces sp. and the mutagenized strain Geomyyces sp.M210. Wherein, the right side of the antagonistic line is the starting strain Geomycins sp.WNF-15A (the culture is white with light pink background color), and the left side is the mutant strain Geomycins sp.M210 (the culture is obviously pink color).
Figure 2, liquid fermentation morphology difference of the antarctic fungus Geomyces sp.and the mutagenic strain Geomyces sp.m 210. Wherein, the left dish is an original strain, and the right dish is a mutant strain.
FIG. 3 shows the yield of the purplish red pigment fermented at 14 ℃ for 0-12 days by the Antarctic fungus Geomycosis sp and the mutant strain Geomycosis sp.M210.
FIG. 4 shows the yield of the magenta pigment obtained by fermenting the Antarctic fungus Geomycins sp and the mutagenic strain Geomycins sp.M210 at 20 ℃ for 0-12 days.
Detailed Description
The inventor screens a mutant strain with greatly improved purplish red pigment yield from a large number of mutant strains of Geomycins sp after large-scale research and screening, and the inventor names the mutant strain Geomycins p.M 210. Compared with the original strain, the strain of the invention greatly improves the yield of the purplish red pigment, shortens the fermentation period to about 2/3 time of the original strain, and can ferment at room temperature and generate relatively higher yield of the purplish red pigment. The Geomyyces sp.M210 strain and the culture, metabolite, culture supernatant or lysate thereof have good industrial application prospects.
Term(s) for
As used herein, the terms "Geomyyces sp. mutant", "Geomyyces sp.M210" or "M210 strain" are used interchangeably and refer to a strain of Geomycosis with a accession number of M2019507.
In the present invention, the term "comprising" means that various ingredients can be used together in the mixture or composition of the present invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising.
As used herein, an "industrially acceptable carrier" or a "microbiologically acceptable carrier" is a medium, solvent, suspending agent or excipient for mixing with the purplish red pigment producing strain or cell culture, cell metabolite, cell culture supernatant or cell lysate of the invention, controllable in toxicity, side effects, environmental friendliness or no harm to humans or animals. The carrier may be a liquid or a solid, and is preferably a carrier capable of maintaining the activity of the purplish red pigment producing strain or cell culture, cell metabolite, cell culture supernatant or cell lysate to a high degree.
Mutagenesis of strains
The Geomycins sp.M210 is a mutant strain obtained by mutagenesis by taking wild Geomycins sp.WNF-15A as an original strain, and is obtained by extensive screening by the inventor. The invention relates to a method for breeding a mutant strain of a Geomyyces sp-WNF-15A, which is a high-yield Antarctic purple-red pigment, by screening, wherein the strain is generated from a wild Geomyyces sp-WNF-15A, and the induced mutation breeding is carried out by adopting a normal-pressure room-temperature plasma (ARTP) induced mutation technology.
In a preferred embodiment of the invention, a mutation breeding method of the Antarctic purpurin high-yield mutant strain is provided, which comprises the following specific steps:
(1) strain activation: inoculating the self-preserved starting strain Geomyyces sp.WNF-15A to a seed liquid culture medium, culturing for 2-5 days at 14-20 ℃, then uniformly coating a proper amount of the seed liquid culture medium on a solid seed culture medium plate, and culturing for 3-5 days in an incubator at 14-20 ℃ to serve as an ARTP mutagenized starting strain.
(2) Preparation of spore suspension: washing the plate-activated 3-5 days Antarctic aspergillus with sterile water, filtering, measuring the spore concentration of the spore suspension by adopting a blood counting plate counting method, and adjusting the spore suspension concentration to 10 by adopting a gradient dilution method6～108one/mL, as spore suspension for subsequent ARTP treatment.
(3) ARTP mutagenesis treatment: uniformly coating 5-15 mu L of diluted spore liquid on the surface of a metal slide, transferring the fungus slide to an objective table by using forceps, and carrying out mutagenesis treatment after setting various parameters.
(4) And (3) culturing after mutagenesis: transferring the slide glass into an EP tube filled with normal saline after the ARTP mutagenesis treatment, shaking and eluting to form new bacterial suspension, diluting the bacterial suspension subjected to the ARTP mutagenesis treatment, coating the bacterial suspension on a seed culture medium plate, and culturing for 3-5 days at 20 ℃.
(5) Acquisition of the lethal curve: the lethality of ARTP treatment was calculated by using a flat viable count method (CFU method) to obtain a lethality curve.
(6) Primary screening by a flat plate: comparing the depth of the purple red pigment produced by single colony of the culture medium, selecting a high-yield strain to be inoculated into a seed culture medium, taking a proper amount of bacterial liquid to be coated on a flat plate, culturing for 3-5 days at 20 ℃, and selecting a strain with good growth condition, wherein the strain has deeper red compared with the original strain culture medium to be cultured for 3 days in a seed liquid culture medium.
(7) Shake flask fermentation and re-screening: and inoculating the preliminarily screened seeds into a 250mL conical flask filled with 50mL of fermentation medium, culturing for 10-12 days at the temperature of 14, 20, 25 and 30 ℃ and under the condition of 130rpm, and taking supernatant to measure the yield of the purple red pigment under the wavelength of 520 nm.
(8) Genetic stability: and (3) continuously passaging the high-yield mutant strain obtained by re-screening verification for 6 times, selecting several generations for shake flask fermentation, and verifying the genetic stability of the mutant strain.
In the screening method, the ARTP processing parameters in the step (3) are set as: the working gas is high-purity helium, the radio frequency power is 40-100W, the gas flow is 8-12 slm, the processing time is 0-300 s, and the irradiation distance is 2-4 mm.
In the screening method, the formula of the seed culture medium in the steps (1), (4), (6) and (7) is as follows: 10g/L glucose, 20g/L mannitol, 20g/L maltose, 10g/L sodium glutamate, 0.3g/L magnesium sulfate heptahydrate, 6g/L yeast extract, 0.5g/L potassium dihydrogen phosphate, and 30g/L agar added into a solid culture medium.
In the screening method, the formula of the fermentation medium in the step (7) is as follows: 28g/L of soluble starch and 1.85g/L of peptone.
In the screening method, the amount of the inoculum in the step (7) is 6% to 10%.
The mutagenized strains are cultured on a flat plate one by one, and the depth, the radius and the hypha radius of the color of the purplish red pigment are generated by comparing the bacterial colony with the wild type strain. And (3) selecting strains with good growth and large pigment radius to carry out shake flask fermentation verification by combining long-term research experience of the inventor, and finally obtaining the mutagenized strains with high-yield Antarctic purplish red pigment.
Geomymyces sp.M210 strain
The invention carries out physiological and biochemical characteristic identification on the Geomymyces sp.M210 strain: it has the following characteristics:
colony color: white;
colony morphology: the center of the bacterial colony is raised, the edge is neat, and hyphae are developed;
aerobic mode: aerobic treatment;
the suitable growth temperature is as follows: 14 to 20 ℃.
On one hand, the Geomymyces sp.M210 of the invention shows that the Geomymyces sp.M210 has obvious common points with the starting strain, including that the partial growth forms of the bacterial colonies are similar and can generate purplish red pigment and the like; however, there are some differences, including that the colony color is easier to produce light red background color than the original strain, the original strain is in pellet shape during culture, and the Geomyes sp.M210 is in velvet ball shape, so that the hyphae are more obvious and the thallus is smaller. Based on the difference, the strain of the present invention can be identified. At the same time, this difference may also prove that the strain has its own specific characteristics.
The strain of the present invention is a living cell, and once the strain of the present invention, Geomymyces sp.M210, is obtained, the strain can be obtained in large quantities by means of inoculation, passaging, regeneration, and the like. This is usually a method of obtaining the living cells of the present invention by inoculating them into a solid plate medium or a liquid medium to perform scale-up culture of the strain. The obtained living cells can be further subjected to laboratory domestication, genetic breeding, molecular genetic manipulation and the like to obtain mutants and transformants. Furthermore, the strains of the invention can also be used as bioengineered host cells for heterologous expression.
Furthermore, the Geomyes sp.M210 strain with high purplish red pigment yield can be used as an initial strain, and is further improved by means of laboratory domestication, genetic breeding, molecular genetic manipulation and the like to obtain a derivative strain with higher yield or more optimized enzyme system. Further screening of the optimized strains by these manual manipulations, using the Geomyes sp.M210 strain of the present invention as the starting strain, should also be included in the overall scope of the present invention.
Methods well known to those skilled in the art can be used to further mutagenize the live strains of the present invention to cause changes in gene coding, enzymatic activity properties, and morphology of the live cells. These methods include physical methods using radiation, particles, laser, ultraviolet light, etc., and chemical mutagenesis methods using alkylating agents, base analogues (base analogues), hydroxylamines (hydroxylamines), acridine pigments, etc. The mutagenesis may be a multiple-generation mutagenesis of the above method or methods and is not limited to these methods. Based on the strain provided by the invention, breeding can be further carried out in a physical and chemical mode, a new purpurin regulatory gene and other related regulatory genes can also be introduced, the enzyme production performance of the obtained mutant and transformant can be further improved, and the breeding method is one or more than one combination.
Methods well known to those skilled in the art can be used to construct expression constructs (vectors) and further engineer the strains of the invention. For example, further improvements (e.g., increased expression of beneficial factors, decreased expression of deleterious factors) have been made in the signaling pathways, and proteins involved in purpurin production that have been or are newly discovered in the strain.
Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. The procedures used are well known in the art.
Cell cultures, cell metabolites, cell culture supernatants or cell lysates
On the basis of obtaining the Geomyyces sp.M210 strain, the invention also provides a cell culture, a cell metabolite, a cell culture supernatant or a cell lysate of the purplish red pigment production strain, which has the following characteristics: contains purple red pigment.
After obtaining the strain of the present invention, one skilled in the art can conveniently obtain a culture thereof, for example, by referring to some of the media or culture processes provided in the specific examples of the present invention, or by using media or culture processes which have been appropriately changed from the examples of the present invention but can also obtain a culture, thereby obtaining a cell culture. The cell culture contains active strains, thereby producing the purplish red pigment.
The cell metabolite is a substance produced or secreted by the strain of the invention in the culture process, and can be directly secreted into a culture medium by cells or separated from the cells after certain treatment. The cell product may be isolated, purified or concentrated.
The cell culture supernatant is a culture solution which is remained after removing cells and solid impurities during or after the process of culturing the strain of the present invention, and may be unconcentrated or concentrated. Typically, cells as well as solid impurities can be removed by means such as centrifugation, filtration, and the like.
The cell lysate is a mixture formed by lysing cells with a cell lysis reagent during or after the culture of the strain of the present invention. The cell lysate may be the product after lysis with solid impurities removed. It may be a purified or concentrated product, as desired.
Application and production process
The invention also provides an application of the purple red pigment production strain, which is used for: producing the purplish red pigment or used for preparing the cell culture, the cell metabolite, the cell culture supernatant or the cell lysate; further, the magenta pigment, cell culture, culture supernatant or metabolite, etc. can be applied to the food industry.
In order to better apply the Geomyyces sp.M210 strain for producing the purple red pigment, the invention provides a production process thereof, which comprises various culture conditions, various components of a culture medium and the like.
Various carbon sources can be used for culturing the strain of the present invention, including soluble starch, glucose, mannose, maltose, etc., and in some preferred embodiments of the present invention, the carbon source is soluble starch. In a further preferable mode, the concentration of the soluble starch is 15-35 g/L; more preferably 20 to 30 g/L.
Various nitrogen sources can be used for culturing the strain of the present invention, and peptone is used as a nitrogen source in a preferred embodiment of the present invention. In a further preferred mode, the concentration of peptone is 1-2.5 g/L, preferably 1.5-2 g/L.
The present inventors found that the culture time of the strain was not as long as possible but fluctuated. The fermentation period of the variant strain Geomyyces sp.M210 is obviously shorter than that of the original strain. Therefore, the culture time of the variant strain Geomyessp.M210 of the present invention may be 6 to 12 days, but is more preferably 7 to 10 days, and still more preferably 7 to 9 days.
The inventors found that the strain of the present invention can be adapted to a relatively wide culture temperature, and thus, as a preferred embodiment of the present invention, the fermentation temperature is 10 to 25 ℃, preferably 12 to 22 ℃, and more preferably 14 to 20 ℃. The original strain can not tolerate the room temperature or the high temperature, the yield of the purple red pigment is very low under the condition of the room temperature of 20 ℃, and the strain of the invention has great improvement on the yield, and the higher yield of the purple red pigment which can meet the requirement of industrial production can be obtained under the condition of the room temperature of 20 ℃.
The Geomymyces sp.M210 strain of the invention can realize long-term growth and stable passage. The medium and the culture method applied to culture the Geomyyces sp.M210 strain of the present invention are not limited to those disclosed above and in the examples, and other media and culture methods conventionally applied to culture a bacterium of the genus Geotrichum may also be applied to the present invention. It will be appreciated that the various medium components described above may be replaced by other components having similar functions, and that in different cases it may be appropriate to add other components or to remove or change the content of some of them depending on the characteristics of the particular strain.
The culture system or the fermentation system of the invention can be enlarged for industrial production, and according to the size of the system, the technicians in the field can make proper adjustment according to the general knowledge grasped so as to be beneficial to the growth or production of the strain.
The invention also provides a method for preparing the food/feed/industrial product composition, which comprises the steps of producing the purple red pigment by the Geomyes sp.M210 strain of the invention, separating and purifying the purple red pigment, and adding the separated and purified purple red pigment into the food/feed/industrial product needing hyperchromic treatment to form the food/feed/industrial product composition containing the purple red pigment. The cell culture, cell metabolite, cell culture supernatant or cell lysate of the purplish red pigment production strain can also be used as a pigment additive to be added into the food/industrial product composition after proper processing, purification and nontoxic treatment.
Under the condition of shake flask fermentation, the OD of the purplish red pigment yield of the strain Geomycins sp.M210 can reach 50.5 in 7-9 days of fermentation, and is improved by 95.7% compared with the OD 25.8 of the initial strain yield in the same time of fermentation, and is improved by 24.4% compared with the maximum OD 40.6 of the initial strain yield in 12 days of fermentation. Meanwhile, the fermentation period is obviously shortened by 4 days. At the normal temperature of 20 ℃, the yield of the Geomycins sp.M210 purple pigment is more than 4 times of that of the original strain, and the Geomycins sp.M210 purple pigment has the normal-temperature fermentation capability which is not possessed by the original strain.
Therefore, the yield of the purple red pigment of the strain Geomymyces sp.M210 is greatly higher than that of the original strain, the production period is obviously shorter than that of the original strain, the production cost of the purple red pigment is greatly reduced, and the strain has wider industrial application value compared with the original strain.
The various culture or assay conditions described in the summary of the invention may be combined or modified according to common knowledge in the art. In order that the invention may be better understood, the invention will now be further described with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.
Example 1 atmospheric pressure Room temperature plasma mutagenesis of Antarctic fungi
In this example, the starting strain was subjected to atmospheric room temperature plasma mutagenesis, and the specific steps were as follows:
1. activation of bacterial strains
Geomycins sp.WNF-15A (Geomycins sp.WNF-15A, obtained from the first Marine institute of Natural resources) was inoculated into a seed liquid medium, cultured at 20 ℃ for 3 days, then applied uniformly to a solid seed medium plate in an appropriate amount, cultured in a 20 ℃ incubator for 3 days, and then used as an ARTP-mutagenized starting strain.
2. Preparation of spore suspension
Washing the plate-activated 3-day-old Antarctic Aspergillus with sterile normal saline, filtering, measuring spore concentration of spore suspension by counting with blood count plate, and adjusting spore suspension concentration to 10 by gradient dilution7one/mL, as spore suspension for subsequent ARTP treatment.
3. ARTP mutagenesis treatment
And (3) uniformly coating 10 mu L of diluted spore liquid on the surface of a metal slide, transferring the fungus slide to an objective table by using forceps, and performing mutagenesis treatment after setting various parameters. Wherein, the mutagenesis conditions are as follows: the working gas is high-purity helium, the radio frequency power is 100W, the gas flow is 10slm, the processing time is 0, 30, 60, 75, 90, 105, 120, 150, 180, 210, 240 and 300s, and the irradiation distance is 2 mm.
4. Post-mutagenesis culture
Transferring the slide glass into an EP tube filled with 0.99mL of physiological saline after the ARTP mutagenesis treatment, shaking and eluting to form new bacterial suspension, diluting the bacterial suspension subjected to the ARTP mutagenesis treatment, coating the bacterial suspension on a seed culture medium plate, and culturing for 3-5 days at 20 ℃.
5. Acquisition of a lethal Curve
The lethality of ARTP treatment was calculated by using a flat viable count method (CFU method) to obtain a lethality curve.
Example 2 screening and identification of high producing strains
After mutagenesis, the mutagenized strains are screened and identified in order to obtain high-producing strains therefrom.
1. Preliminary screening
Comparing the depth of the purple red pigment produced by single colony of the culture medium, selecting a high-yield strain to be inoculated into a seed culture medium, taking a proper amount of bacterial liquid to be coated on a flat plate, culturing for 3-5 days at 20 ℃, and selecting a strain with good growth condition, wherein the strain has deeper red compared with the original strain culture medium to be cultured for 3 days in a seed liquid culture medium.
In the screening method of the strain, the formula of the seed culture medium is as follows: 10g/L glucose, 20g/L mannitol, 20g/L maltose, 10g/L sodium glutamate, 0.3g/L magnesium sulfate heptahydrate, 6g/L yeast extract, 0.5g/L potassium dihydrogen phosphate, and 30g/L agar added into a solid culture medium.
2. Shaking flask fermentation double screen
The seeds obtained by primary screening are inoculated into a 250mL conical flask containing 50mL of fermentation medium, and cultured for 12 days at the temperature of 14 ℃ and 20 ℃ and at the speed of 130rpm respectively, and the supernatant is taken to measure the yield of the purplish red pigment.
In the shake flask fermentation re-screening method, the formula of the fermentation medium is as follows: 28g/L of soluble starch and 1.85g/L of peptone. Preparing a fermentation culture medium: accurately weighing 28g of soluble starch, dissolving and stirring the soluble starch into paste by using a small amount of deionized water, pouring the paste into boiling hot water for gelatinization, accurately weighing 1.85g of peptone after cooling to room temperature, adding the peptone into the gelatinized starch solution for dissolution, and fixing the volume of the culture medium to 1000mL after the peptone is completely dissolved.
The fermentation culture method comprises the following steps: digging a fungus block with the diameter of about 1cm on a fresh flat plate, evenly dividing the fungus block into four small blocks, inoculating into a seed culture medium, culturing in a shaking table with the temperature of 20 ℃ and the speed of 130r/min for 3 days, inoculating into the same fresh seed culture medium according to the inoculum size of 10 percent, culturing in a shaking table with the temperature of 20 ℃ and the speed of 130r/min for 36 hours, inoculating into a fermentation culture medium according to the inoculum size of 6 percent, and culturing in a shaking table with the temperature of 14 ℃ and the temperature of 20 ℃ and the speed of 130r/min for 8-14 days.
Determination of the yield of Antarctic magenta pigment: taking 1mL of the fermentation liquid prepared in the previous step, centrifuging, taking the supernatant, diluting by a proper time, measuring the OD value at 520nm of a spectrophotometer,
3. identification of high-yielding strain
After a plurality of times, through large-scale screening and comprehensive comparison, the inventor obtains a mutant strain with high yield of the fermented purple red pigment and ideal growth performance, which is named as Geomyes sp.M 210.
Example 3 morphological characterization of Geomymyces sp.M210 Strain
In this example, the differences between the Geomyces sp.m210 strain and the original starting strain were identified by antagonism experiments and strain morphology.
1. Physiological and biochemical characteristics of Geomyyces sp.M210 strain
The main physiological and biochemical characteristics of the strain Geomyces sp.m210 include the following:
colony color: white;
colony morphology: the center of the bacterial colony is raised, the edge is neat, and hyphae are developed;
aerobic mode: aerobic treatment;
the temperature adaptability is strong, and the growth temperature is more suitable: 14 to 20 ℃.
2. Antagonism of mutant strains
Inoculating the obtained mutant strain Geomyyces sp.M210 and the original strain into a proper amount of seed culture medium for activation, dipping a small amount of the activated mutant strain Geomyyces sp.M210 and the original strain into a solid plate of the seed culture medium by using a gun head, pouring the activated mutant strain Geomyyces sp.M210 and the original strain onto the solid plate of the seed culture medium at an interval of about 1cm, and inversely placing the activated mutant strain Geomyyces sp. The growth of both strains and whether antagonism was formed were observed.
As a result, as shown in fig. 1, a distinct antagonistic line was formed between the mutant strain Geomyces sp.m210 and the original strain, indicating that the mutant Geomyces sp.m210 is a novel strain that is changed from the original strain.
Observing the growth of the strain in the figure 1, two sides of an antagonistic line can be seen, and a culture of the starting strain Geomyes sp.WNF-15A on the right side is white and slightly provided with light pink ground color; whereas the left mutagenic strain, Geomymyces sp.M210, exhibited a distinct pink color. The apparent color difference that can be measured visually indicates that the mutagenized strain Geomyes sp.M210 has high production activity and stronger purplish red pigment production capacity than the original strain.
3. Comparison of mutant liquid fermentation forms
The mutant strain Geomyyces sp.M210 and an original strain are dug and inoculated into a proper amount of seed culture medium, after being cultured in a shaker at 20 ℃ and 130rpm for 3 days, the second-level seed culture medium is inoculated according to the inoculation amount of 10 percent, after being cultured for 36 hours under the same condition, the fermentation culture medium is inoculated, the fermentation culture is carried out in the shaker at 14 ℃ and 130rpm, samples are taken every 24 hours, and the morphological difference of the two strains is compared.
The results are shown in FIG. 2, with the left dish being the starting strain and the right dish being the mutagenized strain. The starting strain is in the shape of pellet, while the mutagenic strain M210 is in the shape of downy ball, so that hyphae are more obvious and thalli are smaller. Therefore, the two strains have obvious difference in morphology in the liquid fermentation process.
Fig. 2 also shows that the mutant strain Geomyces sp.m210 and the original strain both produce the purplish red pigment, which is uniformly dispersed in the culture dish and has the same color.
Example 4 fermentation culture of Antarctic fungus mutant
The yield of the rhodopsin of the Antarctic fungus mutant strain Geomyyces sp.M210 is identified and compared with that of the starting strain. The fermentation medium and the cultivation process were carried out as described in "2, shaking flask fermentation rescreening" in example 1, on a shaker at 14 ℃ and 20 ℃ at 130 r/min.
1. Fermentation at 14 deg.C
The result of the mutant strain Geomycins sp.M210 cultured at 14 ℃ is shown in FIG. 3, and from the beginning of fermentation, the yield of the purplish red pigment expressed by the mutant strain Geomycins sp.M210 is obviously higher than that of the original strain (Wild type), the yield is the highest on day 8, the OD value reaches 50.5, and the OD value is improved by about 24.4% compared with the highest OD value of 40.6 of the original strain.
Meanwhile, from the fermentation period of shake flask culture, the mutant strain Geomycins sp.M210 can reach the highest peak of the yield of the purplish red pigment only by fermenting for 8 days, while the starting strain reaches relatively high value on day 12, but the high value of the starting strain on day 12 is also lower than the value of the mutant strain Geomycins sp.M210 on day 8. Therefore, the fermentation period of the mutant strain Geomyyces sp.M210 is obviously shortened by 4 days compared with that of the original strain.
2. Fermentation at 20 deg.C
As shown in fig. 4, the result of culturing the mutant strain Geomyces sp.m210 at 20 ℃ at room temperature and 20 ℃ is shown in fig. 4, and from the beginning of fermentation, the yield of the purplish red pigment expressed by the mutant strain Geomyces sp.m210 is remarkably higher than that of the original strain (Wild type), and the yield is stable and is higher in days 7-10, the OD value reaches 32.5, the OD of the original strain only reaches 2.3, the yield of the original strain reaches 14 times or more in the same period, and the highest yield of the original strain is also exceeded, namely the OD is 7.8 (day 12), and reaches 4 times or more of the yield of the original strain.
From the fermentation temperature, the mutant strain Geomyyces sp.M210 can be fermented and cultured at 20 ℃, and the yield of the purplish red pigment of the original strain is very low at normal temperature. Therefore, the mutant strain Geomyyces sp.M210 of the present invention has the ability to ferment at room temperature.
Preservation of biological materials
The strain Geomymyces sp.M210 of the invention is preserved in China center for type culture Collection (Wuhan, Wuhan university, China), and the preservation date is as follows: and 7, 7 and 2 days in 2019, the preservation number is CCTCC NO: M2019507.
It should be understood that the above-described embodiments of the present invention and the technical principles applied thereto are described, and after reading the above teaching of the present invention, those skilled in the art can make various changes or modifications to the present invention without departing from the spirit and scope of the present invention, and these equivalents also fall within the scope of the present invention.
Claims (10)
1. An isolated purplish red pigment producing strain, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2019507.
2. The magenta pigment-producing strain according to claim 1, which is a strain of the genus Geomycosis (Geomycosis sp.); preferably, the strain is obtained by mutation of a strain of the genus Geomycosis (Geomycosis sp.); more preferably, it is obtained by mutation of Geomyes sp.WNF-15A.
3. A cell culture, a cell metabolite, a cell culture supernatant or a cell lysate of the magenta pigment-producing strain according to any one of claims 1 to 2.
4. The cell culture, cell metabolite, cell culture supernatant or cell lysate of a purplish red pigment producing strain according to claim 3, comprising purplish red pigment, or capable of producing purplish red pigment.
5. Use of the magenta pigment-producing strain according to any one of claims 1 to 2 for:
(1) producing the purplish red pigment; or
(2) Preparing a cell culture, a cell metabolite, a cell culture supernatant or a cell lysate.
6. Use of the cell culture, cell metabolite, cell culture supernatant or cell lysate according to claim 3 or 4 for the preparation of a pigment, as a food additive or as an industrial additive.
7. A composition for producing or separating a magenta pigment comprising an ingredient selected from the group consisting of:
the magenta pigment-producing strain according to any one of claims 1 to 2; or
A cell culture, cell metabolite, cell culture supernatant or cell lysate according to any one of claims 3 to 4.
8. A method for producing a purple red pigment by using the purple red pigment production strain of any one of claims 1 to 2, comprising the following steps: the purplish red pigment producing strain was cultured under the following conditions:
the temperature is 10 to 25 ℃, preferably 12 to 22 ℃, more preferably 14 to 20 ℃;
a rotation speed of 50 to 300rpm, preferably 100 to 250rpm, more preferably 100 to 150 rpm;
the cultivation time is 6-12 days, preferably 7-10 days, more preferably 7-9 days.
9. The method of claim 8, wherein soluble starch, glucose, mannose or maltose is used as the carbon source, preferably soluble starch is used as the carbon source; preferably, the concentration of the soluble starch is 15-35 g/L; more preferably 20 to 30 g/L;
peptone is used as a nitrogen source; preferably, the concentration of peptone is 1-2.5 g/L, preferably 1.5-2 g/L.
10. A kit for producing or separating a magenta pigment, comprising: a container, and the magenta pigment-producing strain according to any one of claims 1 to 2 or the cell culture, cell metabolite, cell culture supernatant or cell lysate according to any one of claims 3 to 4 in the container.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116637112A (en) * | 2023-06-09 | 2023-08-25 | 临沂大学 | Application of antarctic red pigment in preparing medicine for treating and preventing hyperuricemia |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104804007A (en) * | 2015-03-14 | 2015-07-29 | 王能飞 | Geomyces sp.wnf-15A(phy) coloring compound |
| WO2016025572A1 (en) * | 2014-08-12 | 2016-02-18 | Georgia State University Research Foundation, Inc. | Volatile organic compounds for inhibiting fungal growth |
| WO2016066896A1 (en) * | 2014-10-27 | 2016-05-06 | Ab Enzymes Oy | Fungal endoglucanase variants, their production and use |
| CN110257253A (en) * | 2019-03-12 | 2019-09-20 | 方龙 | The mould mutant strain of a kind of ground silk and its application |
-
2019
- 2019-10-29 CN CN201911035781.7A patent/CN110643520A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016025572A1 (en) * | 2014-08-12 | 2016-02-18 | Georgia State University Research Foundation, Inc. | Volatile organic compounds for inhibiting fungal growth |
| WO2016066896A1 (en) * | 2014-10-27 | 2016-05-06 | Ab Enzymes Oy | Fungal endoglucanase variants, their production and use |
| CN104804007A (en) * | 2015-03-14 | 2015-07-29 | 王能飞 | Geomyces sp.wnf-15A(phy) coloring compound |
| CN110257253A (en) * | 2019-03-12 | 2019-09-20 | 方龙 | The mould mutant strain of a kind of ground silk and its application |
Non-Patent Citations (4)
| Title |
|---|
| CHEN WH等: "Morphological traits and molecular analysis for Geomyces fujianensis sp. nov. from China", 《MYCOSPHERE》 * |
| 孙云飞: "一株产红色素南极真菌的鉴定及其分泌色素的性质与结构研究", 《中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑》 * |
| 王风芹等: "1株南极真菌的鉴定及其分泌色素性质分析", 《中国食品学报》 * |
| 董龙龙: "南极真菌Geomyces sp.WNF-15A产红色素的发酵条件优化及诱变研究", 《中国优秀硕士学位论文全文数据库（电子期刊）工程科技Ⅰ辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116637112A (en) * | 2023-06-09 | 2023-08-25 | 临沂大学 | Application of antarctic red pigment in preparing medicine for treating and preventing hyperuricemia |
| CN116637112B (en) * | 2023-06-09 | 2023-11-03 | 临沂大学 | Application of antarctic red pigment in preparing medicine for treating and preventing hyperuricemia |
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