CN107513504A - A kind of saccharomyces cerevisiae mutant bacterial and its mutagenesis and screening technique - Google Patents
A kind of saccharomyces cerevisiae mutant bacterial and its mutagenesis and screening technique Download PDFInfo
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Abstract
The invention discloses a kind of saccharomyces cerevisiae mutant bacterial and its mutagenesis and screening technique, saccharomyces cerevisiae mutant bacterial is spherical or oval, 5~10 μm of diameter, rat, milky；Without fungal filament, there is concentric ring, the speed of growth of the mutant strain improves 10 40% compared with starting strain, and the mutant strain can grow under 45 DEG C of high temperature, and preserving number is China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC No.14448.The optimum pH of growth is 4.5 6.0.The mutant strain be starting strain after ground simulation space mutagenesis and/or space mutagenesis test, the fast single bacterium colony of the speed of growth of screening, ground simulation space mutagenesis is vibration test and/or vacuum low energy particle mutagenesis.
Description
Technical field
The invention belongs to microbial technology field, and in particular to a kind of saccharomyces cerevisiae mutant bacterial and its mutagenesis and screening side
Method.
Background technology
At present, saccharomyces cerevisiae (Saccharomyces cerevisiae) category saccharomyces, is the saccharomyces cerevisiae in saccharomyces
Kind.Saccharomyces cerevisiae is most important, most widely used one kind in saccharomycete, in fermentation, functional nutrient source and biological field etc.
Play an important role.Saccharomyces cerevisiae is spherical or oval, and 5~10 μm of diameter, no fungal filament, cell is by repeating budding breeding.
Beta glucan and manna oligosacchride isoreactivity polysaccharide, protein, glutathione isoreactivity small peptide, core are rich in saccharomyces cerevisiae thalline
The trophic function composition such as acid, amino acid, vitamin, these nutriments are constantly researched and developed and applied to medicine, functions
Multiple industries such as food and feed.
S. cervisiae has many containing abundant proteins,vitamins,minerals, polysaccharide and many bioactive substances
Complete enzyme system, and contain 2.5%~10% ribonucleic acid (ribose nucleic acid, RNA).These trophic functions
Composition plays an important role to immune, antitumor, anti-oxidant and digestive function of body etc., be maintain body health it is important into
Point.At present, these functional mass to human body, the effect of animal and mechanism always constantly research and to its development and application.
Brewing yeast cell wall contains abundant beta-1,3-dextran.Beta glucan is by β -1,3/1,6 glycosidic bonds
A kind of structural polysaccharide that mode combines to form, positioned at the internal layer of yeast cell wall, account for the 30%~60% of cell membrane dry mass.β-
Glucan is used widely in food industry, and is immunized with stimulation, is reduced cholesterolemia, antitumor and prevention anthrax etc.
Notable medical efficacy；In addition, beta glucan is respectively provided with significantly in terms of enhancing lysozyme activity, complement activity and bactericidal activity
Effect.TORELLO C O etc. result of study shows, oral β -1,3-D- glucans can promote the cell of delivery of biologically active because
Son, so as to produce marrow immunocompetence and strengthen the resistance of tumor-bearing mice.
Yeast strain ferments produce functional materials, are typically determined by both sides characteristic：First, the growth ability of bacterial strain；
Second, the ability of bacterial strain production functional materials.Because microbial variation is fast, therefore microbial fermentation engineering is constantly subjected to biological work
Cheng Xueke attention.The method of mutagenesis of microorganism is constantly subjected to the attention of numerous scientific research personnel, is microbial fermentation engineering field
One of focus on research direction.Method of mutagenesis is always one only more preferable, without best research topic；Efficiently lure simultaneously
Change method and the bacterial strain that high yield functional materials are filtered out from numerous mutant strains, it is always what microorganism mutagenesis worked
Difficult point.The Space Mutation Technique mutagenesis means new as saccharomycete have the high distinguishing feature of mutagenesis beneficial mutation rate, screen mould
Formula is easy, one of new tool for obtaining high efficient strain of can yet be regarded as.
The content of the invention
It is an object of the invention to provide a kind of saccharomyces cerevisiae mutant bacterial and its mutagenesis and screening technique, there is provided by ground
The speed of growth that face mould intends the saccharomyces cerevisiae mutant bacterial of space mutagenesis and/or space mutagenesis improves compared with starting strain
10-40%, screening technique is simple, solves the problems, such as microorganism mutagenesis operational difficulties.
To achieve these goals, a kind of saccharomyces cerevisiae mutant bacterial provided by the invention, is spherical or oval, diameter
5~10 μm, rat, milky；The mutant strain is yeast strain through ground simulation space mutagenesis and/or space mutagenesis
After experiment, the fast single bacterium colony of the relative growth rate of screening, Classification And Nomenclature：Saccharomyces Cerevisiae in S accharomyces
Cerevisiae, microbial strain：Shenzhou 11-4, preserving number are that China Committee for Culture Collection of Microorganisms is commonly micro-
Bio-Centers CGMCC No.14448.
Preferably, the speed of growth of the mutant strain improves 10-40% compared with yeast strain starting strain, described prominent
Becoming bacterial strain can grow under 45 DEG C of high temperature, and the optimum pH of growth is 4.5-6.0.
The Physiology and biochemistry feature of saccharomyces cerevisiae mutant bacterial is：Growth temperature range is wider than starting strain, high temperature resistant, and 45
DEG C it still is able to grow, optimum growth temperature is 28-30 DEG C, the growth ability marked improvement of mutant strain.The growth of mutant strain
Speed improves 10-40% compared with yeast strain starting strain, and the growth optimum pH of mutant strain is 4.5-6.0, compares mutagenesis
Preceding starting strain accommodation broadens, and the accommodation of starting strain is pH5.0-6.0 before mutagenesis, the growth energy of mutant strain
Power marked improvement.
Preferably, the mutant strain has concentric ring without fungal filament.
Saccharomyces cerevisiae mutant bacterial, there is concentric ring, the yeast strain starting strain before mutagenesis is easily chosen without concentric ring
Take.
Preferably, ground simulation space mutagenesis is vibration test and/or vacuum low energy particle mutagenesis.
Vibration test, the flat board for covering with bacterial strain is fixed on Double earthquakes, vibrated 1 hour under 0-400kN, wherein before
0.5 hour is low frequency sinusoidal vibration test, and latter 0.5 hour is random vibration test；Vacuum low energy particle mutagenesis, bacterial strain will be covered with
Flat board be put into the low energy combined irradlation experimental facilities of space, 10-3-10-6Returned after 30-180 minutes are irradiated under Pa vacuums
Receive, total radiation dose is 1 × 1014eV-4×1014eV。
Preferably, in the space mutagenesis test, space environment is：Microgravity 10-3-10-6G, vibrate 0-400kN；Space
Dose of radiation 0.1-0.9mGy in cabin.
The Wine brewing yeast strain growing more rapidly picked out from ground simulating is seeded on solid medium, will
Finished inclined-plane is put into incubator, is cultivated 3 days, in order to carry out space mutagenesis test.Aseptically, one piece is scraped
Cultured inclined-plane is added in loading pipe, sealed membrane sealing, gives loading pipe to the firing base relevant personnel, is carried out with airship empty
Between mutagenesis testing, finally obtained the mutant strain that the speed of growth improves 10-40% compared with starting strain.Using ground face mould
Intend mutagenesis, the saccharomyces cerevisiae mutant bacteria of growth ability marked improvement can be obtained by then carrying out the associating method of mutagenesis of space mutagenesis
Strain.
The present invention also provides a kind of saccharomyces cerevisiae mutant bacterial in fermentation, biology, medicine, fermentation, functional nutrient source, food
Application in terms of product or feed.
The present invention also provides a kind of method of mutagenesis of saccharomyces cerevisiae mutant bacterial, concretely comprises the following steps：
(1) culture of starting strain：Saccharomyces cerevisiae starting strain is seeded on YEPD solid mediums and cultivated, is trained
Support 1-10 days, it is stand-by to filter out the faster Wine brewing yeast strain of the speed of growth；
(2) mutagenesis：By the bacterial strain of screening through ground simulation space mutagenesis and/or space mutagenesis, it is dilute that mutagenic strain does multiple proportions
Release, take 10-4With 10-5Times dilution carries out culture 2-7 days.
Preferably, ground simulation space mutagenesis is vibration test and/or vacuum low energy particle mutagenesis.
Ground simulation space mutagenesis is first used, the saccharomyces cerevisiae that then associating method of mutagenesis of progress space mutagenesis obtains is dashed forward
Become strain growth more rapidly, the speed of growth of mutant strain improves 10-40% compared with starting strain, and metabolic efficiency is higher；It is resistance to
High temperature, growth adaptation scope broaden, growth ability marked improvement.
The present invention also provides a kind of screening technique of saccharomyces cerevisiae mutant bacterial, concretely comprises the following steps：
(1) screen：Saccharomyces cerevisiae metabolite is added in the YEPD solid mediums prepared, forms screening and culturing medium,
Mutagenic strain is inoculated on screening and culturing medium and cultivated, filter out the speed of growth faster, the bacterial strain that changes greatly of colonial morphology makees
For alternative bacterial strain.
(2) ferment：By alternative inoculation in fluid nutrient medium, shaking table culture fermentation, the final OD for relying on zymotic fluid
Value determines the fast saccharomyces cerevisiae mutant bacterial of speed of production.
Preferably, the saccharomyces cerevisiae metabolite is ethyl acetate, cognac oil, acetic acid, butyric acid and/or caproic acid, institute
The concentration for stating metabolite is 10-3g/L—102g/L
High concentration end product of metabolism is added in commune vanch flat board by this method, based on being metabolized in microbial metabolism approach
Product feedback inhibition principle, the good bacterial strain of upgrowth situation in such flat board, be generally all grow more rapidly, metabolic efficiency more
High strain excellent, such a plate screening method are efficient, easy.This method filters out superior strain using independent culture plate,
Compare with conventional screening methods, reduce workload 50%-100%, and greatly reduce the blindness of screening.This method operates
Process is simple, and required reagent and material are the common general reagent in laboratory.
A kind of saccharomyces cerevisiae mutant bacterial and its mutagenesis and screening technique provided by the invention, have the advantages that：
Ground simulation space mutagenesis is first used, the saccharomyces cerevisiae that then associating method of mutagenesis of progress space mutagenesis obtains is dashed forward
Become strain growth more rapidly, the speed of growth of mutant strain improves 10-40% compared with starting strain, and metabolic efficiency is higher；
The growth temperature range of saccharomyces cerevisiae mutant bacterial is wider than starting strain, high temperature resistant, and 45 DEG C can still grow, mutation
The growth optimum pH of bacterial strain is 4.5-6.0, is broadened than the starting strain accommodation before mutagenesis, and starting strain is suitable before mutagenesis
It is pH5.0-6.0 to answer scope, the growth ability marked improvement of mutant strain；
The production method of saccharomyces cerevisiae mutant bacterial, based on metabolite feedback inhibition principle in microbial metabolism approach,
Such a plate screening method is efficient, easy；
This method filters out superior strain using independent culture plate, and conventional screening methods compare, and reduce workload
50%-100%, and greatly reduce the blindness of screening；
This method operating process is simple, and required reagent and material are the common general reagent in laboratory.
Brief description of the drawings
Fig. 1 is characteristic morphology schematic diagram of the saccharomyces cerevisiae mutant bacterial of the present embodiment on YEPD culture mediums.
Embodiment
In order that those skilled in the art more fully understand the present invention program, with reference to embodiment to this hair
It is bright to be described in further detail.
The present invention provides a kind of more quick saccharomyces cerevisiae (Saccharomyces cerevisiae) of growth
Shenzhou 11-4, there is provided bacterial strain its speed of growth 10-40% is improved compared with starting strain.Correspondingly additionally provide
State the method for mutagenesis and screening technique of mutant strain.
One plant of more quick saccharomyces cerevisiae mutant bacterial of growth, it is named as saccharomyces cerevisiae shenzhou 11-4, the bacterium
Kind is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number on July 20th, 2017
For CGMCC No.14448, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postal
Political affairs coding 100101.
On YEPD solid culture flat boards, morphological feature is Wine brewing yeast strain：Saccharomycete is spherical or oval, diameter
5~10 μm, no fungal filament, rat, milky, smooth moistening, there is concentric ring (without concentric ring before space mutagenesis), easily
Picking.The Physiology and biochemistry feature of the bacterial strain is：Growth temperature range is wider than starting strain, high temperature resistant, and 45 DEG C can still grow, most
Suitable growth temperature is 28-30 DEG C；The optimum pH 4.5-6.0 of growth, broadened than the starting strain accommodation before mutagenesis, mutagenesis
The optimal pH scope of preceding starting strain is pH5.0-6.0.
Starting strain is the original strain for breeding.
The preparation of YEPD culture mediums：Dusty yeast 10g, peptone 20g, glucose 20g, distilled water 1000mL, natural pH, warp
Cross 120-125 DEG C of moist heat sterilization 25min.
A kind of method of mutagenesis for growing more quick saccharomyces cerevisiae shenzhou 11-4, it is first empty using ground simulation
Between mutagenesis, then carry out space mutagenesis associating method of mutagenesis.
Embodiment 1
Strain culturing before ground simulation space mutagenesis：Starting strain is seeded in the flat board (Φ 9cm) of solid medium
On cultivated, it is stand-by after bacterial strain covers with whole flat board.The production method of bacterial strain flat board is as follows：：1) glucose 2%, albumen
Peptone 2%, yeast extract powder 1%, agar 1.5-2.0%, sterile distilled water, natural pH；2) sterilize 25min at 125 DEG C；3) it is every
The individual plate 20-30ml of falling culture medium, after 30-33 DEG C is cultivated 24 hours, sterile plate can use；4) starting strain is inoculated with
It is incubated at 25-35 DEG C afterwards, it is stand-by to filter out within 1-10 days the faster bacterial strain of the speed of growth.
Following set of or several group of screening test is carried out to the Wine brewing yeast strain on flat board, it is faster to filter out the speed of growth
Wine brewing yeast strain：
1st, ground simulation space mutagenesis
(1) vacuum low energy particle mutagenesis：The flat board for covering with Wine brewing yeast strain is put into space low energy combined irradlation experiment
Vacuum low energy particle irradiation test is carried out in equipment.Reclaimed after 1 hour, bacterial strain after irradiation test is added in seed liquor and expanded
Increase culture.Dose of radiation is：5×1010e/cm2× S and 10 × 1010e/cm2× S, exposure time are 1 hour, and irradiation is one small
When accumulated dose be：1.8×1014EV and 3.6 × 1014eV。
(2) cultivate：The ground simulation space mutagenesis sample that culture area is 0.5 square centimeter is aseptically taken, with 5
The sterile water elution thalline of milliliter is inhaled with pipettor into sterile centrifugation tube and breaks uniform thalline eluent, take 0.5 milliliter of bacterium solution to arrive
In 4.5 milliliters of sterilized waters, suction is beaten uniformly, by that analogy, does doubling dilution.Take 10-4With 10-5Dilution is applied to flat board culture
Base, 50-250 microlitres of dilution bacterium solution is applied on every piece of flat board, and each dilution gradient applies polylith flat board.After bacterium solution is cultured base absorption
Culture 2-7 days is inverted in incubator for 25-35 DEG C.
(3) screen：Choose the bacterial strain that growth is rapid, single bacterium colony is big to continue to cultivate, preserve, carry out next step space mutagenesis examination
Test.
2nd, space mutagenesis test
(1) divine boat's ride on Bus No. 11 carries preparation of samples
The inoculation that ground simulation space mutagenesis is filtered out is on solid medium, in order to carry out space mutagenesis examination
Test.Finished inclined-plane is put into incubator, culture 3 days is carried out under the conditions of 25~35 DEG C, in order to carry out space mutagenesis examination
Test.Aseptically, scrape one piece of cultured inclined-plane to add in 2mL loading tubules, sealed membrane sealing, then by loading tubule
It is fitted into the loading big pipe that volume is 30-50mL.Preserved before delivering carrying in 4 DEG C.It is relevant to give loading big pipe to firing base
Personnel, on Shenzhou 11 spacecraft, space mutagenesis test is carried out with airship.This loading pipe is put into " divine boat's ride on Bus No. 11 " and flown
In ship, reclaimed after being flown 33 days with airship in space.
(2) environmental condition of space mutagenesis is implemented
This time space mutagenesis, mainly deploy after divine boat's ride on Bus No. 11 and No. two spacecrafts rendezvous of Heavenly Palace, space environment
Related data is：Apart from 3393 kilometers of ground；Flight 30 days；Level of Microgravity：10-3-10-6g；Dose of radiation test result table
Bright, dose of radiation 0.1-0.9mGy in space capsule, the average daily dose of this flight is in 0.5mGy.The temperature range that Heavenly Palace two：
19-23 DEG C, humidity is 50% or so.Oxygen concentration is consistent with ground.
(3) cultivate：Reclaim loading pipe.The carrying that culture area is 0.5 square centimeter is aseptically taken to reclaim sample,
With 5 milliliters of sterile water elution thalline into sterile centrifugation tube, inhaled with pipettor and break uniform thalline eluent, take 0.5 milliliter of bacterium
Liquid into 4.5 milliliters of sterilized waters, beat uniformly, by that analogy, does doubling dilution by suction.Take 10-4With 10-5Dilution again is applied to
Plating medium, 50-250 microlitres of dilution bacterium solution is applied on every piece of flat board, and each dilution gradient applies polylith flat board.Treat that bacterium solution is cultured
Base absorbs is inverted culture 2-7 days after 25-35 DEG C in incubator.
3rd, screen
(1) making of screening flat board
1) screening flat board is by following several material compositions：Glucose 2%, peptone 2%, yeast extract powder 1%, agar
1.5-2.0%, sterile distilled water, natural pH；1ml " ethyl acetate:Caproic acid=1:10 " mixed liquor, melting concn 10g/L,
Natural pH.
2) 125 DEG C of sterilizing 25min
3) a diameter of 90mm for bacterium of having gone out culture dish is taken, each culture dish 30ml of falling culture medium, 37 DEG C are cultivated 24 hours,
Choose aseptic flat board and enter next working link, pollution flat board sterilizing is destroyed.
(2) it is inoculated with
Select and rapid single bacterium colony is grown after space mutagenesis, method of scoring is inoculated in screening flat board well prepared in advance.
(3) cultivate
By the flat board being inoculated with mold incubator, 30 DEG C incubated 3 days.
(4) bacterium colony is selected
The fast single bacterium colony of the speed of growth on flat board is chosen.Method of scoring is inoculated into No. 1 production medium of oral liquid, you can
Into production fermenting step.
(5) ferment
1) liquid medium is calculated (4 bottles of packing) with every liter
| Liquid medium composition | Liquid Culture liquid hold-up |
| Brewer's wort | 300mL |
| Glucose | 60.8g |
| Calcium carbonate | 0.2g |
| Zinc sulfate | 0.05g |
| Distilled water | 700mL |
Sterilize 20min at 121 DEG C.
2) bacterium is connect
1. take carrying before, carry after single bacterium colony flat board, be inoculated in fluid nutrient medium；
2. every bottle of 1 ring strain of inoculation；
3. it is parallel respectively to do 2-3 groups before carrying, after carrying；
4. blank cultures reserve 2-3 bottles；
Carry front and rear single bacterium colony plating in fluid nutrient medium 5. taking, every bottle connects 1 single bacterium colony, similarly hereinafter 3. -4.；
3) shaking table culture
30℃、200r/min；Blank cultures are cultivated simultaneously with bacterium solution.
4) bacterium is connect to mix (being now culture 0h), be sampled detection at regular intervals
1. detection method
The same time takes blank cultures and bacterium solution to carry out ultraviolet specrophotometer measure, using blank cultures as control,
Wavelength 600nm, determine bacterium solution absorbance；
In the case of the ＜ 1 of absorbance, using detected value；In the case of absorbance ＞ 1, with the undiluted liquid of blank cultures
For control, 10 times, 100 times are carried out to bacterium solution or more high magnification numbe dilutes, to absorbance ＜ 1；
2. testing result
The testing inspection result of embodiment 1
| Numbering | Title | OD600 before carrying | Carry OD600 |
| 1 | Cultivate 0h | 0.449 | 0.452 |
| 2 | Cultivate 5h- | 1.007 | 1.054 |
| 3 | Cultivate 7h | 4.660 | 4.990 |
| 4 | Cultivate 24h | 9.735 | 9.880 |
| 5 | Cultivate 72h | 11.000 | 12.35 |
5) result
Fermented result verification, mutagenic strain improve 12.27% than the growth rate of original strain.
Embodiment 2：
The strain culturing method before space mutagenesis is simulated with embodiment 1.
1st, ground simulation space mutagenesis
(1) vibration test of space flight is simulated：Cultured S. cervisiae flat board (Φ 9cm) is fixed on 400kN
On Double earthquakes, 1h is tested, it is low frequency sinusoidal vibration test to start 0.5 hour, and latter 0.5 hour is random vibration test.This examination
The situation of vibration when testing the transmitting of simulated flight device and returning.
(2) cultivate：The analog sample that culture area is 0.5 square centimeter is aseptically taken, with 5 milliliters of sterile washings
De- thalline is inhaled with pipettor into sterile centrifugation tube and breaks uniform thalline eluent, take 0.5 milliliter of bacterium solution sterile to 4.5 milliliters
In water, suction is beaten uniformly, by that analogy, does doubling dilution.Take 10-4With 10-5Times dilution be applied to plating medium, every piece
50-250 microlitres of dilution bacterium solution is applied on flat board, each dilution gradient applies polylith flat board.Treat that bacterium solution is cultured base and absorbed after culture
Culture 2-7 days is inverted in case for 25-35 DEG C.
(3) screen：Choose the bacterial strain that growth is rapid, single bacterium colony is big to continue to cultivate, preserve, carry out next step space mutagenesis examination
Test.
2nd, space mutagenesis test is same as Example 1.
3rd, screen
(1) making of screening flat board
1) screening flat board is by following several material compositions：1) glucose 2%, peptone 2%, yeast extract powder 1%, agar
1.5-2.0%, natural pH；1ml " cognac oil：Acetic acid：Butyric acid=1：5：1 " mixed liquor, melting concn 100g/L are natural
pH。
2) sterilize 25min at 121 DEG C.
3) a diameter of 90mm for bacterium of having gone out culture dish is taken, each culture dish 30ml of falling culture medium, 37 DEG C are cultivated 24 hours,
Choose aseptic flat board and enter next working link, pollution flat board sterilizing is destroyed.
(2) it is inoculated with
Select and rapid single bacterium colony is grown after space mutagenesis, method of scoring is inoculated in screening flat board well prepared in advance.
(3) cultivate
By the flat board being inoculated with mold incubator, 30 DEG C incubated 3 days.
(4) bacterium colony is selected
The fast single bacterium colony of the speed of growth on flat board is chosen.Method of scoring is inoculated into No. 1 production medium of oral liquid, you can
Into production fermenting step.
Fermentation process is same as Example 1.
The testing inspection result of embodiment 2
| Numbering | Incubation time h | OD600 before carrying | OD600 after carrying |
| 1 | Cultivate 0h | 0.670 | 0.758 |
| 2 | Cultivate 5h | 0.786 | 0.895 |
| 3 | Cultivate 10h | 8.380 | 8.790 |
| 4 | Cultivate 25h | 32.500 | 42.900 |
| 5 | Cultivate 30h | 26.700 | 32.600 |
Fermented result verification, the growth rate of the bacterial strain improve 22.09% than starting strain, and effect is very notable.
Embodiment 3：
The strain culturing method before space mutagenesis is simulated with embodiment 1.
1st, ground simulation space mutagenesis test is same as Example 1.
2nd, space mutagenesis test is same as Example 1.
3rd, screen
(1) making of screening flat board
1) screening flat board is by following several material compositions：1) glucose 2%, peptone 2%, yeast extract powder 1%, agar
1.5-2.0%, natural pH；1ml ethyl acetate, solution concentration 1g/L, natural pH..
2) 121 DEG C of sterilizing 25min
3) a diameter of 90mm for bacterium of having gone out culture dish is taken, each culture dish 30ml of falling culture medium, 37 DEG C are cultivated 24 hours,
Choose aseptic flat board and enter next working link, pollution flat board sterilizing is destroyed.
(2) it is inoculated with
Select and rapid single bacterium colony is grown after space mutagenesis, method of scoring is inoculated in screening flat board well prepared in advance.
(3) cultivate
By the flat board being inoculated with mold incubator, 30 DEG C incubated 3 days.
(4) bacterium colony is selected
The fast single bacterium colony of the speed of growth on flat board is chosen.Method of scoring is inoculated into No. 1 production medium of oral liquid, you can
Into production fermenting step.
(5) ferment same as Example 1.
As a result：The fermented checking of the bacterial strain, the growth rate of the bacterial strain improve 13.19% than starting strain, and effect shows
Write.
Embodiment 4：
The strain culturing method before space mutagenesis is simulated with embodiment 1.
1st, the vibration test of ground simulation space mutagenesis is identical with embodiment 21.
(1) vibration test of space flight is simulated：Test the vibration test with 1, (1) simulation space flight in embodiment 2；
(2) cultivate：Culture experiment after simulation is cultivated with 1, (2) in embodiment 2；
(3) screen：With embodiment 21, (3) screening；
Choose the bacterial strain that growth is rapid, single bacterium colony is big and carry out simulation space mutagenesis test.
2nd, vacuum low energy particle mutagenesis is tested
(1) vacuum low energy particle mutagenesis：With 1, (2) in embodiment 1；
(2) cultivate：With 1 in embodiment 1, (3)；
(3) screen：With 1 in embodiment 1, (4).
3rd, screen
(1) making of screening flat board
1) screening flat board is by following several material compositions：1) glucose 2%, peptone 2%, yeast extract powder 1%, agar
1.5-2.0%, natural pH；1ml " cognac oil：Acetic acid：Butyric acid=1：5：1 " mixed liquor, melting concn 100g/L are natural
pH。
2) 121 DEG C of sterilizing 25min
3) a diameter of 90mm for bacterium of having gone out culture dish is taken, each culture dish 30ml of falling culture medium, 37 DEG C are cultivated 24 hours,
Choose aseptic flat board and enter next working link, pollution flat board sterilizing is destroyed.
(2) it is inoculated with
Select and grown rapidly after space mutagenesis, method of scoring is inoculated in screening flat board well prepared in advance.
(3) cultivate
By the flat board being inoculated with mold incubator, 30 DEG C incubated 3 days.
(4) bacterium colony is selected
The fast single bacterium colony of the speed of growth on flat board is chosen.Method of scoring is inoculated into the production medium of east 1, you can is entered
Enter to produce fermenting step.
(5) ferment same as Example 1.
As a result：The fermented checking of the bacterial strain, growth rate improve 33.78% than starting strain, and effect is very notable.
Specific case used herein is elaborated to inventive concept, and the explanation of above example is only intended to
Help to understand core concept of the invention.It should be pointed out that for those skilled in the art, this is not being departed from
On the premise of inventive concept, any obvious modification, equivalent substitution or other improvement for being made, the present invention should be included in
Protection domain within.
Claims (10)
1. a kind of saccharomyces cerevisiae mutant bacterial, it is characterised in that the mutant strain is that yeast strain lures through ground simulation space
Become and/or space mutagenesis test after, the fast single bacterium colony of the relative growth rate of screening, Classification And Nomenclature：Saccharomyces cerevisiae
Saccharomyces cerevisiae, microbial strain：Shenzhou 11-4, preserving number are Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center CGMCC No.14448.
2. saccharomyces cerevisiae mutant bacterial according to claim 1, it is characterised in that the speed of growth of the mutant strain with
Yeast strain starting strain can grow compared to 10-40%, the mutant strain is improved under 45 DEG C of high temperature, the optimal pH of growth
It is worth for 4.5-6.0.
3. saccharomyces cerevisiae mutant bacterial according to claim 1, it is characterised in that the mutant strain has without fungal filament
Concentric ring.
4. saccharomyces cerevisiae mutant bacterial according to claim 1, it is characterised in that ground simulation space mutagenesis tries for vibration
Test and/or vacuum low energy particle mutagenesis.
5. saccharomyces cerevisiae mutant bacterial according to claim 1, it is characterised in that in the space mutagenesis test, space
Environment is：Level of Microgravity 10-3-10-6G, level of vibration 0-400kN；Dose of radiation 0.1-0.9mGy in space capsule.
6. the saccharomyces cerevisiae mutant bacterial according to claim any one of 1-5 is in biology, medicine, fermentation, functional nutrient
Application in terms of source, food or feed.
A kind of 7. method of mutagenesis of the saccharomyces cerevisiae mutant bacterial described in any one of claim 1-5, it is characterised in that specific step
Suddenly it is：
(1) culture of starting strain：Saccharomyces cerevisiae starting strain is seeded on YEPD solid mediums and cultivated, cultivates 1-
10 days, it is stand-by to filter out the faster Wine brewing yeast strain of the speed of growth；
(2) mutagenesis：The bacterial strain of screening is done into doubling dilution through ground simulation space mutagenesis and/or space mutagenesis, mutagenic strain, taken
10-4With 10-5Times dilution carries out culture 2-7 days.
8. method of mutagenesis according to claim 7, it is characterised in that ground simulation space mutagenesis be vibration test and/or
Vacuum low energy particle mutagenesis.
A kind of 9. screening technique of the saccharomyces cerevisiae mutant bacterial described in any one of claim 1-5, it is characterised in that specific step
Suddenly it is：
(1) screen：Saccharomyces cerevisiae metabolite is added in the YEPD solid mediums prepared, screening and culturing medium is formed, will lure
Become inoculation in being cultivated on screening and culturing medium, filter out the speed of growth faster, the bacterial strain that changes greatly of colonial morphology is as standby
Select bacterial strain.
(2) ferment：By alternative inoculation in fluid nutrient medium, shaking table culture fermentation, the final OD values by zymotic fluid are really
Determine the fast saccharomyces cerevisiae mutant bacterial of speed of production.
10. screening technique according to claim 9, it is characterised in that the saccharomyces cerevisiae metabolite be ethyl acetate,
Cognac oil, acetic acid, butyric acid and/or caproic acid, the concentration of the metabolite is 10-3g/L—102g/L。
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| WO2023039922A1 (en) * | 2021-09-16 | 2023-03-23 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Mutant strain selected using aerospace breeding technology |
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