CN114540340A - Method for improving ultraviolet mutagenesis effect of monascus - Google Patents

Method for improving ultraviolet mutagenesis effect of monascus Download PDF

Info

Publication number
CN114540340A
CN114540340A CN202210400865.1A CN202210400865A CN114540340A CN 114540340 A CN114540340 A CN 114540340A CN 202210400865 A CN202210400865 A CN 202210400865A CN 114540340 A CN114540340 A CN 114540340A
Authority
CN
China
Prior art keywords
monascus
spore suspension
ultraviolet
concentration
mutagenesis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210400865.1A
Other languages
Chinese (zh)
Inventor
付瑞燕
冯婧
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Anhui Agricultural University AHAU
Original Assignee
Anhui Agricultural University AHAU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Anhui Agricultural University AHAU filed Critical Anhui Agricultural University AHAU
Priority to CN202210400865.1A priority Critical patent/CN114540340A/en
Publication of CN114540340A publication Critical patent/CN114540340A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management

Abstract

The invention relates to a method for improving the ultraviolet mutation effect of monascus, belonging to the technical field of microorganism mutation breeding. According to the method, monascus spore powder is inoculated into sterile physiological saline containing tween 80, and is filtered by a 350-400-mesh cell sieve to prepare low-concentration monascus spore suspension; then inoculating into a monascus liquid culture medium, carrying out oscillation heat treatment, and then culturing for 2-4 h at the temperature of 32 +/-2 ℃ to prepare activated low-concentration monascus spore suspension; and (3) carrying out ultraviolet irradiation with the wavelength of 245-260 nm for 60-210 s on the activated low-concentration monascus spore suspension to obtain the irradiated monascus spore suspension, adding a small amount of sterile water into the irradiated monascus spore suspension, and finally, completely inoculating the monascus spore suspension subjected to ultraviolet treatment onto a flat plate without dilution for culture, so that the positive mutation rate of ultraviolet mutation breeding of monascus is improved. The invention obviously improves the ultraviolet mutagenesis effect of the monascus spores and provides conditions for finally obtaining the monascus mutant strain with high color value and low citrinin.

Description

Method for improving ultraviolet mutagenesis effect of monascus
Technical Field
The invention belongs to the technical field of microorganism mutation breeding, and particularly relates to a method for improving the ultraviolet mutation effect of monascus.
Background
Monascus (Monascus) is a small filamentous saprophytic fungus belonging to Ascomycotina (Ascomycotina), Ascomycotina (Plectomycetes), Eurotiales (Eurotiales), Monascus (Monacaceae) and asexual propagation thereof is the final formation of mycelia through the germination of conidia and the branching of filamentous materials. Red yeast rice is red rice yeast which is prepared by using rice as a matrix, artificially culturing mycelia or spores of monascus in a specific ecological environment to enable the mycelia to grow in the rice and finally fermenting. Red yeast rice has been used for a long time to improve the taste and color of food, and also can be used as a traditional medicine for digestion and blood vessel function, and has the effects of strengthening spleen to promote digestion, promoting blood circulation to remove blood stasis and the like. As a natural microbial fermentation product, the monascus pigment is relatively stable in property, safe and non-toxic, can be used as a safe natural pigment in daily life, is more and more widely applied, and can replace nitrite to be used as a colorant of foods such as cakes, fermented bean curd, meat products and the like, and also can be widely applied to cosmetics. After the artificially synthesized pigment is proved to have toxic and side effects in different degrees, the monascus pigment is more and more favored and valued by people as a natural pigment. In addition to producing biologically active substances beneficial to the human body, monascus can also produce a mycotoxin, citrinin. Animal experiments prove that citrinin is a neurotoxic mycotoxin, has teratogenic and even lethal effects on animals, and can cause symptoms of kidney enlargement, urine volume increase, renal tubular dilatation, epithelial cell degeneration necrosis and the like of experimental animals, so that the production of red yeast rice products with low citrinin content becomes a current research hotspot.
The ultraviolet mutagenesis is ultraviolet irradiation with an effective wavelength of 200-300 multiplied by 10nm, most preferably 254nm (which is the absorption peak of nucleic acid). After purine and pyrimidine of DNA and RNA absorb ultraviolet light, DNA molecules form pyrimidine dimers, namely two adjacent pyrimidines are covalently connected, the dimers can weaken the action of hydrogen bonds between double bonds, and cause the distortion of double-stranded structures, prevent normal pairing between bases, and possibly cause mutation or death. In addition, the formation of dimers prevents the unwinding of the double strand, thereby affecting the replication and transcription of DNA. In general, UV radiation can cause base transitions, transversions, frameshift mutations or deletions, so-called mutagenesis. Mutation breeding is an effective way for improving the color value of red yeast rice and reducing the content of citrinin. Ultraviolet mutagenesis is one of the commonly used physical mutagenesis methods, and the operation method and the used equipment are relatively simple, the experimental process is relatively safe, and the mutation efficiency is high, so the ultraviolet mutagenesis method is widely applied to mutation breeding work.
Disclosure of Invention
The invention aims to improve the positive mutation rate of monascus ultraviolet mutation breeding by a simple processing method, so that the monascus spore ultraviolet mutation effect is obviously improved, and conditions are provided for finally obtaining monascus mutant strains with high color value and low citrinin.
A method for improving the ultraviolet mutagenesis effect of monascus comprises the following specific steps:
firstly, monascus spore powder is dissolved in sterile physiological saline containing tween 80, filtered by a 350-400 mesh cell sieve, diluted and mixed uniformly to obtain the monascus spore powder with the concentration of (5.3 +/-0.3) x 102Per mL monascus spore suspension;
secondly, inoculating the monascus spore suspension into a monascus liquid culture medium, performing oscillation heat treatment at the temperature of 45 +/-2 ℃ and the speed of 100-200 r/min, and then culturing for 2-4 hours at the temperature of 32 +/-2 ℃ and the speed of 100-200 r/min to obtain activated monascus spore suspension;
③ ultraviolet mutagenesis: uniformly mixing the activated monascus spore suspension, inoculating the activated monascus spore suspension onto a monascus flat plate, and carrying out ultraviolet irradiation on the monascus spore suspension for 60-210 seconds under the stirring effect and at the wavelength of 245-260 nm to obtain the irradiated monascus spore suspension;
inoculating the irradiated spore suspension to a monascus plate, spreading the monascus plate on a culture medium, and culturing the monascus plate in an environment of 32 +/-2 ℃ to obtain the mutagenized monascus.
Wherein, in the sterile physiological saline containing the Tween 80, the content of the Tween 80 is 0.08-0.12%.
Wherein the time of the oscillating heat treatment is 30-50 min.
Wherein, the monascus liquid culture medium has the following formula: 50-70 g/L of glucose, 5-15 g/L of peptone and 25-35 g/L of soluble starch.
Wherein the volume ratio of the monascus spore suspension to the monascus liquid culture medium is 1: 1.
Wherein the power of the ultraviolet light is 10-20 w.
Advantageous effects
Currently, high-concentration spore suspension is commonly used in monascus ultraviolet mutagenesis research, and the concentration of the spore suspension is generally 106~107Per mL, the concentration of the invention is (5.3 +/-0.3) multiplied by 102Ultraviolet mutagenesis is carried out on monascus spore suspension per mL under specific conditions, all spore suspensions after mutagenesis are inoculated and cultured without dilution, and researches show that the method can obviously improve the positive mutation rate compared with the traditional high-concentration spore ultraviolet mutagenesis method. If the concentration of monascus spore suspension is too low during ultraviolet mutagenesis, the efficiency of ultraviolet irradiation of spores is reduced, the number of obtained effective mutagenized monascus spores is insufficient, and the number of obtained effective mutagenized monascus is small; if the monascus spore suspension is too high during ultraviolet mutagenesis, the spore concentration may be too high, which is unfavorable for the forward mutation of color value/citrinin, and the forward mutation is low, and the specific mechanism is not researched yet. Through multiple experiments, the probability of generating the color value/citrinin forward mutation by the shock heat treatment at 45 ℃ and specific ultraviolet illumination at specific concentration is remarkably increased from 4.68% to 7.33%, and the optimal strain has remarkable performance improvement.
In the ultraviolet mutation breeding process, the most suitable mutation target is a unicellular and mononuclear individual, and in order to make spores which receive ultraviolet irradiation in an ungerminated state but have nuclear materials in a replicating active state, it is tried to activate monascus spores for a short time. In order to determine the time for activating the spores in a short time, the monascus spores are inoculated into a monascus liquid culture medium, heat treatment is carried out at 45 ℃ for 30min, then culture is continued at 32 ℃ to excite the activity of the spores, and samples are taken for microscopic examination after different culture times. The spore has stronger refractivity and no dyeability before being treated at 45 ℃, and the dyeability is increased after being treated at 45 ℃, and the difference is very obvious under a microscope. As shown in FIG. 1, no spores germinated when cultured at 32 ℃ for 4 h or before, while at 5h, the spores began to grow out of germ tubes, and germination was presumed, and since spores were probably undergoing nuclear division in 4 h and were not haploid, subsequent UV mutagenesis was performed by using spore suspension cultured for 3 h in an activated manner.
If the mould mycelium is directly treated by the mutagen, genetic instability and screening difficulty are caused due to more nuclei in mycelium cells. Every conidia of the mold has only one single nucleus, so that the problems described above can be avoided by subjecting the conidia to ultraviolet mutagenesis. If ultraviolet rays are directly irradiated to the spore suspension, the mutagenesis effect is not good because the spores are a structure for terminating vegetative growth of filamentous fungi into a dormant state, genetic materials are inactive, the mutagenesis is not facilitated, and the germination rate of the spores is not high, which means that even mutated spores may not grow into colonies. The method of the invention firstly adopts the method of reducing the spore concentration during ultraviolet mutagenesis and activating the spores in a short time to carry out ultraviolet mutagenesis on the monascus so as to obviously improve the positive mutation rate, thereby obviously improving the ultraviolet mutagenesis effect of the monascus spores and providing conditions for finally obtaining the monascus mutant strain with high color value and low citrinin.
Drawings
FIG. 1 is a photograph of microscopic examination of spores after heat treatment at 45 ℃ and cultivation at 32 ℃ for 1-5 h, wherein A, B, C, D, E shows activation for 1 h, 2 h, 3 h, 4 h and 5h in sequence;
FIG. 2 is a graph showing the lethality of three UV mutagenesis methods;
FIG. 3 is a graph comparing the positive mutation rate of three UV mutagenesis modes;
FIG. 4 is a histogram of color number and citrinin content of UV-mutagenized mutants;
FIG. 5 is a histogram of color number and citrinin content of the low concentration spore suspension UV-mutagenized mutants;
FIG. 6 is a bar graph of color number and citrinin content of the UV-mutagenized mutants from low-concentration spore suspensions after activation.
Detailed Description
A specific embodiment of the present invention is described in detail below, but it should be understood that the scope of the present invention is not limited by the specific embodiment.
Example 1
The embodiment provides a method for improving the ultraviolet mutagenesis effect of monascus, and the specific mutagenesis method is as follows:
preparing a low-concentration monascus spore suspension: using inoculating loop to scrape monascus spore powder on a loop plate, dissolving the monascus spore powder in 50 mL of sterile physiological saline added with 0.1% Tween 80, sucking 20 mL of the monascus spore powder, filtering the monascus spore powder by a 375-mesh cell sieve, and blowing and sucking the obtained monascus spore liquid for 7-8 times by using a liquid transfer gun to uniformly mix the spore liquid to obtain the monascus spore powder with the concentration of (5.3 +/-0.3) multiplied by 102And (4) preparing monascus spore suspension per mL for later use.
Ultraviolet mutagenesis: adding 0.5 mL of sterile water into 0.5 mL of monascus spore suspension, sucking by a pipette again for 7-8 times, uniformly mixing, completely inoculating the monascus spore suspension to a monascus plate, slightly tilting by hand to enable the spore suspension to be flatly laid on the monascus plate, and setting three parallel monascus spore suspensions as blank control groups. Preheating for 20 min in a clean bench by a magnetic stirrer, placing 3 mL of the monascus spore suspension spore liquid in an aseptic plate, respectively irradiating 4 parts of 3 mL of the monascus spore suspension spore liquid with ultraviolet rays with the power of 15w for 253.7nm in 60 s, 150 s, 210s and 300 s under the action of magnetic stirring, inoculating the irradiated spore suspension to the monascus plate according to 1 mL under a red light, slightly obliquely shaking by hands to enable the spore suspension to be tiled on a culture medium, and arranging three groups in parallel for each experimental group. After culturing for 6 days at 32 ℃ in a dark place, plates with non-overlapping single colonies on the plates are selected for counting. The number of colonies per three parallel controls was averaged and the number of viable bacteria per mL in the placebo group = mean number of colonies × 2 (CFU/mL).
The number of viable bacteria per mL in the experimental group is the average number of colonies × 1 (CFU/mL).
Lethality = (number of colonies of blank control group-number of colonies of experimental group)/number of colonies of blank control group × 100%.
Positive mutation rate = number of darker colored colonies on experimental group plates/number of colonies of blank control group x 100%.
Example 2
The embodiment provides a method for improving the ultraviolet mutagenesis effect of monascus, and the specific mutagenesis method is as follows:
in this example, the ingredients and formulation (g/L) of the monascus liquid medium: glucose 60, peptone 20, soluble starch 30.
Preparing a low-concentration monascus spore suspension: using inoculating loop to scrape monascus spore powder on a loop plate, dissolving the monascus spore powder in 50 mL of sterile physiological saline added with 0.1% Tween 80, sucking 20 mL of the monascus spore powder, filtering the monascus spore powder by a 375-mesh cell sieve, and blowing and sucking the obtained monascus spore liquid for 7-8 times by using a liquid transfer gun to uniformly mix the spore liquid to obtain the monascus spore powder with the concentration of (5.3 +/-0.3) multiplied by 102And (4) per mL of monascus spore suspension.
Activation of low-concentration monascus spore suspension: inoculating the obtained monascus spore suspension into a monascus liquid culture medium according to the volume ratio of 1:1, performing oscillation heat treatment at 45 ℃ and 150 r/min for 30min, and then culturing at 32 ℃ and 150 r/min for 3 h to obtain the activated low-concentration monascus spore suspension.
Ultraviolet mutagenesis: taking 0.5 mL of activated low-concentration monascus spore suspension, sucking and blowing the suspension for 7-8 times by using a pipette gun again, uniformly mixing the suspension, completely inoculating the suspension onto a monascus plate, slightly tilting and shaking the suspension by hand to enable the spore suspension to be flatly laid on the monascus plate, and arranging three parallel monascus spore suspensions to serve as a blank control group. Preheating for 20 min in an ultra-clean workbench by using a magnetic stirrer, placing 3 mL of the activated low-concentration monascus spore suspension in an aseptic plate, respectively carrying out 253.7nm irradiation with ultraviolet rays at the power of 15w on 4 parts of 3 mL of the activated low-concentration monascus spore suspension for 60 s, 150 s, 210s and 300 s under the action of magnetic stirring, inoculating the irradiated spore suspension to a monascus plate according to 1 mL under a red light, slightly tilting and shaking by hand to enable the spore suspension to be tiled on a culture medium, and arranging three groups in parallel in each experiment. After culturing for 6 days at 32 ℃ in a dark place, plates with non-overlapping single colonies on the plates are selected for counting. The number of colonies per three parallel controls was averaged and the number of viable bacteria per mL in the placebo group = mean number of colonies × 2 (CFU/mL).
The number of viable bacteria per mL in the experimental group is the average number of colonies × 1 (CFU/mL).
Lethality = (number of colonies of blank control group-number of colonies of experimental group)/number of colonies of blank control group × 100%.
Positive mutation rate = number of darker colored colonies on experimental group plates/number of colonies of blank control group x 100%.
Comparative example 1
In this example, monascus spores were treated by traditional ultraviolet mutagenesis, which was performed as follows:
preparation of spore suspension: adding 5 mL of sterile normal saline containing 0.1% Tween 80 into a plate with good spore production, scraping and washing the plate by scraping the surface of the plate with a coating rod, filtering the washed spore liquid with a 375-mesh cell sieve, filtering into an empty conical bottle, counting the filtrate with a blood counting plate, and diluting the spore suspension with normal saline into a sterile centrifuge tube to (6.5 +/-0.2) multiplied by 106The cells are ready for use after being treated with one/mL.
Ultraviolet mutagenesis: taking 15 mL of spore suspension into a sterilized hollow conical bottle, shaking uniformly, dividing into 5 parts, each of which is 3 mL, preheating the spore suspension in a super clean workbench for 20 min by using a magnetic stirrer, taking 3 mL of the spore solution, placing the spore solution into an aseptic plate, and respectively carrying out 253.7nm ultraviolet irradiation on 4 parts of the spore solution under the action of magnetic stirring for 60 s, 150 s, 210s and 300 s at the power of 15w, wherein the spore suspension which is not subjected to ultraviolet treatment, namely the spore suspension with the irradiation time of 0s, is used as a blank control. Sequentially diluting 0.5 mL of spore suspension subjected to mutagenesis in different time lengths by 10 times and 4.5 mL of physiological saline under a red light, and diluting by 10 times-3、10-4After gradient, stock solution and 10-3、10-4And (3) inoculating 200 mu L of diluted spore liquid to an monascus plate by a cross mixing method, setting three parallel controls for each gradient treatment, culturing for 6 days at 32 ℃ in a dark place, and selecting the plates with non-overlapping single colonies on the plates for counting.
Examples 1 and 2 and comparative example 1, and the results of the mutant strains obtained by the three ultraviolet mutagenesis modes for fermenting red yeast rice are compared.
Selecting colony from the plate obtained by mutagenesis according to the obvious difference between the obtained colony and the original strain color, separating and purifying by three-zone scribing method, selecting spore powder on the single colony after purification, inoculating to liquid culture medium, shaking and culturing at 32 ℃ for 6 d at 150 r/min, filtering by using a 375-mesh cell sieve, counting the filtrate by a blood counting plate, diluting the spore liquid concentration of all target strains to (4.0 +/-0.2) multiplied by 106one/mL. Use ofWeighing 30 g of indica rice by a balance, placing the indica rice in a 250 mL beaker, adding 100 mL of water with the pH value of 3.5, soaking for 10 h, filtering by using gauze after soaking, draining, determining the weight until the water content is 45%, sealing by using 6 layers of gauze and 2 layers of newspaper, sterilizing for 20 min at 121 ℃, stirring by using a sterile glass rod in a workbench while the rice is hot after sterilizing, dispersing the rice grains, inoculating 2 mL of monascus monospore suspension after the rice grains are cooled to room temperature, adding 2 mL of sterile water every 24 h for 1-2 d, adding water, stirring and stirring by using the sterile glass rod in a super clean workbench, stirring uniformly, placing the mixture at 32 ℃ for continuous culture, adding 4 mL of sterile water every 24 h for 3 d, and fermenting for 9 d in total.
And drying the red yeast rice at 60 ℃ for 48 h after fermentation is completed, crushing the dried red yeast rice by using a crushing and grinding machine, sieving by using a 60-mesh sieve, and collecting for later use. Weighing 0.2 g of the red yeast rice powder which is uniformly mixed after being crushed, dissolving the red yeast rice powder by using 100 mL of 70% ethanol solution, transferring the solution into a 100 mL volumetric flask, and fixing the volume to a scale mark. Placing in a constant temperature water bath at 60 ℃ for 1 h, taking out, cooling to room temperature, continuously adding 70% ethanol solution to the scale mark, shaking up, and filtering with filter paper. The absorbance A of the sample was measured at a wavelength of 505 nm with reference to a 70% ethanol solution. The color value X (U/g) of the monascus pigment extracting solution is calculated according to the following formula:
X=A×100/m×b
in the formula: a is the absorbance of the sample;
m is the weight of the sample, and the unit is gram (g);
b-dilution factor.
Weighing 2.0 g of the red yeast rice powder which is crushed and uniformly mixed into a 50 mL centrifuge tube, adding 8 mL of 70% methanol solution for dissolving, carrying out vortex oscillation and shaking for 3 min, then using ultrasonic oscillation for 30min for dissolving assistance, centrifuging at 8000 r/min for 10 min, collecting supernatant, continuously adding 8 mL of 70% methanol into the precipitate, repeating the operation once, finally enriching the supernatant obtained in two times, and drying at 60 ℃ for 3 d. Adding 20 mL of methanol solution with pH of 1.5 into the centrifuge tube after drying for redissolution, stirring uniformly after dissolution to dilute 600 and 700 times, and measuring the concentration of the solution by using a fluorescence spectrophotometer under the conditions that the excitation wavelength is lambda Ex =330 nm, the emission wavelength is lambda Em =485 nm, the slit width is Ex =10 nm and the Em = 5nmFluorescence, which was brought to the citrinin standard curve equation y =1865.4x +2.0566 (R)2=0.999) to yield the citrinin content.
In the ultraviolet mutation breeding process, the most suitable mutation target is a unicellular and mononuclear individual, and in order to make spores which receive ultraviolet irradiation in an ungerminated state but have nuclear materials in a replicating active state, it is tried to activate monascus spores for a short time. The monascus spores are inoculated into a monascus liquid culture medium, heat treatment is carried out for 30min at 45 ℃, then the monascus spores are transferred to 32 ℃ for continuous culture, so as to excite the activity of the spores, and the samples are sampled for microscopic examination after different times of culture. The spore has strong refractivity before being activated, has no dyeability, has increased dyeability after being activated properly, and has obvious difference under a microscope. As shown in FIG. 1, no spore germinates during and before 4 h of culture at 32 ℃, while at the 5h, the spore begins to grow out of a germ tube, and the germination phenomenon is assumed, and the spore probably has nuclear division and is not haploid already at the 4 h, so that the subsequent ultraviolet mutagenesis is carried out by using the spore suspension which is activated and cultured for 3 h. If the activation time is not within this range, the experiment shows that the number of positively mutated strains is significantly reduced.
FIG. 2 is a lethality curve of three UV mutagenesis methods, as shown in FIG. 2, the lethality of the conventional UV mutagenesis, the UV mutagenesis of the low concentration spore suspension and the UV mutagenesis of the activated low concentration Monascus spore suspension of comparative example 1 showed a consistent trend with the increase of the UV irradiation time, and the lethality was 77.3%, 78.34% and 77.69% respectively at the irradiation time of 150 s. The lethal effect of ultraviolet on the monascus spores is shown to be not changed along with the change of the concentration and the activity of the spores, but to present certain stability.
Fig. 3 is a comparison of positive mutation rates of three ultraviolet mutagenesis modes, and as shown in fig. 3, the positive mutation rates of the three ultraviolet mutagenesis modes are all the highest when the ultraviolet irradiation is carried out for 150 s, at this time, the mortality rate is about 78%, and the positive mutation rates are respectively 4.68%, 6.79% and 7.33%, wherein the ultraviolet mutagenesis positive mutation rate of the low-concentration monascus spore suspension after activation is the highest and is obviously higher than that of the traditional method and the ultraviolet mutagenesis of the low-concentration spore suspension before activation.
The applicant compares the effects of the mutant strains obtained by three ultraviolet mutagenesis modes on fermenting red yeast rice
And (3) carrying out co-separation and purification after traditional ultraviolet mutagenesis to obtain 6 mutant strains, fermenting the red yeast rice by using spore liquid of the 6 mutant strains respectively, and measuring the color value and the citrinin content of the red yeast rice. As shown in FIG. 4, in the 6 strains, the color value of the Z9 strain reaches 3806 +/-35.553U/g, the content of citrinin is 91.116 +/-1.485 mg/kg, and the color value/citrinin reaches 41.771; the blank control group without mutagenesis has the color value of 2849 +/-29.962U/g, the content of citrinin is 90.568 +/-1.597 mg/kg, and the color value/citrinin is 31.457. The color value of the Z9 strain is increased by 33.60 percent compared with that of a blank control, and the color value/citrinin is increased by 32.79 percent compared with that of the blank control.
Co-separating and purifying after ultraviolet mutagenesis by using low-concentration spore suspension to obtain 3 mutant strains, fermenting red yeast rice by using spore liquid of the 3 mutant strains respectively, and measuring the color value and the citrinin content of the red yeast rice. As shown in FIG. 5, the color number of the D5 strain reaches 4247.500 + -27.839U/g, the content of citrinin is 75.312 + -1.308 mg/kg, and the color number/citrinin reaches 56.399; the blank control group without mutagenesis has the color value of 2929.056 +/-21.718U/g, the content of citrinin of 89.762 +/-1.538 mg/kg and the color value/citrinin of 32.632. Compared with a blank control group, the color value of the D5 strain is improved by 45.01%, the color value of the citrinin is reduced by 16.10%, and the color value/citrinin is improved by 72.83% compared with the blank control group, which shows that mutagenesis sites in the method are likely to be dispersed, so that good mutagenesis effects of color value increase and citrinin reduction can be achieved simultaneously.
Carrying out ultraviolet mutagenesis on the activated low-concentration spore suspension, carrying out co-separation and purification to obtain 5 mutant strains, fermenting the red yeast rice by using spore liquid of the 5 mutant strains respectively, and determining the color value and the citrinin content of the red yeast rice. As shown in FIG. 6, the DH3 strain had a color number of 5021.611 + -51.496U/g and a citrinin content of 106.419 + -3.807 mg/kg, while the non-mutagenized control group had a color number of 2995.056 + -55.254U/g and a citrinin content of 95.626 + -6.274 mg/kg. The color value of the DH3 strain is increased by 67.66% compared with that of a blank control, and the color value increase amplitude is obviously increased compared with that before spore activation (45.01%), which indicates that the main mutagenesis site is positioned in a gene related to the color value after the spore activation.
The comparison results of the mutagenesis effects of the three ultraviolet mutagenesis modes are shown in table 1, and compared with the traditional ultraviolet mutagenesis method, the ultraviolet mutagenesis method of the low-concentration spore suspension and the ultraviolet mutagenesis method of the activated low-concentration spore suspension can respectively improve the positive mutation rate of monascus ultraviolet mutagenesis by 45.1% and 56.6%; the ultraviolet mutagenesis method of the low-concentration spore suspension is beneficial to improving the color value/citrinin ratio of the red yeast rice by 35.02 percent compared with the traditional ultraviolet mutagenesis method, and the ultraviolet mutagenesis method of the activated low-concentration spore suspension is beneficial to improving the color value of the red yeast rice by 31.94 percent compared with the traditional ultraviolet mutagenesis method. Therefore, the method for reducing the spore concentration and activating the spores in a short time can simply and efficiently obtain the citrinin monascus mutant strain with high color value and low cost.
TABLE 1 comparison of the mutagenic effects of the three mutagenesis modes
Parameter(s) Comparative example 1 Example 1 Example 2
Highest positive mutation rate (%) 4.68 6.79 7.33
Optimum mutant color number content (U/g) 3806 4247.5 5021.611
Optimal mutant color number increase compared to control (%) 33.60 45.01 67.66
Optimal mutant color number/citrinin ratio 41.771 56.399 47.187
Optimal mutant color number/citrinin increase compared to control (%) 32.79 72.83 50.66
In order to test the genetic stability of the optimal mutant strain obtained by the 3 mutagenesis methods of traditional ultraviolet mutagenesis, low-concentration spore suspension ultraviolet mutagenesis and activated low-concentration spore suspension ultraviolet mutagenesis, the 3 strains are subcultured, spores of the mutant strain are used for fermenting red yeast rice after 5 generations of culture, the color value/citrinin ratio is taken as an index and compared with the red yeast rice fermented by the strain of the 1 st generation, the result is shown in table 2, the color value/citrinin ratio of the 3 strains after 5 consecutive generations is less than 5% compared with that of the strain of the 1 st generation, and the mutant strain obtained by the 3 mutagenesis methods has better genetic stability.
TABLE 2 comparison of color values/citrinin ratios at Generation 1 and Generation 5 of the optimal mutant strains
Mutant strain Generation 1 5 th generation
Z9 41.77 41.82
D5 56.40 54.87
DH3 47.24 47.70
Determination of genetic stability of mutants
Inoculating monascus mutant strains Z9, D5, DH3, H7 and F2 into a monascus liquid culture medium, performing shake culture at a constant temperature of 150 r/min at 32 ℃ for 2D to serve as a 1 st generation, sucking the bacterial liquid of the 1 st generation, transferring the bacterial liquid into a fresh monascus liquid culture medium, performing shake culture at a constant temperature of 150 r/min at 32 ℃ for 2D to serve as a 2 nd generation, and repeating the steps until the 5 th generation. Inoculating indica rice with monascus mutant strains which are continuously passed for 5 generations to ferment the monascus rice, detecting the contents of monascus pigment and citrinin after fermenting for 9 days, calculating the ratio of color value to citrinin, and comparing the ratio with the fermentation level of the mutant strains of the 1 st generation so as to detect whether the mutant strains can be stably inherited.
Example 3
The embodiment provides a method for improving the ultraviolet mutagenesis effect of monascus, and the specific mutagenesis method comprises the following steps:
in this example, the ingredients and formulation (g/L) of the monascus liquid medium: glucose 50, peptone 15, soluble starch 35.
Preparing a low-concentration monascus spore suspension: an inoculating ring is used for scraping monascus spore powder on a ring plate to be inoculatedDissolving in 50 mL sterile physiological saline containing 0.12% Tween 80, sucking 20 mL, filtering with 350 mesh cell sieve, and sucking the obtained Monascus purpureus spore solution with pipette 7-8 times to obtain spore solution with concentration of (5.3 + -0.3) × 102And (4) one/mL of monascus spore suspension (low-concentration monascus spore suspension).
Activation of low-concentration monascus spore suspension: inoculating the low-concentration monascus spore suspension into a monascus liquid culture medium according to the volume ratio of 1:1, performing oscillating heat treatment at 45 +/-2 ℃ and 100 r/min for 50 min, and then transferring the monascus spore suspension to 32 +/-2 ℃ and 100 r/min for culture for 4 h to obtain the activated low-concentration monascus spore suspension.
③ ultraviolet mutagenesis: taking 0.5 mL of activated low-concentration monascus spore suspension, blowing and sucking by using a pipette gun again for 7-8 times, uniformly mixing, then completely inoculating the activated low-concentration monascus spore suspension to a monascus plate, slightly obliquely shaking by hand to enable the spore suspension to be tiled on the monascus plate, preheating by a magnetic stirrer in a clean bench for 20 min, then taking 3 mL of the activated low-concentration monascus spore suspension, placing the activated low-concentration monascus spore suspension in a sterile flat dish, irradiating the activated low-concentration monascus spore suspension by ultraviolet rays with the power of 20w for 210s under the action of magnetic stirring, inoculating the irradiated spore suspension to the monascus plate according to 1 mL under a red light, slightly obliquely shaking by hand to enable the spore suspension to be tiled on a culture medium, and culturing in the environment of 32 +/-2 ℃ to obtain the mutagenized monascus.
Example 4
The embodiment provides a method for improving the ultraviolet mutagenesis effect of monascus, and the specific mutagenesis method is as follows:
in this example, the ingredients and formulation (g/L) of the monascus liquid medium: glucose 70, peptone 5, soluble starch 25.
Preparing a low-concentration monascus spore suspension: inoculating Monascus spore powder on a ring plate with an inoculating ring, scraping, dissolving in 50 mL sterile physiological saline containing 0.08% Tween 80, sucking 20 mL, filtering with 400 mesh cell sieve, and sucking the obtained Monascus spore liquid with a liquid transfer gun for 7-8 times to mix the spore liquid uniformly to obtain the product with concentration of (5.3 + -0.3) × 102Monascus spores suspension/mL (Low concentration Red Rice)A suspension of mold spores).
Activation of low-concentration monascus spore suspension: inoculating the low-concentration monascus spore suspension into a monascus liquid culture medium according to the volume ratio of 1:1, performing oscillation heat treatment at 45 +/-2 ℃ and 200 r/min for 30min, and then transferring the monascus spore suspension to 32 +/-2 ℃ and 200 r/min for culture for 2 h to obtain the activated low-concentration monascus spore suspension.
③ ultraviolet mutagenesis: taking 0.5 mL of activated low-concentration monascus spore suspension, blowing and sucking by using a liquid transfer gun for 7-8 times again, uniformly mixing, then completely inoculating the activated low-concentration monascus spore suspension to a monascus plate, slightly shaking by hand to enable the spore suspension to be tiled on the monascus plate, preheating for 15-30 min by a magnetic stirrer in an ultra-clean workbench, then taking 2-4 mL of the activated low-concentration monascus spore suspension, placing the activated low-concentration monascus spore suspension in an aseptic plate, irradiating the activated low-concentration monascus spore suspension for 260nm of 60 s under the action of magnetic stirring with ultraviolet with the power of 10w, inoculating the irradiated spore suspension to the monascus plate according to 1 mL under a red light, slightly shaking by hand to enable the spore suspension to be tiled on a culture medium, and culturing in an environment of 32 +/-2 ℃ to obtain the mutagenized monascus.

Claims (6)

1. A method for improving the ultraviolet mutagenesis effect of monascus is characterized by comprising the following specific steps:
firstly, monascus spore powder is dissolved in sterile physiological saline containing tween 80, filtered by a 350-400 mesh cell sieve, diluted and mixed uniformly to obtain the monascus spore powder with the concentration of (5.3 +/-0.3) x 102Per mL monascus spore suspension;
secondly, inoculating the monascus spore suspension into a monascus liquid culture medium, performing oscillation heat treatment at the temperature of 45 +/-2 ℃ and the speed of 100-200 r/min, and then culturing for 2-4 hours at the temperature of 32 +/-2 ℃ and the speed of 100-200 r/min to obtain activated low-concentration monascus spore suspension;
③ ultraviolet mutagenesis: uniformly mixing the activated low-concentration monascus spore suspension, inoculating the activated low-concentration monascus spore suspension to a monascus flat plate, and irradiating the monascus spore suspension with ultraviolet rays with the wavelength of 245-260 nm for 60-210 seconds under the action of stirring to obtain the irradiated monascus spore suspension;
inoculating the irradiated spore suspension to an monascus plate, laying the monascus plate on a culture medium, and culturing the monascus plate in an environment of 32 +/-2 ℃ to obtain the monascus subjected to mutagenesis.
2. The method for improving ultraviolet mutation effect of monascus according to claim 1, wherein the tween 80 is contained in the sterile physiological saline containing tween 80 in an amount of 0.08-0.12%.
3. The method for improving ultraviolet mutation effects of monascus according to claim 1, wherein the shaking heat treatment time is 30-50 min.
4. The method for improving the ultraviolet mutation effect of monascus according to claim 1, wherein the monascus liquid culture medium has a formula: 50-70 g/L of glucose, 5-15 g/L of peptone and 25-35 g/L of soluble starch.
5. The method for improving the ultraviolet mutation effect of monascus according to claim 1, wherein the volume ratio of the monascus spore suspension to the monascus liquid medium is 1: 1.
6. The method for improving the ultraviolet mutation effect of monascus according to claim 1, wherein the power of the ultraviolet light is 10-20 w.
CN202210400865.1A 2022-04-18 2022-04-18 Method for improving ultraviolet mutagenesis effect of monascus Pending CN114540340A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210400865.1A CN114540340A (en) 2022-04-18 2022-04-18 Method for improving ultraviolet mutagenesis effect of monascus

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210400865.1A CN114540340A (en) 2022-04-18 2022-04-18 Method for improving ultraviolet mutagenesis effect of monascus

Publications (1)

Publication Number Publication Date
CN114540340A true CN114540340A (en) 2022-05-27

Family

ID=81666837

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210400865.1A Pending CN114540340A (en) 2022-04-18 2022-04-18 Method for improving ultraviolet mutagenesis effect of monascus

Country Status (1)

Country Link
CN (1) CN114540340A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5429943A (en) * 1992-10-26 1995-07-04 Lotte Confectionery Co., Ltd. Monascus strain, Monascus anka 732Y3 (KCCM 10014) producing high amount of monascus pigments
US20060210584A1 (en) * 2005-03-18 2006-09-21 Chiu Siu W Method for preparing citrinin-free Monascus biomass and use of citrinin-free Monascus biomass
CN104893983A (en) * 2014-12-25 2015-09-09 东莞市天益生物发酵技术有限公司 Liquid state fermentation lower citrinin and high color value monascus red pigment preparing method and product

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5429943A (en) * 1992-10-26 1995-07-04 Lotte Confectionery Co., Ltd. Monascus strain, Monascus anka 732Y3 (KCCM 10014) producing high amount of monascus pigments
US20060210584A1 (en) * 2005-03-18 2006-09-21 Chiu Siu W Method for preparing citrinin-free Monascus biomass and use of citrinin-free Monascus biomass
CN104893983A (en) * 2014-12-25 2015-09-09 东莞市天益生物发酵技术有限公司 Liquid state fermentation lower citrinin and high color value monascus red pigment preparing method and product

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
方春玉等: "高产红曲色素的紫红红曲霉诱变育种技术研究", 中国酿造, no. 23, pages 19 - 21 *

Similar Documents

Publication Publication Date Title
Ammar Cultivation of Microalgae Chlorella vulgaris in Airlift photobioreactor for Biomass Production using commercial NPK Nutrients
CN108034591B (en) Monascus purpureus and application thereof
CN103146624A (en) Mixed liquid fermentation process of three plants of bacillus licheniformis
US20160230192A1 (en) Triterpenoids high yielding strain of antrodia cinnamomea and use thereof
CN110408545A (en) A kind of purplish red aspergillus kind and the method and product that Hongqu powder (red colouring agent) is prepared by it
CN102286378B (en) Composite probiotics for inhabiting aspergillus flavus growth and degrading aflatoxin and application thereof
CN110122164B (en) Yellow needle mushroom strain and cultivation method thereof
CN113773963A (en) High-density high-yield porphyridium culture method
CN114540340A (en) Method for improving ultraviolet mutagenesis effect of monascus
CN103392512B (en) Antrodia cinnamomea high yield triterpene bacterial strain and application thereof
CN106190898B (en) Industrial liquid fermentation method of bacillus cereus JZBC1 for dissolving pond dinoflagellate
CN116121078B (en) Method for combined preparation of high protein powder and high nucleic acid wall-broken mycelium
CN101928702A (en) Method for preparing cellulase
CN108913607B (en) Preparation method and application of wild russula filiformis mycelium with efficient dye removal function
CN110885764A (en) Eurotium cristatum selenium-rich strain and domestication and fermentation method
CN114276944A (en) Selenium-rich yeast new strain and production method of yeast selenium
CN107879483A (en) A kind of preparation method of breeding water body reparation microorganism formulation
CN100368553C (en) Production bacterial strain of abamectin with high yield and high secreation rate and new method of extracting AVM
CN108575556B (en) Hericium erinaceus strain and breeding method thereof
CN115287280A (en) Method for enhancing ultraviolet mutagenesis effect of monascus
CN115418339B (en) Method for improving fermentation activity of monascus spores
CN104694607A (en) Method for preparing beta-carotin by solid fermentation
CN111172042B (en) Laccase-producing mutant bacterium trametes sanguinea and application thereof
CN115873727B (en) Rosa roxburghii Shan Ninggao effective degrading bacterium SL006 and application thereof
CN109161490B (en) Candida tropicalis HY4-17-21 for producing nucleic acid and preparation method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination