CN110643519A - Antarctic fungus mutant strain with high purplish red pigment yield, breeding method and application thereof - Google Patents

Antarctic fungus mutant strain with high purplish red pigment yield, breeding method and application thereof Download PDF

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CN110643519A
CN110643519A CN201911035568.6A CN201911035568A CN110643519A CN 110643519 A CN110643519 A CN 110643519A CN 201911035568 A CN201911035568 A CN 201911035568A CN 110643519 A CN110643519 A CN 110643519A
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red pigment
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蔡孟浩
黄河舟
丁璐璐
张元兴
王能飞
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East China University of Science and Technology
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Abstract

The invention provides a rhodochrous pigment high-yield Antarctic fungus mutant strain, a breeding method and application thereof. The inventor screens a mutant strain with purplish red pigment yield greatly improved compared with the original strain from a large number of mutant strains of Geomyces sp.M300. 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 50 percent of the original strain, and has obviously higher capability of producing the purplish red pigment by fermenting at room temperature than the original strain. The Geomyyces sp.M300 strain and the culture, metabolite, culture supernatant or lysate thereof have very ideal industrial application value.

Description

Antarctic fungus mutant strain with high purplish red pigment yield, breeding method and application thereof
Technical Field
The invention belongs to the technical field of microbial strain breeding, and particularly relates to a purple-red pigment high-yield Antarctic fungus mutant strain, a breeding method and application thereof.
Background
Pigments have an indispensable role in life, and the position of pigments in the industrial fields such as chemistry and food additives is also important. At present, most of the pigments approved to be produced by the country are basically natural pigments of plant sources. However, the raw material planting of the pigment is affected by climate, region, scale and cost, the processing technology of the pigment often causes differences among product batches, and the natural pigment from the plant has generally poor stability and weak tolerance to acid-base environment, oxidation, temperature, insolation and the like. In addition to the gradual rise of 'green economy', microbial pigments enter the sight of people. Fungi, on the other hand, can produce pigments of a large variety of colors, making fungal pigments superior to microbial pigments.
Fungi have been utilized in the art to produce pigments. The fungal pigments are numerous and numerous, and at present, more than 600 fungal pigments are recorded in the book, but only a few fungal pigments actually realizing industrial scale production, such as Monascus flavin (Ankaflavin), Monascorubramin (Monascorubramin), erythemin (Rubropunctatin), Anthraquinone pigments (Anthraquinone) produced by Penicillium oxyalicum, Riboflavin (Riboflavin) produced by Ashbya gossypii, beta-carotene (beta-carone) produced by Blakeslea trispora, and the like. At present, monascus pigment from monascus, namely polyketides, is the most commonly used pigment in asian regions, but researches show that monascus produces Citrinin (Citrinin), and the toxin can produce certain toxic effects on human animals, so that the monascus produced purpurin is limited by host pathogenicity in application.
The Geomyyces sp.WNF-15A is an excellent strain capable of secreting a large amount of purple red pigment, the color value and the stability of the pigment in all aspects are superior to those of monascus pigment, and the pigment is non-toxic and harmless to human bodies, so that the Geomyyces sp.WNF-15A is an industrial strain with great potential. However, the yield of the purplish red pigment of Geomymyces sp.WNF-15A strain is difficult to improve and far from the industrial requirement. Another disadvantage of this strain in industrial applications is that it requires low temperature cultivation, which not only increases the difficulty of the fermentation operation but also consumes energy, which to some extent limits the possibility of further development and utilization of the strain.
Therefore, the search for new purple red pigment substitutes is urgent by obtaining mutant strains with high yield and adaptive to production temperature by effective means.
Disclosure of Invention
The invention aims to overcome the defects of the existing original strain and provide a mutagenic strain of the extracellular mauve pigment high-yield Antarctic fungus Geomyyces sp.WNF-15A; the invention also aims to provide a breeding method of the geotrichum strain with high extracellular purplish red pigment yield; the invention also aims to provide application of the mutant strain of the extracellular mauve pigment high-yield Antarctic fungus Geomyyces sp.WNF-15A.
In the first aspect of the invention, an isolated purplish red pigment producing strain is provided, and the strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2019508.
In a preferred embodiment, the strain is a strain of the genus geotrichum (Geomyces 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.
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, a cell metabolite, a cell culture supernatant or a 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 (a pigment exhibiting a purple-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 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 25 ℃, more preferably 14 to 20 ℃;
a rotation speed of 50 to 180rpm, preferably 100 to 160rpm, more preferably 120 to 140 rpm;
the cultivation time is 5 to 12 days, preferably 6 to 10 days, more preferably 6 to 7 days;
carrying out aerobic fermentation.
In a preferred embodiment, the method uses starch, glucose, mannose or maltose as a carbon source, preferably uses starch as the carbon source; preferably, the concentration of the starch is 15-35 g/L; more preferably 20 to 30 g/L.
In another preferred example, the method uses peptone as nitrogen source; preferably, the concentration of peptone is 1-2.5 g/L, preferably 1.5-2 g/L.
In another preferred example, the purple red pigment producing strain further comprises, before being inoculated into a fermentation medium: the strain is activated and expanded in advance, and then inoculated into a fermentation medium.
In another preferred example, the method further comprises: purifying or separating the purplish red pigment from the culture product (fermentation liquor); preferably, the method comprises: and (4) carrying out solid-liquid separation on the fermentation liquor after fermentation is finished, and taking liquid to obtain the extracellular water-soluble mauve pigment.
In another preferred embodiment, the solid-liquid separation method is centrifugation.
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.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
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FIG. 1, left panel, is a photograph of a seed plate of a mutagenized strain M300 of Geomyes sp.WNF-15A cultured at 20 ℃ for 7 days; the right panel shows the colony antagonistic morphology of mutant strain M300 and the starting strain of Geomyyces sp.WNF-15A.
FIG. 2 is a diagram showing the results of detecting conventional fermentation extracellular purple red pigment of mutant strains and original strains at different temperatures.
FIG. 3 is a graph showing the results of fermentation of mutagenized strain M300 of Geomyes sp.WNF-15A and the starting strain at different temperatures for 12 days.
Detailed Description
The inventor screens a mutant strain with purplish red pigment yield greatly improved compared with the original strain from a large number of mutant strains of Geomyces sp through large-scale research and screening, and the inventor names the mutant strain as Geomyes sp.M300. 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 50 percent of the original strain, and has obviously higher capability of producing the purplish red pigment by fermenting at room temperature than the original strain. The Geomyyces sp.M300 strain and the culture, metabolite, culture supernatant or lysate thereof have very ideal industrial application value.
Term(s) for
As used herein, the terms "mutant strain Geomycins sp.M300", "Geomycins sp.mutant strain", "Geomycins sp.M300", "purplish red pigment producing strain Geomycins sp.M300" or "M300 strain" are used interchangeably and refer to a strain of Geomycinia with a accession number of CCTCC NO: M2019508.
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 red pigment-producing strain or cell culture, cell metabolite, cell culture supernatant or cell lysate of the present invention, which is controllable in toxicity, side effects, environment-friendly or harmless to humans and animals. The carrier may be a liquid or a solid, and is preferably a carrier capable of maintaining the activity of the red pigment-producing strain or cell culture, cell metabolite, cell culture supernatant or cell lysate to a high degree.
Mutagenesis of strains
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. Therefore, the Geomycins sp.M300 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.
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) spore suspension preparation: inoculating the starting strain on a seed solid culture medium, culturing for 4-5 days at 15-20 ℃, washing a strain plate by using sterile normal saline to obtain a suspension containing spores, and properly diluting according to the growth condition; in a preferred mode, the concentration of spores in the spore-containing suspension is preferably 107An order of magnitude of one/mL; in a preferred mode, the composition of the seed culture medium is as follows: 1g of glucose, 2g of mannitol, 2g of maltose, 1g of sodium glutamate, 0.6g of yeast extract and MgSO4·7H2O 0.03g，KH2PO40.05g of agar powder and 3g of agar powder, and the volume is determined to be 100mL by using distilled waterH is natural.
(2) ARTP mutagenesis: placing the suspension containing the spores obtained in the step (1) into an ARTP mutagen according to an operation procedure, and carrying out mutagenesis on the strain by treating for a certain time under the conditions of 80-120W of power, 5-10L/min of air flow and 1-3 mm of distance between a plasma emission source and a sample;
(3) separating and screening a flat plate: then diluting the suspension liquid after mutagenesis in the step (2) for a certain gradient, coating the suspension liquid on a seed plate, carrying out constant-temperature dark inversion culture at 15-20 ℃, after the bacterial colony grows out, selecting a positive mutant strain with good bacterial colony morphology and larger pigment circle diameter, carrying out single bacterial colony streaking separation, and repeatedly separating and purifying to obtain a purer mutagenic strain; in a preferred embodiment, the colony morphology includes colony size, color, and hyphae; the purification operation is as follows: streaking colonies on a seed plate for culture until the morphologies of the colonies streaked from the same colony are similar;
(4) fermentation and breeding: preparation of seed liquid: inoculating the mutagenic strain obtained after purification in the step (3) into a sterilized seed culture medium for culture and proliferation, digging and inoculating 1.7-2 cm-diameter seed flat plate bacterium blocks for 4-7 days in culture in each 50mL of the seed culture medium, and culturing at 130-200 rpm for 2-3 days to ensure that the seed culture medium has mature spores, thereby obtaining a primary seed solution; inoculating the second-level seeds with the inoculation amount of 5-10% by volume, and culturing at 130-200 rpm for 24-36 h to obtain a second-level seed solution with high activity; ② fermentation culture: inoculating the seed solution into 50mL of fermentation medium according to the inoculation amount (dry weight of the thallus) of 6-10 mg, performing fermentation culture for 8-9 days, centrifuging to obtain a supernatant containing extracellular purple red pigment, and detecting the yield of the extracellular pigment to obtain a high-yield purple red pigment Antarctic fungus Geomyyces sp.WNF-15A mutant strain; in a preferred mode, the composition of the seed culture medium is as follows: 1g of glucose, 2g of mannitol, 2g of maltose, 1g of sodium glutamate, 0.6g of yeast extract, 0.03g of MgSO4 & 7H2O 0.03 and KH2PO40.05g, wherein the volume is fixed to 100mL by using distilled water, and the pH is natural; in a preferred mode, the composition of the fermentation medium is: 2.8g of starch and 0.185g of peptone, and distilled water is used for fixing the volume to 100mL, and the pH value is natural.
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.M300 strain
The Geomyyces sp.M300 and the starting strain have obvious common points in morphology, and can generate the purplish red pigment. The invention identifies the morphological characteristics of the Geomyyces sp.M300 strain, which mainly comprises the following characteristics: culturing on a seed flat plate for 7 days, wherein the colony shape is large, the diameter of the colony is 15-16 mm, the colony is milky white, and the back surface is tawny; the colony edge is complete and round; in the later growth stage, radial cracks are often accompanied, white villous aerial hyphae grow, and the aerial hyphae are compact; the hypha in the medium is sparse, and the purple pigment is seeped out of the medium. The hyphae are transparent, thin-walled and provided with a partition; the spores are spherical or ellipsoidal.
The strain of the present invention is a living cell, and once the strain of the present invention, Geomymyces sp.M300, 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.M300 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 with the Geomyes sp.M300 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.
The strain Geomyyces sp.M300 according to the invention is particularly suitable for the production of purplish red pigment, in a preferred embodiment of the invention, the production method comprises:
I. inoculating the Geomymyces sp.M300 strain into a fermentation medium, and performing aerobic fermentation to enable the mutagenized strain M300 to be in a logarithmic growth phase or a stationary phase;
II. And carrying out solid-liquid separation on the fermented fermentation liquor, and taking the liquid to obtain the extracellular water-soluble purple red pigment.
In a preferred embodiment, the composition of the basic fermentation medium described in step (I) is as follows: 2.8g of starch and 0.185g of peptone, and distilled water is used for fixing the volume to 100mL, and the pH value is natural.
In a preferred embodiment, the strain of Geomycins sp.M300 described in step (I) is activated and expanded when inoculated into a fermentation medium, and then inoculated into the fermentation medium.
The activation is carried out by conventional means, such as plating the mutagenized species on a plate for culture.
The amplification culture is a conventional means, and a preferred operation method comprises the following steps: inoculating activated mutagenic strains into a seed culture medium from a flat plate for culture and proliferation, digging every 50mL of the seed culture medium to inoculate seed flat plate bacterium blocks with the diameter of 1.7-2 cm and cultured for 4-7 days, and culturing at 14-25 ℃ and 100-200 rpm for 2-3 days to ensure that the seed culture medium has mature spores to obtain a primary seed solution; inoculating the second-level seeds with the inoculation amount of 5-10% by volume, and culturing at 130-200 rpm for 24-36 h to obtain the second-level seed liquid with high activity.
The step of the scale-up culture is more preferably as follows: inoculating the activated mutagenic strain on a flat plate into a seed culture medium for culture and proliferation, digging and inoculating a seed flat plate with the diameter of 1.7cm and cultured for 6 days into each 50mL of the seed culture medium, and culturing at 20 ℃ and 130rpm for 3 days to ensure that the seed culture medium has mature spores to obtain a primary seed solution; inoculating the second-level seeds with the inoculation amount of 6 percent by volume, and culturing for 36h at 130rpm to obtain the second-level seed liquid with high activity.
The inventor verifies the yield and the production period of the purplish red pigment through liquid fermentation culture and compares the purplish red pigment with the original strain. In the fermentation culture process at 14 ℃, the yield of the extracellular purple pigment of the Geomyyces sp.M300 strain is improved by 43.73 percent compared with that of the original strain. The yield of the extracellular purple red pigment of the strain is improved by 86.67 percent compared with the original strain when the strain is cultured under the condition (20 ℃) of exceeding the optimal production temperature (14 ℃). The fermentation period of the mutant strain is shortened by 50 percent compared with the original strain by culturing at the optimum production temperature.
The inventors also determined the strain Geomyces sp.m300 and compared it with the starting strain. The dry weight of the fermented thallus under different conditions is cultured at 14 ℃, and the growth (dry weight) of the mutant strain is improved by 5.28 percent compared with the growth (dry weight) of the original strain; the growth (dry weight) of the mutant strain is improved by 33.3 percent compared with the original strain when the mutant strain is cultured at the temperature of 20 ℃; when cultured at 30 ℃, the growth (dry weight) of the mutant strain is improved by 57.68 percent compared with the original strain.
The yield of the purple red pigment of the strain Geomymyces sp.M300 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 more ideal functions compared with the original strain. Therefore, the Geomyyces sp.M300 strain can be applied to producing extracellular purple red pigment, is beneficial to large-scale industrial production, and has important significance for the research of an extreme environment adaptation mechanism of Antarctic fungi and the safe and nontoxic industrial production application of the purple red pigment.
Cell cultures, cell metabolites, cell culture supernatants or cell lysates
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 on the basis of obtaining the Geomyes sp.M300 strain: 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 bacterial strain Geomyes sp.M300 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 starch, glucose, mannose, maltose, etc., and in some preferred embodiments of the present invention, the carbon source is starch. In a further preferable mode, the concentration of the 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 variant strain Geomyyces sp.M300 has a fermentation period which is obviously shorter than that of an original strain, and can achieve the mass production of the purplish red pigment in a short time after the fermentation is started. Therefore, the culture time of the variant strain Geomycins sp.M300 of the present invention can be 5 to 12 days, preferably 6 to 10 days, and more preferably 6 to 7 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 25 ℃, and more preferably 14 to 20 ℃. The original strain can not tolerate room temperature or high temperature, the yield of the purple red pigment is very low at the room temperature of 20 ℃, but 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 at the room temperature of 20 ℃; it can grow and proliferate well even at 30 ℃. It has more superiority in growth at 30 deg.C than wild type, and has better growth at 25 deg.C.
The Geomymyces sp.M300 strain of the invention can realize long-term growth and stable passage. The medium and the culture method applied to culture the Geomyyces sp.M300 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 Geomyces sp.M300 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.
Compared with the prior art, the invention has the following advantages and effects:
(1) according to the high-yield extracellular purpurin geotrichum strain provided by the invention, the yield of the extracellular purpurin of the high-yield extracellular purpurin geotrichum strain is improved by 43.73% compared with that of the original strain; the yield of the extracellular purple red pigment of the strain is improved by 86.67 percent compared with the original strain when the strain is cultured under the condition (20 ℃) of exceeding the optimal production temperature (14 ℃). The fermentation period of the mutant strain is shortened by 50 percent compared with the original strain by culturing at the optimum production temperature; the growth (dry weight) of the mutant strain is improved by 5.28 percent compared with the original strain when the mutant strain is cultured at the temperature of 14 ℃; the growth (dry weight) of the mutant strain is improved by 33.3 percent compared with the original strain when the mutant strain is cultured at the temperature of 20 ℃; when cultured at 30 ℃, the growth (dry weight) of the mutant strain is improved by 57.68 percent compared with the original strain.
(2) The breeding method provided by the invention has the advantages of mild breeding conditions, simple operation, less investment and easy mastering.
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 acquisition of Geotrichum strains with high yields of extracellular purplish Red pigment
In this example, the starting strain was subjected to normal pressure room temperature plasma mutagenesis, which specifically comprises the following steps:
1. spore suspension preparation
Inoculating starting strain (Geomycins sp. WNF-15A, obtained from the first Marine research institute of Nature resources department) on seed solid culture medium, culturing at 20 deg.C for 5 days, washing strain plate with 5mL sterile normal saline to obtain spore-containing suspension, diluting appropriately according to growth conditions, and making into spore-containing 107Spore suspensions of the order of one/mL.
2. ARTP mutagenesis
Putting the suspension containing the spores obtained in the step (1) into an ARTP mutagen according to the operating protocol, and carrying out reaction at the power of 120W; performing mutagenesis by treating for 0, 30, 60, 90, 120, 150, 180, 210, 240 and 300s under the conditions that the gas amount is 10L/min and the distance between a plasma emission source and the sample is 2 mm.
3. Plate separation screening
Taking the mutagenized bacteria liquid, adding sterile normal saline for gradient dilution (10 times, 100 times and 1000 times), then taking a proper amount of the bacteria liquid, coating the bacteria liquid on a seed plate (glucose 1g, mannitol 2g, maltose 2g, sodium glutamate 1g, yeast extract 0.6g and MgSO 2)4·7H2O 0.03g，KH2PO40.05g of agar powder and 3g of agar powder, wherein the volume is fixed to 100mL by using distilled water, the pH is natural), the agar powder is inversely cultured for 48 hours at the constant temperature of 20 ℃ in a dark place, after the bacterial colony grows out, the positive mutant bacterial strain which has better state and larger diameter of a pigment circle and changes the form of the bacterial colony (including the size, the color and the aerial hyphae) is selected, the single bacterial colony is streaked and separated, and the pure mutant bacterial strain is obtained by repeated separation and purification and is preserved at the temperature of 4 ℃.
4. Fermentation and breeding
Taking the mutagenic strain separated and purified in the step 3, and carrying out fermentation experiment, namely inoculating the mutagenic strain into a seed culture medium for culture and proliferation, wherein each 50mL of the seed culture medium (1 g of glucose, 2g of mannitol, 2g of maltose, 1g of sodium glutamate, 0.6g of yeast extract and MgSO4·7H2O 0.03g，KH2PO40.05g, distilled water to 100mL, natural pH) was inoculated into a 6-day-culture slab with a diameter of 1.7cm, and the slab was cultured for 72 hours on a shaker at 130rpm, so that the seed medium had mature spores, to obtain a primary seed solution.
Inoculating the seed solution at an inoculum size of 10% (V/V) into 50mL seed culture medium (glucose 1g, mannitol 2g, maltose 2g, sodium glutamate 1g, yeast extract 0.6g, MgSO4·7H2O 0.03g，KH2PO40.05 percent, distilled water is used for fixing the volume to 100mL, the pH is natural), and the secondary seed solution is obtained after fermentation culture is carried out for 36 hours under the condition of shaking table and at the temperature of 20 ℃;
inoculating the seed solution into 50mL of fermentation medium (2.8 g of starch, 0.185g of peptone, distilled water to 100mL of constant volume and natural pH) according to the inoculation amount of 10mg, controlling 130rpm under the condition of a shaking table and 14 ℃ for fermentation culture for 6 days, then centrifuging the fermentation liquor to obtain a supernatant containing the extracellular purple red pigment, and carrying out multiple subculture verification on a target strain by detecting the yield of the extracellular pigment so as to find the geotrichum strain capable of highly producing the extracellular purple pigment.
Example 2 screening and morphological characterization of Geomymyces sp.M300 Strain
After a plurality of times, through large-scale screening and comprehensive comparison, the inventor obtains a Geomyces strain with high extracellular purplish red pigment yield, which is named as Geomyes sp.M300.
1. Morphological identification
The variant strain Geomyyces sp.M300 has the following characteristics: culturing on a seed flat plate for 7 days, wherein the colony shape is large, the diameter of the colony is 15-16 mm, the colony is milky white, and the back surface is tawny; the colony edge is complete and round; in the later growth stage, radial cracks are often accompanied, white villous aerial hyphae grow, and the aerial hyphae are compact; the hypha in the medium is sparse, and the purple pigment is seeped out of the medium. The hyphae are transparent, thin-walled and provided with a partition; the spores are spherical or ellipsoidal.
2. Antagonism experiment
And inoculating the obtained mutant strain Geomycins sp.M300 and the starting strain Geomycins sp.WNF-15A into a proper amount of seed culture medium for activation, dipping a small amount of the activated mutant strain Geomycins sp.WNF-15A on a solid plate of the seed culture medium by using a gun head, pouring the activated mutant strain Geomycins sp.M300 and the starting strain Geomycins sp.WNF-15A on the solid plate of the seed culture medium at an interval of about 1cm, and inversely. The growth of both strains and whether antagonism was formed were observed.
As a result, as shown in the right panel of fig. 1, a distinct antagonistic line was formed between the mutant strain Geomyces sp.m300 (left side culture in the figure) and the original strain (right side culture in the figure), indicating that the mutant Geomyces sp.m300 is a novel strain that is changed from the original strain.
Example 3 preparation of magenta pigment Using Geomymyces sp.M300 Strain according to conventional method
In this example, the purplish red pigment was prepared by a conventional Geomyces sp.
1. Preparation of seed liquid
The obtained mutant strain and original strain were inoculated into sterilized seed medium (1 g of glucose, 2g of mannitol, 2g of maltose, 1g of sodium glutamate, 0.6g of yeast extract, MgSO 1mL of seed medium) for culture and proliferation4·7H2O 0.03g，KH2PO40.05%, distilled water to 100mL, natural pH) and digging and inoculating a seed flat plate with the diameter of 1.7cm and culturing for 6 days, culturing for 3 days at 130rpm to ensure that the seed culture medium has mature spores, and obtaining a primary seed solution; inoculating the second-level seeds with the inoculation amount of 10 percent by volume, and culturing for 36h at 130rpm to obtain a second-level seed liquid with high activity.
2. Fermentation culture and determination of purplish red pigment
Inoculating the seed liquid into 50mL of basic fermentation medium (2.8 g of starch, 0.185g of peptone, distilled water for fixing the volume to 100mL and natural pH) according to the inoculation amount of 10mg (dry weight of thalli), controlling the conditions of a shaking table at 130rpm and the temperature at 14 ℃ or 20 ℃ for fermentation culture, centrifuging the fermentation liquid to obtain a supernatant containing the extracellular rhodochrous pigment, and measuring the pigment yield of the extracellular fermentation liquid at a time point needing to be measured, the dry weight of the thalli and other indexes.
The method for measuring the purplish red pigment comprises the following steps: centrifuging 1mL fermentation liquid at 12000rpm for 10min, collecting supernatant, diluting by a certain multiple (30 times), and detecting absorbance value of the filtrate at 520nm absorption wavelength by using spectrophotometer.
Method for measuring dry weight of cells: taking 10mL of fermentation liquor, carrying out suction filtration on the thalli onto a filter paper sheet with a weighed mass in advance by suction filtration, and repeatedly washing with deionized water until the filtrate is colorless; and taking down the filter paper, putting the filter paper into an oven for drying (2h), then putting the filter paper into a dryer for cooling to room temperature, weighing the total weight of the filter paper and the thalli by using a precision analytical balance, and finally calculating the dry weight of the thalli.
3. Yield of 14 ℃ fermentation culture
The production of the purplish red pigment in the fermentation culture process at 14 ℃ is shown in the left graph of figure 2, at the fermentation temperature of 14 ℃, the Geomymyces sp.M300 strain which is the beginning of the fermentation shows higher yield than the original strain, and the highest absorbance at 520nm of the mutant strain fermentation extracellular fluid is 44.73 by using an ultraviolet spectrophotometer; the highest absorbance of the extracellular fluid fermented by the starting strain at 520nm is 26.37 by using an ultraviolet spectrophotometer.
Therefore, in the fermentation culture process at 14 ℃, the yield of the mutant strain extracellular purple red pigment is improved by 43.73 percent compared with the original strain.
From the fermentation period, the highest peak of the yield of the purplish red pigment can be achieved when the Geomycins sp.M300 strain is cultured in the fermentation process to about day 6, the starting strain is relatively higher at day 12, and the high value of the starting strain at day 12 is lower than that of the mutant Geomycins sp.M300 strain at day 6 and later. Therefore, under the condition of fermentation culture at 14 ℃, the fermentation period of the mutant strain Geomymyces sp.M300 is obviously shortened by 50 percent compared with the original strain.
4. Yield of fermentation culture at room temperature of 20 DEG C
The production of the purplish red pigment in the fermentation culture process at 20 ℃ is shown in the right graph of figure 2, and the highest absorbance at 520nm of the extracellular fluid fermented by the Geomyes sp.M300 mutant strain is 15.96 by using an ultraviolet spectrophotometer at the fermentation temperature of 20 ℃; the absorbance of the extracellular fluid fermented by the starting strain at 520nm is measured by an ultraviolet spectrophotometer to be 8.55 at most.
It can be seen that the extracellular purple pigment yield of the Geomymyces sp.M300 mutant strain is improved by 86.67% compared with the original strain at the room temperature of 20 ℃.
The mutant strain of Geomycins sp.M300 showed a greater decrease in the yield of purplish red pigment than the starting strain at 20 ℃ at room temperature, indicating that the optimum temperature for the Geomycins sp.is not 20 ℃ at room temperature, more preferably 14 ℃. However, compared with the original strain, the yield of the purplish red pigment of the Geomyyces sp.M300 mutant strain is improved by 86.67 percent, so that the mutant strain has more ideal temperature adaptability and has the potential of fermentation at room temperature to a certain extent.
5. Comparison of the Dry weights of the cells fermented at different temperatures
The measurement results of the dry weight of the bacteria are shown in FIG. 3, fermentation is carried out for 12 days at different temperatures of 14 ℃, 20 ℃ and 30 ℃ to obtain fermentation liquor, and the measured dry weight values of the Geomyes sp.M300 mutant strain are 7.97g/L, 11.73g/L and 8.83g/L respectively; the measured dry weight values of the original strain are 7.57g/L, 8.8g/L and 5.6g/L respectively.
Therefore, when the mutant strain is cultured at 14 ℃, the growth (dry weight) of the mutant strain is improved by 5.28 percent compared with the original strain; the growth (dry weight) of the mutant strain is improved by 33.3 percent compared with the original strain when the mutant strain is cultured at the temperature of 20 ℃; when cultured at 30 ℃, the growth (dry weight) of the mutant strain is improved by 57.68 percent compared with the original strain.
This result indicates that the mutant strain of Geomymyces sp.M300 has more ideal temperature adaptability, can grow well under the temperature condition of 30 ℃, and thus has the capability of fermentation at higher temperature. However, at 30 ℃, the growth of M300 is better than that of the wild strain although the growth is strongly inhibited.
Preservation of biological materials
The strain Geomymyces sp.M300 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: M2019508.
The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. It will be appreciated that various alterations and modifications of the invention will occur to those skilled in the art upon reading the above teachings, and that such equivalents are intended to fall within the scope of the invention as defined by the appended claims.

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: M2019508.
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 25 ℃, more preferably 14 to 20 ℃;
a rotation speed of 50 to 180rpm, preferably 100 to 160rpm, more preferably 120 to 140 rpm;
the cultivation time is 5 to 12 days, preferably 6 to 10 days, more preferably 6 to 7 days;
carrying out aerobic fermentation.
9. The method of claim 8, wherein starch, glucose, mannose or maltose is used as the carbon source, preferably starch is used as the carbon source; preferably, the concentration of the 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.
CN201911035568.6A 2019-10-29 2019-10-29 Antarctic fungus mutant strain with high purplish red pigment yield, breeding method and application thereof Pending CN110643519A (en)

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