CN107868778A - A kind of preparation method of Se-enriched yeast - Google Patents
A kind of preparation method of Se-enriched yeast Download PDFInfo
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Abstract
The present invention relates to fermentation industry technical field, and in particular to a kind of preparation method of Se-enriched yeast.The present invention selects Bama County of Guangxi pedotheque, Se content in bar horse soil, cereal is higher than more than 10 times of average national level, by to barms screening and breeding in pedotheque, culture obtains the yeast seeds of selenium-rich, plasma selenium concentration, condition of culture are optimized, improve the raising of saccharomycete Se content；Yeast rich in selenium of the invention by being obtained to separation screening in selenium-rich soil, by using the domestication of the sodium selenite of gradient concentration, the high yeast colony of anti-selenium ability is screened, then carry out ultraviolet mutagenesis, strengthen its selenium rich ability, obtain obtaining the yeast that biomass is high, selenium rich ability is strong.
Description
Technical field
The present invention relates to fermentation industry technical field, and in particular to a kind of preparation method of Se-enriched yeast.
Background technology
It is well known that selenium is a kind of essential trace element in humans and animals body, it is organism glutathion inside peroxidating
The important component of compound, there are many biological functions, wherein most important biological function is to remove in organism to produce
Raw excessive active oxygen radical, thus have the function that to improve organism immune power, anti-aging, anticancer.In addition, in Environmental Arsenic
Poison is a worldwide problem, and its harm is not showed only as recent arsenic and the health of humans and animals is harmful to, and more seriously draws
Play human body long term malignization change-canceration, mutation and distortion.According to arsenic-selenium can antidote each other medicine principle, in addition
Selenium has the biological function suppressed with anticancer change, anti-mutation and anti-distortion, and the yeast rich in selenium is in prevention and treatment region
Obvious action will be played in terms of poisoning.In addition, yeast cells must also containing abundant protein, nucleic acid, vitamin and body
The 14 kinds of trace elements needed, to the healthy and beneficial of body.
Selenium is the essential trace elements of the human body.The nutrition of selenium is mainly particularly combined performance with zymoprotein by protein
Antioxidation.Se-enriched yeast refers to add selenium element during culture yeasts, has absorbed selenium during yeast growth, has made
Selenium is combined with the protein in yeast and polysaccharide organic is converted into Biological Selenium, so as to eliminate chemical selenium（Such as sodium selenite）It is right
The toxicity and stomach of human body stimulate, and enable that selenium is more efficient, be more safely absorbed by the body utilization.Se-enriched yeast is by human body
After absorption, free radical and peroxidating in human body cell can effectively be removed by the synergy with other zymoproteins and vitamin
Thing, so as to delay the aging of cell, prevention and suppression tumour, anti-aging, the normal structure of cardiovascular system and function are maintained, in advance
Anti- artery and hardening and the appearance of coronary heart disease.Yeast has the selenium rich ability of height, inorganic selenium can be converted into Organic Selenium, therefore
Se-enriched yeast is widely used in the source as microelement-supplementing selenium in food, health products and feedstuff industry.
Currently, the Se-enriched yeast having had a large amount in variety on the market, its production technology is different, richness of the prior art
Selenium yeast is mainly enlarged culture by raw material of molasses, but its damage ratio is more serious using molasses as raw material so that environmentally friendly to take
With height, and the Se content in obtained Se-enriched yeast only has 1000~2000ppm, and the content of selenium is relatively low, is insufficient for
Human wants, and its maximum economic benefit can not be played.
In summary, the Se content of Se-enriched yeast is relatively low mainly following 2 reasons：One is not to barms
Any screening and breeding are carried out, at random with a kind of barms, because different barmses have larger difference to the resistance of selenium, from
It is and also very big to the absorption and conversion capability difference of plasma selenium.The second is not to producing the culture medium of fermented and cultured Se-enriched yeast
Plasma selenium concentration, condition of culture optimize, although therefore the yeast of culture be referred to as " Se-enriched yeast ", it is this be referred to as it is " rich
The Se content of selenium yeast " cell is relatively low, and the biomass of yeast cells is relatively low.
Method substantially has three kinds with being referred to as " Se-enriched yeast " cell for production in the prior art, and the first is to use mixed processing
Method, a certain amount of selenate or selenite are mixed with yeast cells.Its major defect is：It is not by inorganic selenium
Organic Selenium is transformed into, this selenate or selenite can make one with so-called " Se-enriched yeast " that yeast cells mixes
Very low to its absorptivity with animal, caused biological action is poor, is not real Se-enriched yeast.
Second is to use simple adsorption method, by the Saccharomyces cerevisiae of in the market or beer yeast cells be suspended in containing
Certain time is handled in the aqueous solution of selenate or selenite, plasma selenium is adsorbed in yeast cell surface, its major defect
It is：This processing still can not transform into inorganic selenium Organic Selenium, and the yeast of this absorption selenate or selenite is thin
Difference of the born of the same parents with above-mentioned first method without essence, can make human or animal very low to its absorptivity, caused biology is made
Equally it is not real Se-enriched yeast with poor.
The third is the culture yeasts cell in the culture medium containing plasma selenium, but barms is not carried out any
Screening and breeding, are with a kind of barms, its major defect at random：Due to different barmses have to the resistance of selenium it is larger
Difference, so as to which the absorption and conversion capability to plasma selenium is widely different.In addition, this technology is to the plasma selenium concentration of culture medium, training
Foster condition is not optimized, therefore using in the yeast of this technology culture, organic selenium content is relatively low, and every gram of dry yeast cell contains
Below the microgram of Organic Selenium 500, cellular biomass it is also low, so as to economic benefit it is also very low.
Therefore it provides the preparation method of a kind of efficient and easy Se-enriched yeast, is adapted to the demand of society with this, is
One thing being extremely necessary.
The content of the invention
The technical problems to be solved by the invention：For carrying out any screening and breeding currently without to barms, with
A kind of barms of machine, the problem of not optimized to the selenium rich ability of Se-enriched yeast for producing fermented and cultured, there is provided
A kind of preparation method of Se-enriched yeast.
In order to solve the above technical problems, the present invention is using technical scheme as described below：
A kind of preparation method of Se-enriched yeast, the preparation method comprise the following steps：
（1）Soil sample is added in saccharomycete fluid nutrient medium, culture, Yeast Cultivation liquid is obtained, takes Yeast Cultivation liquid mass fraction
Gradient dilution is carried out to 10 for 0.9% physiological saline-6Level is diluted, yeast bacteria culture fluid after must diluting, yeast bacteria culture fluid is applied
It is distributed on saccharomycete plating medium, cultivates, the saccharomycete single bacterium colony plate streaking of picking bacterium footpath maximum 2 ~ 3 times, obtains after purification
Saccharomycete single bacterium colony；
（2）The above-mentioned single bacterium colony of saccharomycete after purification of picking is seeded to saccharomycete fluid nutrient medium, activation culture, obtains saccharomycete activation
Culture, by sodium selenite in mass ratio 1:1000 add domestication and screening and culturing medium mixing, obtain one-level domestication and screening and culturing
Base, saccharomycete activation culture thing is seeded to one-level domestication and screening and culturing medium, culture, obtains saccharomycete bacterium colony after one-level domestication,
By sodium selenite in mass ratio 1:500 add domestication and screening and culturing medium mixing, obtain two level domestication and screening and culturing medium, picking are tamed and dociled
Change saccharomycete colony inoculation to two level domestication and screening and culturing medium, culture, obtain secondary yeast fermented liquid, secondary yeast bacterium is sent out
Zymotic fluid is diluted to 10 with sterilized water-3Dilution level, by sodium selenite in mass ratio 1:300 add domestication and screening and culturing medium
Mixing, three-level domestication and screening and culturing medium are obtained, dilution after fermentation liquid is coated on three-level domestication and screening and culturing medium culture, obtains three
Saccharomycete bacterium colony after level domestication, three-level Yeast culture is repeated into coating culture 3 ~ 4 times, obtains saccharomycete bacterium after the domestication of selenium resistance
Fall；
（3）Saccharomycete colony inoculation activation culture, obtains activated yeast into saccharomycete plating medium after the domestication of picking selenium resistance
Bacterium colony, take sterile water washing activated yeast to fall, pour into the container for filling bead, vibrate, and in being shaken on shaking table, obtain
Unicellular bacteria suspension, is counted with blood counting chamber, and it is 10 to be adjusted unicellular bacteria suspension to cell concentration with sterilized water-6~10-7
Individual/mL, uviol lamp preheating, takes the unicellular bacteria suspension prepared to be placed in sterilized petri dishes, puts and stirred on magnetic stirring apparatus, ultraviolet
Light irradiation, the bacteria suspension of processing is obtained, take the bacteria suspension 0.1mL of processing to be coated on saccharomycete plating medium, cultivated, pick out bacterium
The maximum single bacterium colony in footpath is seeded to kind in fermentation medium, culture, obtains yeast bacteria culture fluid after ultraviolet mutagenesis, dries, obtains purple
Yeast strain after outer mutagenesis；
（4）By sodium selenite in mass ratio 1:250 add fermentation medium mixing, Selenium-enriched fermentation culture medium are obtained, after taming mutagenesis
Yeast strain is seeded to Selenium-enriched fermentation culture medium, culture, must tame mutagenesis Yeast culture, centrifuges, precipitation is taken, with distillation
Water washing precipitates, and adds distilled water, centrifuges, except supernatant, this step 2 ~ 3 times repeatedly, obtains nutrient solution wet yeast, will be wet
Yeast drying, cooling, obtains Se-enriched yeast powder.
The step（1）Middle pedotheque is Bama County of Guangxi pedotheque, soil sample and the matter of saccharomycete fluid nutrient medium
Amount is than being 4:9, condition of culture is 30 ~ 35 DEG C of 18 ~ 24h of 200r/min shaking table cultures.
The step（1）The formula of middle saccharomycete plating medium is according to the mass fraction, to take 12 ~ 15 parts of glucose, ferment
Female 8 ~ 10 parts of cream, 5 ~ 7 parts of peptone, 0.02 ~ 0.05 part of magnesium sulfate, 15 ~ 20 parts of agar, pH value is naturally, 121 DEG C of sterilizing 20min；
Saccharomycete fluid nutrient medium：Remove the agar in the formula of saccharomycete plating medium, other components are constant.
The step（2）In the mass ratio of saccharomycete single bacterium colony and saccharomycete fluid nutrient medium after purification be 1:9, one-level is tamed and dociled
Sodium selenite and the mass ratio of domestication and screening and culturing medium are 1 in change and screening and culturing medium:1000, saccharomycete activation culture thing with
One-level is tamed and the mass ratio of screening and culturing medium is 1:8, sodium selenite and domestication and screening in two level domestication and screening and culturing medium
The mass ratio of culture medium is 1:500, sodium selenite and domestication and the quality of screening and culturing medium in three-level domestication and screening and culturing medium
Than for 1:300；The formula of domestication and screening and culturing medium takes 8 ~ 10 parts of glucose for according to the mass fraction, 15 ~ 18 parts of yeast extract,
5 ~ 7 parts of peptone, 0.02 ~ 0.05 part of magnesium sulfate, 15 ~ 20 parts of agar, 10 ~ 12 parts of sodium selenite, pH value is naturally, 121 DEG C of sterilizings
20min。
The step（3）The condition of middle ultraviolet mutagenesis is that 180s is irradiated at 15W uviol lamp 25cm distances.
The step（4）The formula of middle fermentation medium takes 10 ~ 12 parts of glucose for according to the mass fraction, and yeast extract 18 ~
20 parts, 1 ~ 2 part of dipotassium hydrogen phosphate, 10 ~ 15 parts of beef extract, 8 ~ 10 parts, pH4.8 ± 0.2,121 DEG C sterilizing 20min of peptone.
The step（4）The mass ratio of middle yeast strain and Selenium-enriched fermentation culture medium is 1:9, precipitate the matter with distilled water
Amount is than being 1:50.
Compared with other method, advantageous effects are the present invention：
（1）The present invention selects Bama County of Guangxi pedotheque, and the Se content in bar horse soil, cereal is higher than average national level 10
More than times, by the way that to barms screening and breeding in pedotheque, culture obtains the yeast seeds of selenium-rich, dense to plasma selenium
Degree, condition of culture optimize, and improve the raising of saccharomycete Se content；
（2）Yeast rich in selenium of the invention by being obtained to separation screening in selenium-rich soil, by using gradient concentration
The domestication of sodium selenite, the high yeast colony of anti-selenium ability is screened, then carry out ultraviolet mutagenesis, strengthened its selenium rich ability, obtain
To the yeast that acquisition biomass is high, selenium rich ability is strong.
Embodiment
Raw material：Bama County of Guangxi soil.
Saccharomycete plating medium：According to the mass fraction, 12 ~ 15 parts of glucose, 8 ~ 10 parts of yeast extract, peptone 5 ~ 7 are taken
Part, 0.02 ~ 0.05 part of magnesium sulfate, 15 ~ 20 parts of agar, pH value is naturally, 121 DEG C of sterilizing 20min.
Saccharomycete fluid nutrient medium：Remove the agar in saccharomycete plating medium, other components are constant.
Domestication and screening and culturing medium：As mass fraction, 8 ~ 10 parts of glucose, 15 ~ 18 parts of yeast extract, peptone 5 ~ 7 are taken
Part, 0.02 ~ 0.05 part of magnesium sulfate, 15 ~ 20 parts of agar, 10 ~ 12 parts of sodium selenite, pH value is naturally, 121 DEG C of sterilizing 20min.
Fermentation medium：According to the mass fraction, 10 ~ 12 parts of glucose, 18 ~ 20 parts of yeast extract, dipotassium hydrogen phosphate 1 ~ 2 are taken
Part, 10 ~ 15 parts of beef extract, 8 ~ 10 parts of peptone, 10 ~ 12 parts, pH4.8 ± 0.2,121 DEG C sterilizing 20min of sodium selenite.
A kind of preparation method of Se-enriched yeast, comprises the following steps：
（1）By Bama County of Guangxi pedotheque in mass ratio 4:9 add in saccharomycete fluid nutrient medium, 200r/ at 30 ~ 35 DEG C
Min 18 ~ 24h of shaking table culture, obtain Yeast Cultivation liquid, take Yeast Cultivation liquid to carry out gradient with the physiological saline that mass fraction is 0.9%
It is diluted to 10-6Level is diluted, yeast bacteria culture fluid after must diluting, yeast bacteria culture fluid is coated on saccharomycete plating medium,
36 ~ 48h is cultivated in 30 ~ 35 DEG C of constant incubators, the saccharomycete single bacterium colony plate streaking of picking bacterium footpath maximum 2 ~ 3 times, is obtained pure
Saccharomycete single bacterium colony after change；
（2）The above-mentioned single bacterium colony of saccharomycete after purification in mass ratio 1 of picking:9 are seeded to saccharomycete fluid nutrient medium, 25 ~ 30 DEG C of work
Change 18 ~ 24 h of culture, saccharomycete activation culture thing is obtained, by sodium selenite in mass ratio 1:1000 add domestication and screening and culturing medium
Mixing, one-level domestication and screening and culturing medium are obtained, saccharomycete activation culture thing is seeded to one-level domestication and screening and culturing medium, by ferment
Female bacterium activation culture thing in mass ratio 1:8 are seeded to the domestication of one-level and screening and culturing medium, 25 ~ 33 DEG C of 36 ~ 48h of culture, obtain one
Saccharomycete bacterium colony after level domestication, by sodium selenite in mass ratio 1:500 add domestication and screening and culturing medium mixing, obtain two level domestication
And screening and culturing medium, picking domestication saccharomycete colony inoculation is tamed to two level and screening and culturing medium, and 25 ~ 30 DEG C of shaking table cultures 36 ~
48h, secondary yeast fermented liquid is obtained, secondary yeast fermented liquid is diluted to 10-3 dilution level with sterilized water, by Asia
Sodium selenate in mass ratio 1:300 add domestication and screening and culturing medium mixing, obtain three-level domestication and screening and culturing medium, will be sent out after dilution
Zymotic fluid is coated on three-level domestication and screening and culturing medium culture, obtains three-level Yeast culture, three-level Yeast culture is repeated
Coating culture 3 ~ 4 times, obtain saccharomycete bacterium colony after the domestication of selenium resistance；Overcoating cloth culture 3 ~ 4 times, obtain saccharomycete bacterium after the domestication of selenium resistance
Fall；
（3）Saccharomycete colony inoculation is into saccharomycete plating medium after the domestication of picking selenium resistance, 25 ~ 30 DEG C of constant temperature activation cultures
12 ~ 18h, activated yeast bacterium colony is obtained, take sterile water washing activated yeast to fall, pour into the container for filling bead, vibrated
30min, and in shaking 60 ~ 70min on shaking table, obtain unicellular bacteria suspension, counted with blood counting chamber, will be slender with sterilized water
It is 10-6 ~ 10-7/mL that born of the same parents' bacteria suspension, which is adjusted to cell concentration, uviol lamp preheating 30min, takes the unicellular bacteria suspension prepared
It is placed in sterilized petri dishes, puts and stirred on magnetic stirring apparatus, 180s is irradiated at 15W uviol lamp 25cm distances, obtains the bacteria suspension of processing,
Take the bacteria suspension 0.1ml of processing to be coated on saccharomycete plating medium, after 25 ~ 30 DEG C are cultivated 72h, pick out the maximum list in bacterium footpath
Colony inoculation 25 ~ 30 DEG C, 200r/min shaken cultivation 48h, obtains saccharomycete culture after ultraviolet mutagenesis to planting in fermentation medium
Liquid, dry, obtain yeast strain after ultraviolet mutagenesis；
（4）By sodium selenite in mass ratio 1:250 add fermentation medium mixing, obtain Selenium-enriched fermentation culture medium, will tame mutagenesis
Yeast strain in mass ratio 1 afterwards:9 are seeded to Selenium-enriched fermentation culture medium, shaken cultivation, must tame mutagenesis Yeast culture,
Centrifugation, takes precipitation, is precipitated with distillation water washing, then in mass ratio 1:50 add distilled water, centrifugation, except supernatant, this step are anti-
It is multiple 2 ~ 3 times, nutrient solution wet yeast is obtained, wet yeast is placed in 100 DEG C of baking oven and dries 6h, room temperature is cooled to, obtains selenium-rich
Dusty yeast.
Example 1
Saccharomycete plating medium：According to the mass fraction, 12 parts of glucose, 8 parts of yeast extract, 5 parts of peptone, magnesium sulfate 0.02 are taken
Part, 15 parts of agar, pH value is naturally, 121 DEG C of sterilizing 20min.
Saccharomycete fluid nutrient medium：Remove the agar in saccharomycete plating medium, other components are constant.
Domestication and screening and culturing medium：As mass fraction, 8 parts of glucose, 15 parts of yeast extract, 5 parts of peptone, magnesium sulfate are taken
0.02 part, 15 parts of agar, 10 parts of sodium selenite, pH value is naturally, 121 DEG C of sterilizing 20min.
Fermentation medium：According to the mass fraction, 10 parts of glucose, 18 parts of yeast extract, 1 part of dipotassium hydrogen phosphate, beef extract are taken
10 parts, 8 parts of peptone, 10 parts, pH4.8 ± 0.2,121 DEG C sterilizing 20min of sodium selenite.
Raw material：Bama County of Guangxi soil.
A kind of preparation method of Se-enriched yeast, comprises the following steps：
（1）By Bama County of Guangxi pedotheque in mass ratio 4:9 add in saccharomycete fluid nutrient medium, and 200r/min shakes at 30 DEG C
Bed culture 18h, obtains Yeast Cultivation liquid, takes Yeast Cultivation liquid to carry out gradient dilution extremely with the physiological saline that mass fraction is 0.9%
10-6Level is diluted, yeast bacteria culture fluid after must diluting, yeast bacteria culture fluid is coated on saccharomycete plating medium, at 30 DEG C
36h is cultivated in constant incubator, the saccharomycete single bacterium colony plate streaking of picking bacterium footpath maximum 2 times, obtains saccharomycete single bacterium after purification
Fall；
（2）The above-mentioned single bacterium colony of saccharomycete after purification in mass ratio 1 of picking:9 are seeded to saccharomycete fluid nutrient medium, 25 ~ 30 DEG C of work
Change 18 ~ 24 h of culture, saccharomycete activation culture thing is obtained, by sodium selenite in mass ratio 1:1000 add domestication and screening and culturing medium
Mixing, one-level domestication and screening and culturing medium are obtained, saccharomycete activation culture thing is seeded to one-level domestication and screening and culturing medium, by ferment
Female bacterium activation culture thing in mass ratio 1:8 are seeded to the domestication of one-level and screening and culturing medium, 25 ~ 33 DEG C of 36 ~ 48h of culture, obtain one
Saccharomycete bacterium colony after level domestication, by sodium selenite in mass ratio 1:500 add domestication and screening and culturing medium mixing, obtain two level domestication
And screening and culturing medium, picking domestication saccharomycete colony inoculation is tamed to two level and screening and culturing medium, and 25 ~ 30 DEG C of shaking table cultures 36 ~
48h, secondary yeast fermented liquid is obtained, secondary yeast fermented liquid is diluted to 10-3 dilution level with sterilized water, by Asia
Sodium selenate in mass ratio 1:300 add domestication and screening and culturing medium mixing, obtain three-level domestication and screening and culturing medium, will be sent out after dilution
Zymotic fluid is coated on three-level domestication and screening and culturing medium culture, obtains three-level Yeast culture, three-level Yeast culture is repeated
Coating culture 3 ~ 4 times, obtain saccharomycete bacterium colony after the domestication of selenium resistance；
（3）Saccharomycete colony inoculation is into saccharomycete plating medium after the domestication of picking selenium resistance, 25 DEG C of constant temperature activation cultures
12h, activated yeast bacterium colony is obtained, takes sterile water washing activated yeast to fall, pour into the container for filling bead, vibrate 30min,
And in shaking 60min on shaking table, obtain unicellular bacteria suspension, counted with blood counting chamber, with sterilized water by unicellular bacteria suspension
It is 10-6 ~ 10-7/mL to adjust to cell concentration, uviol lamp preheating 30min, takes the unicellular bacteria suspension prepared to be placed in sterile
In plate, put and stirred on magnetic stirring apparatus, 180s is irradiated at 15W uviol lamp 25cm distances, the bacteria suspension of processing is obtained, takes processing
Bacteria suspension 0.1ml is coated on saccharomycete plating medium, after 25 DEG C are cultivated 72h, picks out the maximum single bacterium colony in bacterium footpath and is seeded to
Kind 25 DEG C, 200r/min shaken cultivation 48h, obtains yeast bacteria culture fluid after ultraviolet mutagenesis in fermentation medium, dries, obtains purple
Yeast strain after outer mutagenesis；
（4）By sodium selenite in mass ratio 1:250 add fermentation medium mixing, obtain Selenium-enriched fermentation culture medium, will tame mutagenesis
Yeast strain in mass ratio 1 afterwards:9 are seeded to Selenium-enriched fermentation culture medium, shaken cultivation, must tame mutagenesis Yeast culture,
Centrifugation, takes precipitation, is precipitated with distillation water washing, then in mass ratio 1:50 add distilled water, centrifugation, except supernatant, this step are anti-
It is multiple 2 ~ 3 times, nutrient solution wet yeast is obtained, wet yeast is placed in 100 DEG C of baking oven and dries 6h, room temperature is cooled to, obtains selenium-rich
Dusty yeast.
Embodiment 2
Saccharomycete plating medium：According to the mass fraction, 13 parts of glucose, 9 parts of yeast extract, 6 parts of peptone, magnesium sulfate 0.03 are taken
Part, 17 parts of agar, pH value is naturally, 121 DEG C of sterilizing 20min.
Saccharomycete fluid nutrient medium：Remove the agar in saccharomycete plating medium, other components are constant.
Domestication and screening and culturing medium：As mass fraction, 9 parts of glucose, 16 parts of yeast extract, 6 parts of peptone, magnesium sulfate are taken
0.03 part, 17 parts of agar, 11 parts of sodium selenite, pH value is naturally, 121 DEG C of sterilizing 20min.
Fermentation medium：According to the mass fraction, 11 parts of glucose, 19 parts of yeast extract, 1 part of dipotassium hydrogen phosphate, beef extract are taken
13 parts, 9 parts of peptone, 11 parts, pH4.8 ± 0.2,121 DEG C sterilizing 20min of sodium selenite.
Raw material：Bama County of Guangxi soil.
A kind of preparation method of Se-enriched yeast, comprises the following steps：
（1）By Bama County of Guangxi pedotheque in mass ratio 4:9 add in saccharomycete fluid nutrient medium, and 200r/min shakes at 33 DEG C
Bed culture 22h, obtains Yeast Cultivation liquid, takes Yeast Cultivation liquid to carry out gradient dilution extremely with the physiological saline that mass fraction is 0.9%
10-6Level is diluted, yeast bacteria culture fluid after must diluting, yeast bacteria culture fluid is coated on saccharomycete plating medium, at 32 DEG C
42h is cultivated in constant incubator, the saccharomycete single bacterium colony plate streaking of picking bacterium footpath maximum 2 times, obtains saccharomycete single bacterium after purification
Fall；
（2）The above-mentioned single bacterium colony of saccharomycete after purification in mass ratio 1 of picking:9 are seeded to saccharomycete fluid nutrient medium, 25 ~ 30 DEG C of work
Change 18 ~ 24 h of culture, saccharomycete activation culture thing is obtained, by sodium selenite in mass ratio 1:1000 add domestication and screening and culturing medium
Mixing, one-level domestication and screening and culturing medium are obtained, saccharomycete activation culture thing is seeded to one-level domestication and screening and culturing medium, by ferment
Female bacterium activation culture thing in mass ratio 1:8 are seeded to the domestication of one-level and screening and culturing medium, 25 ~ 33 DEG C of 36 ~ 48h of culture, obtain one
Saccharomycete bacterium colony after level domestication, by sodium selenite in mass ratio 1:500 add domestication and screening and culturing medium mixing, obtain two level domestication
And screening and culturing medium, picking domestication saccharomycete colony inoculation is tamed to two level and screening and culturing medium, and 25 ~ 30 DEG C of shaking table cultures 36 ~
48h, secondary yeast fermented liquid is obtained, secondary yeast fermented liquid is diluted to 10-3 dilution level with sterilized water, by Asia
Sodium selenate in mass ratio 1:300 add domestication and screening and culturing medium mixing, obtain three-level domestication and screening and culturing medium, will be sent out after dilution
Zymotic fluid is coated on three-level domestication and screening and culturing medium culture, obtains three-level Yeast culture, three-level Yeast culture is repeated
Coating culture 3 ~ 4 times, obtain saccharomycete bacterium colony after the domestication of selenium resistance；
（3）Saccharomycete colony inoculation is into saccharomycete plating medium after the domestication of picking selenium resistance, 27 DEG C of constant temperature activation cultures
15h, activated yeast bacterium colony is obtained, takes sterile water washing activated yeast to fall, pour into the container for filling bead, vibrate 30min,
And in shaking 65min on shaking table, obtain unicellular bacteria suspension, counted with blood counting chamber, with sterilized water by unicellular bacteria suspension
It is 10-6 ~ 10-7/mL to adjust to cell concentration, uviol lamp preheating 30min, takes the unicellular bacteria suspension prepared to be placed in sterile
In plate, put and stirred on magnetic stirring apparatus, 180s is irradiated at 15W uviol lamp 25cm distances, the bacteria suspension of processing is obtained, takes processing
Bacteria suspension 0.1ml is coated on saccharomycete plating medium, after 27 DEG C are cultivated 72h, picks out the maximum single bacterium colony in bacterium footpath and is seeded to
Kind 27 DEG C, 200r/min shaken cultivation 48h, obtains yeast bacteria culture fluid after ultraviolet mutagenesis in fermentation medium, dries, obtains purple
Yeast strain after outer mutagenesis；
（4）By sodium selenite in mass ratio 1:250 add fermentation medium mixing, obtain Selenium-enriched fermentation culture medium, will tame mutagenesis
Yeast strain in mass ratio 1 afterwards:9 are seeded to Selenium-enriched fermentation culture medium, shaken cultivation, must tame mutagenesis Yeast culture,
Centrifugation, takes precipitation, is precipitated with distillation water washing, then in mass ratio 1:50 add distilled water, centrifugation, except supernatant, this step are anti-
It is multiple 2 ~ 3 times, nutrient solution wet yeast is obtained, wet yeast is placed in 100 DEG C of baking oven and dries 6h, room temperature is cooled to, obtains selenium-rich
Dusty yeast.
Embodiment 3
Saccharomycete plating medium：According to the mass fraction, 15 parts of glucose, 10 parts of yeast extract, 7 parts of peptone, magnesium sulfate are taken
0.05 part, 20 parts of agar, pH value is naturally, 121 DEG C of sterilizing 20min.
Saccharomycete fluid nutrient medium：Remove the agar in saccharomycete plating medium, other components are constant.
Domestication and screening and culturing medium：As mass fraction, 10 parts of glucose, 18 parts of yeast extract, 7 parts of peptone, sulfuric acid are taken
0.05 part of magnesium, 20 parts of agar, 12 parts of sodium selenite, pH value is naturally, 121 DEG C of sterilizing 20min.
Fermentation medium：According to the mass fraction, 12 parts of glucose, 20 parts of yeast extract, 2 parts of dipotassium hydrogen phosphate, beef extract are taken
15 parts, 10 parts of peptone, 12 parts, pH4.8 ± 0.2,121 DEG C sterilizing 20min of sodium selenite.
Raw material：Bama County of Guangxi soil.
A kind of preparation method of Se-enriched yeast, comprises the following steps：
（1）By Bama County of Guangxi pedotheque in mass ratio 4:9 add in saccharomycete fluid nutrient medium, and 200r/min shakes at 35 DEG C
Bed culture 24h, obtains Yeast Cultivation liquid, takes Yeast Cultivation liquid to carry out gradient dilution extremely with the physiological saline that mass fraction is 0.9%
10-6Level is diluted, yeast bacteria culture fluid after must diluting, yeast bacteria culture fluid is coated on saccharomycete plating medium, at 35 DEG C
48h is cultivated in constant incubator, the saccharomycete single bacterium colony plate streaking of picking bacterium footpath maximum 3 times, obtains saccharomycete single bacterium after purification
Fall；
（2）The above-mentioned single bacterium colony of saccharomycete after purification in mass ratio 1 of picking:9 are seeded to saccharomycete fluid nutrient medium, 25 ~ 30 DEG C of work
Change 18 ~ 24 h of culture, saccharomycete activation culture thing is obtained, by sodium selenite in mass ratio 1:1000 add domestication and screening and culturing medium
Mixing, one-level domestication and screening and culturing medium are obtained, saccharomycete activation culture thing is seeded to one-level domestication and screening and culturing medium, by ferment
Female bacterium activation culture thing in mass ratio 1:8 are seeded to the domestication of one-level and screening and culturing medium, 25 ~ 33 DEG C of 36 ~ 48h of culture, obtain one
Saccharomycete bacterium colony after level domestication, by sodium selenite in mass ratio 1:500 add domestication and screening and culturing medium mixing, obtain two level domestication
And screening and culturing medium, picking domestication saccharomycete colony inoculation is tamed to two level and screening and culturing medium, and 25 ~ 30 DEG C of shaking table cultures 36 ~
48h, secondary yeast fermented liquid is obtained, secondary yeast fermented liquid is diluted to 10-3 dilution level with sterilized water, by Asia
Sodium selenate in mass ratio 1:300 add domestication and screening and culturing medium mixing, obtain three-level domestication and screening and culturing medium, will be sent out after dilution
Zymotic fluid is coated on three-level domestication and screening and culturing medium culture, obtains three-level Yeast culture, three-level Yeast culture is repeated
Coating culture 3 ~ 4 times, obtain saccharomycete bacterium colony after the domestication of selenium resistance；
（3）Saccharomycete colony inoculation is into saccharomycete plating medium after the domestication of picking selenium resistance, 30 DEG C of constant temperature activation cultures
18h, activated yeast bacterium colony is obtained, takes sterile water washing activated yeast to fall, pour into the container for filling bead, vibrate 30min,
And in shaking 70min on shaking table, obtain unicellular bacteria suspension, counted with blood counting chamber, with sterilized water by unicellular bacteria suspension
It is 10-6 ~ 10-7/mL to adjust to cell concentration, uviol lamp preheating 30min, takes the unicellular bacteria suspension prepared to be placed in sterile
In plate, put and stirred on magnetic stirring apparatus, 180s is irradiated at 15W uviol lamp 25cm distances, the bacteria suspension of processing is obtained, takes processing
Bacteria suspension 0.1ml is coated on saccharomycete plating medium, after 30 DEG C are cultivated 72h, picks out the maximum single bacterium colony in bacterium footpath and is seeded to
Kind 30 DEG C, 200r/min shaken cultivation 48h, obtains yeast bacteria culture fluid after ultraviolet mutagenesis in fermentation medium, dries, obtains purple
Yeast strain after outer mutagenesis；
（4）By sodium selenite in mass ratio 1:250 add fermentation medium mixing, obtain Selenium-enriched fermentation culture medium, will tame mutagenesis
Yeast strain in mass ratio 1 afterwards:9 are seeded to Selenium-enriched fermentation culture medium, shaken cultivation, must tame mutagenesis Yeast culture,
Centrifugation, takes precipitation, is precipitated with distillation water washing, then in mass ratio 1:50 add distilled water, centrifugation, except supernatant, this step are anti-
It is multiple 2 ~ 3 times, nutrient solution wet yeast is obtained, wet yeast is placed in 100 DEG C of baking oven and dries 6h, room temperature is cooled to, obtains selenium-rich
Dusty yeast.
Comparative example：The Se-enriched yeast of Zhengzhou food science and technology Co., Ltd production
Method：Take the Se-enriched yeast as embodiment and prepared by comparative example of equivalent, detection wherein Se content.
The specific detection case of Se-enriched yeast such as table 1：
Table 1
| Detection project | Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example |
| Se content（mg/kg） | 113.5 | 127.6 | 143.8 | 58.5 |
From the foregoing, it will be observed that the Se-enriched yeast Se content prepared by the present invention is high, it is a kind of preparation side of safe and efficient Se-enriched yeast
Method, it is worthy to be popularized and uses.
Claims (7)
1. a kind of preparation method of Se-enriched yeast, it is characterised in that the preparation method comprises the following steps：
（1）Soil sample is added in saccharomycete fluid nutrient medium, culture, Yeast Cultivation liquid is obtained, takes Yeast Cultivation liquid mass fraction
Gradient dilution is carried out to 10 for 0.9% physiological saline-6Level is diluted, yeast bacteria culture fluid after must diluting, yeast bacteria culture fluid is applied
It is distributed on saccharomycete plating medium, cultivates, the saccharomycete single bacterium colony plate streaking of picking bacterium footpath maximum 2 ~ 3 times, obtains after purification
Saccharomycete single bacterium colony；
（2）The above-mentioned single bacterium colony of saccharomycete after purification of picking is seeded to saccharomycete fluid nutrient medium, activation culture, obtains saccharomycete activation
Culture, by sodium selenite in mass ratio 1:1000 add domestication and screening and culturing medium mixing, obtain one-level domestication and screening and culturing
Base, saccharomycete activation culture thing is seeded to one-level domestication and screening and culturing medium, culture, obtains saccharomycete bacterium colony after one-level domestication,
By sodium selenite in mass ratio 1:500 add domestication and screening and culturing medium mixing, obtain two level domestication and screening and culturing medium, picking are tamed and dociled
Change saccharomycete colony inoculation to two level domestication and screening and culturing medium, culture, obtain secondary yeast fermented liquid, secondary yeast bacterium is sent out
Zymotic fluid is diluted to 10 with sterilized water-3Dilution level, by sodium selenite in mass ratio 1:300 add domestication and screening and culturing medium
Mixing, three-level domestication and screening and culturing medium are obtained, dilution after fermentation liquid is coated on three-level domestication and screening and culturing medium culture, obtains three
Saccharomycete bacterium colony after level domestication, three-level Yeast culture is repeated into coating culture 3 ~ 4 times, obtains saccharomycete bacterium after the domestication of selenium resistance
Fall；
（3）Saccharomycete colony inoculation activation culture, obtains activated yeast into saccharomycete plating medium after the domestication of picking selenium resistance
Bacterium colony, take sterile water washing activated yeast to fall, pour into the container for filling bead, vibrate, and in being shaken on shaking table, obtain
Unicellular bacteria suspension, is counted with blood counting chamber, and it is 10 to be adjusted unicellular bacteria suspension to cell concentration with sterilized water-6~10-7
Individual/mL, uviol lamp preheating, takes the unicellular bacteria suspension prepared to be placed in sterilized petri dishes, puts and stirred on magnetic stirring apparatus, ultraviolet
Light irradiation, the bacteria suspension of processing is obtained, take the bacteria suspension 0.1mL of processing to be coated on saccharomycete plating medium, cultivated, pick out bacterium
The maximum single bacterium colony in footpath is seeded to kind in fermentation medium, culture, obtains yeast bacteria culture fluid after ultraviolet mutagenesis, dries, obtains purple
Yeast strain after outer mutagenesis；
（4）By sodium selenite in mass ratio 1:250 add fermentation medium mixing, Selenium-enriched fermentation culture medium are obtained, after taming mutagenesis
Yeast strain is seeded to Selenium-enriched fermentation culture medium, culture, must tame mutagenesis Yeast culture, centrifuges, precipitation is taken, with distillation
Water washing precipitates, and adds distilled water, centrifuges, except supernatant, this step 2 ~ 3 times repeatedly, obtains nutrient solution wet yeast, will be wet
Yeast drying, cooling, obtains Se-enriched yeast powder.
2. the preparation method of fruit Se-enriched yeast according to claim 1, it is characterised in that the step（1）Middle soil-like
Product are Bama County of Guangxi pedotheque, and the mass ratio of soil sample and saccharomycete fluid nutrient medium is 4:9, condition of culture is 30 ~ 35 DEG C
18 ~ 24h of 200r/min shaking table cultures.
3. the preparation method of fruit Se-enriched yeast according to claim 1, it is characterised in that the step（1）Middle saccharomycete
The formula of plating medium is according to the mass fraction, to take 12 ~ 15 parts of glucose, 8 ~ 10 parts of yeast extract, 5 ~ 7 parts of peptone, sulfuric acid
0.02 ~ 0.05 part of magnesium, 15 ~ 20 parts of agar, pH value is naturally, 121 DEG C of sterilizing 20min；Saccharomycete fluid nutrient medium：Remove saccharomycete
Agar in the formula of plating medium, other components are constant.
4. the preparation method of fruit Se-enriched yeast according to claim 1, it is characterised in that the step（2）In after purification
The mass ratio of saccharomycete single bacterium colony and saccharomycete fluid nutrient medium is 1:9, one-level domestication and screening and culturing medium in sodium selenite and
The mass ratio of domestication and screening and culturing medium is 1:1000, saccharomycete activation culture thing is tamed with one-level and the quality of screening and culturing medium
Than for 1:8, sodium selenite and the mass ratio of domestication and screening and culturing medium are 1 in two level domestication and screening and culturing medium:500, three-level
Sodium selenite and the mass ratio of domestication and screening and culturing medium are 1 in domestication and screening and culturing medium:300；Domestication and screening and culturing medium
Formula for according to the mass fraction, take 8 ~ 10 parts of glucose, 15 ~ 18 parts of yeast extract, 5 ~ 7 parts of peptone, magnesium sulfate 0.02 ~ 0.05
Part, 15 ~ 20 parts of agar, 10 ~ 12 parts of sodium selenite, pH value is naturally, 121 DEG C of sterilizing 20min.
5. the preparation method of fruit Se-enriched yeast according to claim 1, it is characterised in that the step（3）In ultraviolet lure
The condition of change is irradiation 180s at 15W uviol lamp 25cm distances.
6. the preparation method of fruit Se-enriched yeast according to claim 1, it is characterised in that the step（4）Middle fermentation training
The formula for supporting base is according to the mass fraction, to take 10 ~ 12 parts of glucose, 18 ~ 20 parts of yeast extract, 1 ~ 2 part of dipotassium hydrogen phosphate, beef extract
10 ~ 15 parts, 8 ~ 10 parts, pH4.8 ± 0.2,121 DEG C sterilizing 20min of peptone.
7. the preparation method of fruit Se-enriched yeast according to claim 1, it is characterised in that the step（4）Middle saccharomycete
The mass ratio of bacterial strain and Selenium-enriched fermentation culture medium is 1:9, it is 1 to precipitate with the mass ratio of distilled water:50.
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| CN109182128A (en) * | 2018-10-16 | 2019-01-11 | 广西壮族自治区农业科学院农业资源与环境研究所 | A kind of strain culturing method of Efficient Conversion Selenium in Soil |
| CN109362948A (en) * | 2018-10-23 | 2019-02-22 | 南宁市黄陈生猪养殖场 | A method of Se-enriched feedstuff additive is prepared using Moringa waste wood |
| CN109762775A (en) * | 2019-03-20 | 2019-05-17 | 山西龙盘微生物科技有限公司 | Compound rich strontium bacterial manure of one kind and preparation method thereof |
| CN114410490A (en) * | 2022-01-25 | 2022-04-29 | 淮阴工学院 | Production method of selenium-enriched yeast with high biomass |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109182128A (en) * | 2018-10-16 | 2019-01-11 | 广西壮族自治区农业科学院农业资源与环境研究所 | A kind of strain culturing method of Efficient Conversion Selenium in Soil |
| CN109362948A (en) * | 2018-10-23 | 2019-02-22 | 南宁市黄陈生猪养殖场 | A method of Se-enriched feedstuff additive is prepared using Moringa waste wood |
| CN109762775A (en) * | 2019-03-20 | 2019-05-17 | 山西龙盘微生物科技有限公司 | Compound rich strontium bacterial manure of one kind and preparation method thereof |
| CN114410490A (en) * | 2022-01-25 | 2022-04-29 | 淮阴工学院 | Production method of selenium-enriched yeast with high biomass |
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