CN110249913A - The method of the preparation and mycelia liquid deep layer fermenting of selenium-enriched edible mushroom fluid nutrient medium - Google Patents
The method of the preparation and mycelia liquid deep layer fermenting of selenium-enriched edible mushroom fluid nutrient medium Download PDFInfo
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- CN110249913A CN110249913A CN201810237398.9A CN201810237398A CN110249913A CN 110249913 A CN110249913 A CN 110249913A CN 201810237398 A CN201810237398 A CN 201810237398A CN 110249913 A CN110249913 A CN 110249913A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/50—Inoculation of spawn
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/60—Cultivation rooms; Equipment therefor
- A01G18/64—Cultivation containers; Lids therefor
- A01G18/68—Cultivation bottles
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Abstract
The invention belongs to the cultural methods of hypha of edible fungus, and in particular to the method for selenium-enriched edible mushroom liquid deep layer fermenting mycelia further relates to the preparation of selenium-enriched edible mushroom fermentation medium.A kind of cheap edible fungus liquid fermentation medium is developed, supplement selenium source production hypha of edible fungus is necessary in culture medium.Raw material of the present invention is mainly crops primary processed items and by-product, saves cost, and the carbon source and nitrogen source for being easy to absorb rich in edible mushroom through processed fermentation liquid also contain microelement and vitamin, be a kind of all nutrition type culture medium.Strain is screened through the resistance to selenium of overactivation, and the selenium in culture medium enhances strain to the adaptive faculty and tolerance of selenium by pretreatment.
Description
Technical field:
The invention belongs to the cultural methods of hypha of edible fungus, and in particular to the side of selenium-enriched edible mushroom liquid deep layer fermenting mycelia
Method further relates to the preparation of selenium-enriched edible mushroom fermentation medium.
Background technique:
There are mainly three types of the production ways of edible mushroom: one, artificial cultivation, its advantage is that it is simple and easy, it is at low cost.Its disadvantage
It is that cultivation period is long, the entire production cycle month is differed from 1 to 12.Two, solid fermentation, with solid medium culture edible fungi
Filament, to obtain secondary metabolite, mycelia, which covers with culture medium about, to be needed 30 days, and mycoplasma is dug out, and is dried for extracting.It excellent
Point: 1. culture medium is simple and from a wealth of sources, cheap；2. fermentation process does not need stringent sterile working generally；3. cultivating
Process oxygen supply and temperature can be controlled using direct air plenum；It, can be directly as 4. the processing of fermentation residue is simple
Feed or fertilizer.But its disadvantage is obvious: 1. culture medium water activity is low, and culture medium is uneven and is not easy to stir, the growth of thallus,
The secretion of absorption and metabolite to nutriment is all uneven, so that fermentation parameter detects and controls difficulty, but also
It operates continuously and automates and is highly difficult；2. microbial respiratory and the temperature control for being metabolized produced heat are extremely difficult, because solid
The heat conductivity of body culture medium is poor；3. cultural method is mostly based on empirical data due to solid factor understanding is influenced not enough
With the experience of operation, compared with liquid deep layer fermenting, large labor intensity is taken up a large area, miscellaneous bacteria easy to pollute.Three, deep liquid
Fermentation is reported in late 1980s earliest, and research contents includes nutritional need, environmental factor, zymotechnique etc., to obtain
Optimum operation condition produces a large amount of mycelium and metabolite within a short period of time.Advantage: it 1. can carry out industrializing continuous
Production, by control optimum condition come cultured mycelia, production technology specification；2. it is extensive to cultivate raw material sources, cheap；③
The devices such as air agitation system, humidity control system, pH value regulating system and culture medium make-up system by control fermentor,
Condition of culture is best, and fermentation period is short, high production efficiency.(Yang Hailong etc..Medicinal fungi submerged fermentation production technology, chemical work
Industry publishing house, 2009, p14-17, p212).But the current liquid submerged fermentation method (preparation of selenium enriched oyster mushroom powder
Method, application number: 200610135227.2；A kind of selenium-rich peace Lip river Marasmius fungus extract preparation and preparation method thereof, application number:
201210014153.2) the very big dependence glucose of raw material sources, peptone wait end products, and to add in right amount micro-
Secondary element and vitamin, operation cost are higher.
Selenium is a kind of chemical element,Chemical symbolIt is Se, period 4 VI A race is located in the periodic table of chemical element, is
It is a kind of nonmetallic.It may be used as nutrient necessary to light-sensitive material, electrolytic manganese industry catalyst, animal body and plant be beneficial
Nutrient etc..The existing way of selenium is divided into three kinds: selenium inorganic compound, organic selenium and elemental selenium.Inorganic selenium refers generally to industry selenium
Sour sodium and sodium selenate have biggish toxicity, and are not easy to be absorbed, and are not suitable for humans and animals and use.Organic selenium passes through bioconversion
Be combined into amino acid, generally in the form of selenomethionine exist, biological active selenium be human and animal allow using selenium
Source.
Therefore, a kind of cheap edible fungus liquid fermentation medium is developed, supplement selenium source produces edible fungi in culture medium
Silk is necessary.
Summary of the invention:
The raw material of edible fungus liquid culture medium of the present invention is mainly crops primary processed items and by-product, mainly includes
Broken rice, broken corn, wheat shorts, wheat bran, rape cake, rice bran, soybean powder, potato and corresponding enzyme preparation, including amylase and
Protease, amylase can be alpha-amylase, beta amylase, and protease can be papain, bromelain, fig
Protease.
It is an object of the invention to prepare selenium-enriched edible mushroom fluid nutrient medium and selenium-enriched edible mushroom mycelia.
To achieve the goals above the present invention include strain selenium-rich mutagenesis and purifying, strain resistance to selenium domestication and screening,
Five shake-flask seed culture, the preparation of fermentation medium, inoculated and cultured and fermentation liquid post-processing steps, in order to understand this five
Step is described below:
The selenium-rich mutagenesis and purifying of 1 strain
Aseptically, homogenizing fluid is made in scraping strain, removes cell wall through snail enzyme hydrolysis, suitable gradient is taken to dilute
Liquid program request is inoculated in selenium-rich culturing base culture dish, and through ultraviolet radiation mutagenesis, colonial morphology is observed in thermophilic culture.Choose energy
The colony inoculation of normal growth is to No.1 test tube stock culture medium, thermophilic culture, after inclined-plane is paved with mycelia, aseptically,
Take soya bean size edible bacterial vaccine inoculation into No. two activation test tube stock culture mediums, thermophilic culture, until mycelium germination prolongs
Stretch, cut mycelia tip tube and be inoculated into No. three test tube stock culture mediums, thermophilic culture, cut tip tube mode repeat with
Upper step obtains No. four activation test tube stocks, No. five activation test tube stocks, No. six test tube stocks, when test tube stock culture medium quilt
Mycelia is completely covered as purifying strain.Content is exposed after hypha,hyphae breaking-wall cell, and inhereditary material is in ultraviolet light spoke
Lower mutagenesis recombination is penetrated, activates to obtain dissociant by resistance to selenium.Since mycelia tip growth speed is quicker than virus infection speed,
By test tube species of transferring, mycelia gradually disengages virus and is purified, and the selection for purifying strain can be No. two test tube stocks to six
One in number test tube stock.The source of the edible fungus species is bought from qualified R&D institution or through national professional machine
Structure identifies the edible mushroom that can do safe edible, and the mode of self-carry can be taken to obtain edible fungus species.The side of edible fungus species self-carry
Formula can be the separation of fruit body of edible fungi tissue, Spore cultivation, pedo relict hypha separation culture.The cultivation temperature is 10
Degree Celsius to thermophilic culture between 30 degrees Celsius.The No.1 is identical to No. six test tube stock culture medium preparation methods: weighing
200 grams of potato, distilled water is added after cleaning, being cut into small pieces, boiling water boiling 30 minutes, takes its liquor that 30 grams of glucose, albumen is added
3 grams of peptone, 1.2 grams of potassium dihydrogen phosphate, 0.5 gram of magnesium sulfate, 18~20 grams of agar, vitamin B12~5 milligram, add distilled water constant volume
To 1000 milliliters, pH naturally, 121 DEG C high pressure sterilization 25~30 minutes, take out fixed contra bevel after pressure zero, be down to temperature
It is used after 30 DEG C or less.The selenium-rich culturing base sodium selenite additive amount 0.01g-0.5g, remaining component and test tube stock culture
Base phase is same, and configuration method is identical.
The resistance to selenium of 2 strains is tamed and screening
Aseptically purifying strain is inoculated into ten identical No.1 selenium-rich test tube stock culture mediums, thermophilic training
It supports, observation is chosen the most fast tube of growth rate and is inoculated into No. two selenium-rich test tube stock culture mediums, thermophilic culture, more than repetition
Step obtains No. three selenium-rich test tube stocks, No. four selenium-rich test tube stocks, No. five selenium-rich test tube stocks, No. six selenium-rich parent species, works as richness
Selenium test tube stock culture medium is completely covered by mycelia as selenium-rich test tube stock.Sodium selenite has an impact to mycelia physiological activity,
Gradually progressive addition can mitigate the stress reaction of mycelia to sodium selenite content from low to high in cultural hypha base.It selects each time
The unstable strain of inhereditary feature can be eliminated, by test tube species of transferring, mycelia gradually adapts to the culture medium of selenium-rich, selenium-rich test tube
The selection of parent species strain can be one of No. two test tube stocks into No. six test tube stocks.The cultivation temperature is 10 Celsius
It spends to thermophilic culture between 30 degrees Celsius.The No.1 to No. six test tube stock culture medium additive amounts are prepared in addition to sodium selenite
Method is identical: weighing 200 grams of potato, distilled water is added after cleaning, being cut into small pieces, boiling water boiling 30 minutes, takes its liquor and Portugal
Grape sugar 30 grams, 3 grams of peptone, 1.2 grams of potassium dihydrogen phosphate, 0.5 gram of magnesium sulfate, 18~20 grams of agar, vitamin B12~5 milligram,
Add distilled water to be settled to 1000 milliliters, pH naturally, 121 DEG C high pressure sterilization 25~30 minutes, pressure zero after take out fixation it is oblique
Face uses after temperature is down to 30 DEG C or less.Sodium selenite additive amount 0.001g-0.5g makes its concentration from No.1 to No. six test tubes
It is incremented by successively.
3 shake-flask seed cultures
Strain after resistance to selenium is tamed and is screened is taken into soya bean size strain with inoculation shovel under aseptic processing environment
Being inoculated into 250ML conical flask Shake flask medium makes its floating, and after thermophilic culture -24 hours 12 hours, strain is sprouted, and starting is shaken
Bottle culture rocking equipment, 100-160 revs/min of revolving speed continue culture 3 to 7 days until generate be uniformly distributed grain of rice size bacterium ball or
Grain of rice length mycelia.The cultivation temperature is thermophilic culture between 10 degrees Celsius to 30 degrees Celsius.The Shake flask medium
Preparation method is: weigh 200 grams of potato, after cleaning, being cut into small pieces plus distilled water, boiling water boiling 30 minutes, take its liquor and
30 grams of glucose, 3 grams of peptone, 1.2 grams of potassium dihydrogen phosphate, 0.5 gram of magnesium sulfate, 18~20 grams of agar, vitamin B12~5 millis
Gram, sodium selenite additive amount 0.05g-0.5g (with resistance to selenium screening after select the selenium concentration of parent species it is consistent), add distilled water to be settled to
1000 milliliters, import 100ml-120ml/ bottle of 250ml conical flask constant volume, clogged with tampon, brown paper wrapping bottleneck, pH naturally,
121 DEG C high pressure sterilization 25~30 minutes, pressure zero after take out, used after temperature is down to 30 DEG C or less.
The preparation of 4 fermentation mediums
Saccharification treatment fluid and protein hydrolyzate are warming up to 2-5 minutes high-temperature inactivations of 100 degrees Celsius of maintenances, filtering filter respectively
Liquid imports the empty fermentor to disappear and adds distilled water constant volume, maintains steam pressure 0.1-0.12MPa in-situ sterilization 45 minutes, pressure is returned
It forces to be cooled to 18 to 26 degrees Celsius after zero to be fermentation medium.The saccharification handles liquid making method: broken rice, broken jade
Rice, wheat shorts are weighed in proportion, add the water of 5 to 20 times of quality, and sodium selenite, which is added, makes its mass fraction 0.002%-
0.02%, 100 degrees Celsius are boiled 1 hour, and greenhouse cooling is kept between 55-65 degrees Celsius to 55-65 degrees Celsius of addition amylase
- 2 hours 30 minutes, the amylase of addition can beAlpha-amylaseWithBeta amylaseIt is one or two kinds of.The protein hydrolyzate
Preparation method: wheat bran, rape cake, rice bran, soybean powder are weighed in proportion, add the water of 5 to 20 times of quality, and sodium selenite, which is added, to be made
Its mass fraction 0.003%-0.03% is heated to 55 degrees Celsius of addition protease, and 30 points are kept between 55 to 65 degrees Celsius
Clock by 4 hours, the protease of addition can be one kind of papain, ficin and bromelain, two kinds or
Three kinds.Broken rice, broken corn, wheat shorts starch rich in can obtain hypha of edible fungus by amylorrhexis and be easy to inhale
The carbon source of receipts.Wheat bran, rape cake, rice bran, soybean powder protein rich in and vitamin and minerals, through protease hydrolytic energy
It obtains hypha of edible fungus and is easy the nitrogen source absorbed.Sodium selenite addition ultimate density is consistent with shake-flask seed culture medium, selenous acid
Sodium can reduce toxicity by culture medium pretreatment, be easy to the absorption of hypha of edible fungus.
5 inoculations, culture
The shake-flask seed of healthy and strong pollution-free even suspension is chosen, hair is added by 4%-10% volume additive amount with sterile working
In ferment culture medium, thermophilic culture between 10 to 30 degrees Celsius is passed through pure air culture 5-10 days.
6 fermentation liquid post-processings
Sampling analysis is centrifuged with unit volume and surveys dry matter weight and solution fermentation liquid PH judgement culture reaction end.With 4
Mycelia and filtrate, mycelia heated-air drying are separated by the way of vacuum filtration to 10 layers of gauze, filtrate spray drying is wrapped respectively
Dress.
Embodiment one
In order to better understand the present invention, it is described as follows by taking northern Chinese caterpillar Fungus as an example:
The selenium-rich mutagenesis and purifying of 1 strain
Aseptically, scraping strain is added buffer and homogenizing fluid is made, and hydrolyzes removal cell wall through snail enzymatic treatment,
Ten negative one power to ten minus ten powers gradient dilution liquid program request is inoculated in selenium-rich culturing base culture dish, through 20w ultraviolet lamp
Ultraviolet light 30CM radiates 5s-20s mutagenesis, and colonial morphology is observed in thermophilic culture.Choose can normal growth colony inoculation to No.1
Test tube stock culture medium, 22-25 degrees Celsius is protected from light culture, after inclined-plane is paved with mycelia, aseptically, takes soya bean size food
It is inoculated into No. two activation test tube stock culture mediums with bacterium strain, thermophilic culture, until mycelium germination extension, cuts mycelia tip
Tube is inoculated into No. three test tube stock culture mediums, and thermophilic culture, the mode for cutting tip tube repeats above step, obtains No. four
Test tube stock, No. five activation test tube stocks, No. six test tube stocks are activated, chooses No. six test tube stocks as purifying strain.The food
Source with bacterium strain is Wuhan City, Hubei Province Central China edible fungus culturing research institute.The No.1 is to No. six test tube stock culture mediums
Preparation method is identical: weighing 200 grams of potato, distilled water is added after cleaning, being cut into small pieces, boiling water boiling 30 minutes, its liquor is taken to add
Enter 30 grams of glucose, 3 grams of peptone, 1.2 grams of potassium dihydrogen phosphate, 0.5 gram of magnesium sulfate, 18~20 grams of agar, 5 milli of vitamin B1
Gram, add distilled water to be settled to 1000 milliliters, pH naturally, 121 DEG C high pressure sterilization 25~30 minutes, take out after pressure zero it is fixed fall
Inclined-plane uses after temperature is down to 30 DEG C or less.The selenium-rich culturing base sodium selenite additive amount 0.05-0.2g, remaining component
Identical with test tube stock culture medium, configuration method is identical.
The resistance to selenium of 2 strains is tamed and screening
Aseptically purifying strain is inoculated into ten identical No.1 selenium-rich test tube stock culture mediums, thermophilic training
It supports, observation is chosen the most fast tube of growth rate and is inoculated into No. two selenium-rich test tube stock culture mediums, 22-25 degrees Celsius of culture,
Above step is repeated, No. three selenium-rich test tube stocks, No. four selenium-rich test tube stocks, No. five selenium-rich test tube stocks, No. six selenium-rich are obtained
Parent species, when selenium-rich test tube stock culture medium is completely covered by mycelia as selenium-rich test tube stock.The No.1 is to No. six test tubes
Mother culture media additive amount preparation method in addition to sodium selenite is identical: weighing 200 grams of potato, after cleaning, being cut into small pieces plus steams
Distilled water boiling water boiling 30 minutes, takes its liquor and 30 grams of glucose, 3 grams of peptone, 1.2 grams of potassium dihydrogen phosphate, magnesium sulfate 0.5
Gram, 18~20 grams of agar, vitamin B12~5 milligram, plus distilled water be settled to 1000 milliliters, pH is naturally, 121 DEG C of high pressure sterilizations
25~30 minutes, pressure took out fixed contra bevel after being zeroed, and used after temperature is down to 30 DEG C or less.Sodium selenite additive amount
0.005g-0.2g keeps its concentration incremented by successively from No.1 to No. six test tubes.
3 shake-flask seed cultures
Strain after resistance to selenium is tamed and is screened is taken into soya bean size strain with inoculation shovel under aseptic processing environment
Being inoculated into 250ML conical flask Shake flask medium makes its floating, is protected from light after 22-25 degrees Celsius is cultivated 24 hours, and strain is sprouted,
Start shaking flask culture rocking equipment, 140 revs/min of revolving speed are continued culture generation in 4 days and are uniformly distributed grain of rice size bacterium ball.Described
Shake flask medium preparation method is: weighing 200 grams of potato, distilled water is added after cleaning, being cut into small pieces, boiling water boiling 30 minutes, is taken
Its liquor and 30 grams of glucose, 3 grams of peptone, 1.2 grams of potassium dihydrogen phosphate, 0.5 gram of magnesium sulfate, 18~20 grams of agar, dimension life
Plain B12~5 milligram, sodium selenite additive amount 0.05g-0.5g (selecting the selenium concentration of parent species consistent with after the screening of resistance to selenium), add steaming
Distilled water is settled to 1000 milliliters, imports 100ml/ bottles of 250ml conical flask constant volume, is clogged with tampon, and brown paper wraps up bottleneck, and pH is certainly
So, 121 DEG C high pressure sterilization 30 minutes, pressure are taken out after being zeroed, and are down to 22-25 degrees Celsius of use to temperature.
The preparation of 4 fermentation mediums
Saccharification treatment fluid and protein hydrolyzate are warming up to 100 degrees Celsius of maintenances, 2 minutes high-temperature inactivations, filter liquor respectively
Importing the empty 10L fermentor to disappear adds distilled water to be settled to 6L, maintains steam pressure 0.12MPa in-situ sterilization 45 minutes, pressure
It forces to be cooled to 24 degrees Celsius after returning to zero to be fermentation medium.The saccharification handles liquid making method: broken rice, wheat shorts
It weighs in 200g: 200g ratio, adds the water of 2L, sodium selenite 0.2g is added, 100 degrees Celsius are boiled 1 hour, and greenhouse cooling is extremely
55-65 degrees Celsius of addition alpha-amylase is kept for 1 hour between 55-65 degrees Celsius.The proteolysis liquid making method:
Wheat bran, soybean powder are weighed in 50g: 150g ratio, add the water of 2L, and sodium selenite 0.4g is added, and are heated to 55 degrees Celsius of additions
Papain is kept for 2 hours between 55 to 65 degrees Celsius.
5 inoculations, culture
The shake-flask seed of healthy and strong pollution-free even suspension is chosen, is added in fermentation filling with 5% volume additive amount, 24 degrees Celsius
It is protected from light constant temperature and is passed through pure air culture.
6 fermentation liquid post-processings
Sampling analysis is centrifuged with unit volume and surveys dry matter weight and solution fermentation liquid PH judgement culture reaction end.With 6
Layer gauze separates mycelia and filtrate, mycelia heated-air drying by the way of vacuum filtration, and filtrate spray drying is packed respectively.
Embodiment two
In order to better understand the present invention, it is described as follows by taking Morchellaconica as an example:
The selenium-rich mutagenesis and purifying of 1 strain
Aseptically, scraping strain is added buffer and homogenizing fluid is made, and hydrolyzes removal cell wall through snail enzymatic treatment,
Ten negative one power to ten minus ten powers gradient dilution liquid program request is inoculated in selenium-rich culturing base culture dish, through 20w ultraviolet lamp
Ultraviolet light 30CM radiates 2s-30s mutagenesis, and colonial morphology is observed in thermophilic culture.Choose can normal growth colony inoculation to No.1
Test tube stock culture medium, 15-18 degrees Celsius is protected from light culture, after inclined-plane is paved with mycelia, aseptically, takes soya bean size food
It is inoculated into No. two activation test tube stock culture mediums with bacterium strain, 15-18 degrees Celsius of culture is cut until mycelium germination extension
Mycelia tip tube is inoculated into No. three test tube stock culture mediums, 15-18 degrees Celsius culture, cut tip tube mode repeat with
Upper step obtains No. four activation test tube stocks, No. five activation test tube stocks, chooses No. five test tube stocks as purifying strain.It is described
The source of edible fungus species is Wuhan City, Hubei Province Central China edible fungus culturing research institute.The No.1 is to No. six test tube stock cultures
Base preparation method is identical: weighing 200 grams of potato, distilled water is added after cleaning, being cut into small pieces, boiling water boiling 30 minutes, takes its liquor
30 grams of glucose, 3 grams of peptone, 1.2 grams of potassium dihydrogen phosphate, 0.5 gram of magnesium sulfate, 18~20 grams of agar, vitamin B1 5 is added
Milligram, add distilled water to be settled to 1000 milliliters, pH naturally, 121 DEG C high pressure sterilization 25~30 minutes, pressure zero after take out fix
Contra bevel uses after temperature is down to 20 DEG C or less.The selenium-rich culturing base sodium selenite additive amount 0.05-0.2g, remaining group
Point identical with test tube stock culture medium, configuration method is identical.
The resistance to selenium of 2 strains is tamed and screening
Aseptically purifying strain is inoculated into ten identical No.1 selenium-rich test tube stock culture mediums, thermophilic training
It supports, observation is chosen the most fast tube of growth rate and is inoculated into No. two selenium-rich test tube stock culture mediums, 15-18 degrees Celsius of culture,
Above step is repeated, No. three selenium-rich test tube stocks, No. four selenium-rich test tube stocks, No. five selenium-rich test tube stocks, No. six selenium-rich are obtained
Parent species, when selenium-rich test tube stock culture medium is completely covered by mycelia as selenium-rich test tube stock.The No.1 is to No. six test tubes
Mother culture media additive amount preparation method in addition to sodium selenite is identical: weighing 200 grams of potato, after cleaning, being cut into small pieces plus steams
Distilled water boiling water boiling 30 minutes, takes its liquor and 30 grams of glucose, 3 grams of peptone, 1.2 grams of potassium dihydrogen phosphate, magnesium sulfate 0.5
Gram, 18~20 grams of agar, vitamin B12~5 milligram, plus distilled water be settled to 1000 milliliters, pH is naturally, 121 DEG C of high pressure sterilizations
25~30 minutes, pressure took out fixed contra bevel after being zeroed, and used after temperature is down to 20 DEG C or less.Sodium selenite additive amount
0.005g-0.2g keeps its concentration incremented by successively from No.1 to No. six test tubes.
3 shake-flask seed cultures
Strain after resistance to selenium is tamed and is screened is taken into soya bean size strain with inoculation shovel under aseptic processing environment
Being inoculated into 250ML conical flask Shake flask medium makes its floating, is protected from light after 16 degrees Celsius are cultivated 24 hours, and strain is sprouted, and opens
Shake bottle culture rocking equipment, 135 revs/min of revolving speed are continued culture generation in 3 days and are uniformly distributed mycelia.The Shake flask medium
Preparation method is: weigh 200 grams of potato, after cleaning, being cut into small pieces plus distilled water, boiling water boiling 30 minutes, take its liquor and
30 grams of glucose, 3 grams of peptone, 1.2 grams of potassium dihydrogen phosphate, 0.5 gram of magnesium sulfate, 18~20 grams of agar, vitamin B12~5 millis
Gram, sodium selenite additive amount 0.05g-0.5g (with resistance to selenium screening after select the selenium concentration of parent species it is consistent), add distilled water to be settled to
1000 milliliters, 100ml/ bottles of 250ml conical flask constant volume are imported, is clogged with tampon, brown paper wraps up bottleneck, and pH is naturally, 121 DEG C of height
Pressure sterilizing 30 minutes, pressure is taken out after being zeroed, and is down to 15-18 degrees Celsius of use to temperature.
The preparation of 4 fermentation mediums
Saccharification treatment fluid and protein hydrolyzate are warming up to 100 degrees Celsius of maintenances, 2 minutes high-temperature inactivations, filter liquor respectively
Importing the empty 10L fermentor to disappear adds distilled water to be settled to 6L, maintains steam pressure 0.12MPa in-situ sterilization 45 minutes, pressure
It forces to be cooled to 24 degrees Celsius after returning to zero to be fermentation medium.The saccharification handles liquid making method: broken rice, wheat shorts
It weighs in 200g: 200g ratio, adds the water of 2L, sodium selenite 0.2g is added, 100 degrees Celsius are boiled 1 hour, and greenhouse cooling is extremely
55-65 degrees Celsius of addition alpha-amylase is kept for 1 hour between 55-65 degrees Celsius.The proteolysis liquid making method:
Wheat bran, soybean powder are weighed in 50g: 150g ratio, add the water of 2L, and sodium selenite 0.4g is added, and are heated to 55 degrees Celsius of additions
Papain is kept for 2 hours between 55 to 65 degrees Celsius.
5 inoculations, culture
The shake-flask seed of healthy and strong pollution-free even suspension is chosen, is added in fermentation filling with 6% volume additive amount, 15 degrees Celsius
It is protected from light constant temperature and is passed through pure air culture.
6 fermentation liquid post-processings
Sampling analysis is centrifuged with unit volume and surveys dry matter weight and solution fermentation liquid PH judgement culture reaction end.With 5
Layer gauze separates mycelia and filtrate, mycelia heated-air drying by the way of vacuum filtration, and filtrate spray drying is packed respectively.
Beneficial effect
The present invention uses agricultural and sideline product to save cost for primary raw material, through the fermentation liquid richness crossed with proteolytic treatment that is saccharified
It is easy to the carbon source and nitrogen source absorbed containing edible mushroom, also contains microelement and vitamin, be a kind of all nutrition type culture medium.Strain
Through the resistance to selenium screening of overactivation, the selenium in culture medium passes through pretreatment, enhances strain to the adaptive faculty and tolerance of selenium.
Claims (5)
1. the preparation of selenium-enriched edible mushroom fluid nutrient medium and the method for mycelia liquid deep layer fermenting include strain selenium-rich mutagenesis and
Purifying, the resistance to selenium domestication of strain and screening, shake-flask seed culture, the preparation of fermentation medium, inoculated and cultured and fermentation liquid later period
Handle five steps.
2. according to claims 1 it is characterized in that the raw material for preparing of fermentation medium is mainly crops primary processed items and pair
Product, mainly include broken rice, broken corn, wheat shorts, wheat bran, rape cake, rice bran, soybean powder, potato and corresponding enzyme preparation,
Including amylase and protease, amylase can be alpha-amylase, beta amylase, and protease can be papain, pineapple
Protease, ficin.
3. according to saccharification described in the preparation of 1 fermentation medium of claims handle liquid making method: broken rice, broken corn,
Wheat shorts are weighed in proportion, add the water of 5 to 20 times of quality, and sodium selenite, which is added, makes its mass fraction 0.002%-0.02%,
100 degrees Celsius are boiled 1 hour, greenhouse cooling to 55-65 degrees Celsius of addition amylase, kept between 55-65 degrees Celsius 30 minutes-
2 hours, the amylase of addition can be alpha-amylase and beta amylase is one or two kinds of.
4. according to proteolysis liquid making method described in the preparation of 1 fermentation medium of claims: wheat bran, rape cake, rice
Chaff, soybean powder are weighed in proportion, add the water of 5 to 20 times of quality, and sodium selenite, which is added, makes its mass fraction 0.003%-
0.03%, 55 degrees Celsius of addition protease are heated to, are kept for 30 minutes to 4 hours between 55 to 65 degrees Celsius, the egg of addition
White enzyme can be one kind of papain, ficin and bromelain, two or three.
5. being centrifuged survey dry matter weight according to 1 fermentation liquid post-processing of claims with unit volume and solution fermentation liquid PH sentencing
Surely reaction end is cultivated.Mycelia and filtrate, mycelia heated-air drying, filter are separated by the way of vacuum filtration with 4 to 10 layers of gauze
Liquid spray drying, is packed respectively.
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