CN108271620A - A kind of fermentation process of high density liquid strain - Google Patents
A kind of fermentation process of high density liquid strain Download PDFInfo
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- CN108271620A CN108271620A CN201810042630.3A CN201810042630A CN108271620A CN 108271620 A CN108271620 A CN 108271620A CN 201810042630 A CN201810042630 A CN 201810042630A CN 108271620 A CN108271620 A CN 108271620A
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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
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05B—PHOSPHATIC FERTILISERS
- C05B17/00—Other phosphatic fertilisers, e.g. soft rock phosphates, bone meal
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
- C05D5/00—Fertilisers containing magnesium
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
Abstract
The invention discloses a kind of fermentation process of high density liquid strain, belong to planting almond abalone mushroom technical field, in liquid seeds, shake-flask seed, in seeding tank seed and each stage culture medium of fermentor liquid strain science compounding can be improved strain resist it is cold, heat stress, the Chinese herbal medicine extract of immunity and anti-miscellaneous bacteria ability, science, which has compounded, simultaneously both has adaptability to raw material, wood dust with antifoaming agent function again, and by the way of additional inoculation and staged variable temperature fermentation, shorten strain cell age and proliferation algebraic step away from, stream adds the chitosan with specific function after coating in due course, it is sturdy to turn out fermented hypha, it is active high, mycelia bulb diameter is small, density is big, the liquid spawn being evenly distributed, its mycelium pellet density is 4.5 5.5 X 105A/L, a diameter of 0.5 0.7mm of mycelium pellet, mycelium pellet dry weight are 90 100g/L, and sprout time is 4 5h, and exocellular polysaccharide is 3.0 3.5g/L.
Description
The application is the divisional application of following application：The applying date is on 06 11st, 2015, application No. is
201510316761.2 a kind of entitled high density liquid strain and its fermentation process.
Technical field
The present invention relates to planting almond abalone mushroom technical fields, and in particular to a kind of fermentation process of high density liquid strain.
Background technology
Pleurotus eryngii (PleurotuseryngiiQuel.) also known as pleurotus eryngii, it is excellent to have that bacterial context is plump, quality is tender and crisp etc.
It is point, especially its stem dense structure, solid, milky white, tasty and refreshing, be referred to as " oyster mushroom king ", " dried scallop mushroom ", have almond flavor and
The mouthfeel of abalone is suitble to fresh-keeping, processing, firmly gets liking for people, full of nutrition, rich in protein, carbohydrate, dimension life
Element and the minerals such as calcium, magnesium, copper, zinc, can improve immune function of human body, to human body have anticancer, reducing blood lipid, ease constipation stomach and
The effects that beauty, has high cultivating value and commercial value.
Domestic and international planting almond abalone mushroom is based on solid spawn at present, and liquid spawn has production week relative to solid spawn
Phase is short, cell age is consistent, fruiting is neat, convenient for management, strain is at low cost, inoculation is convenient and it is quick the advantages that, be conducive to edible mushroom give birth to
Scale, the batch production of production.It is improved in recent years due to the fast development of industrial cultivation technique to shorten strain manufacturing cycle
Strain quality and production efficiency, domestic and international manufacturer research and development liquid spawn are universal in current Pleurotus eryngii production for substituting
The solid spawn used, and start to apply in actual production.
Pleurotus eryngii liquid strain is to produce great-hearted Pleurotus eryngii mycelium by liquid fermentation process.Currently, the country is still
Do not promulgate that unified national standard comes the quality and quality of specification pleurotus eryngii liquid strain, but people in actual application
It summarizes application effect and has worked out some region standards, such as Liaoning Province's provincial standard《DB21/T 1693-2008 edible fungus liquids
Strain production technology regulation》Deng clearly stipulate that qualified edible fungi liquid strain should be " visible distinctive in these standards
Hypha form, spherical and plexi mycelium are largely distributed, and mycelia is sturdy, and plasm is evenly distributed in mycelia " " bacterium solution is slightly sticky thick,
Have a large amount of sheets or it is spherical it is mycelium suspended, be evenly distributed ", that is to say, that excellent liquid spawn mycelium pellet density is high, diameter
It is small, it is evenly distributed；On the other hand, pleurotus eryngii liquid strain is to carry out machinery by production line inoculation device in production application
Change inoculation operation, since inoculation device spout is narrow, to prevent bacterium solution from blocking pipeline, it is also necessary to increase pre- before liquid spawn inoculation
Treatment process (general to make the mycelia bulb diameter in bacterium solution become smaller using the method for mechanical crushing, bacterium solution is made to become uniform).Therefore, apricot
The physical characteristics such as size, density, the uniformity of abalone mushroom liquid fermentation mycelium pellet are the key that high-quality liquid spawn Con trolling index.
Currently, research report and patented technology in relation to Pleurotus eryngii Liquid Culture are largely focused on shaking flask level, scale
Change the less of fermentation research, it is especially more in terms of the improvement of fermentation medium in terms of the optimization for concentrating on zymotechnique substantially：
Application No. is Pleurotus eryngii liquid fermentation medium and utilizations that 201410568173.3 Chinese patent discloses a kind of improvement
It cultivates the method for pleurotus eryngii liquid strain, in the fermentation medium one in the gelatin of addition 0.1-0.3g/100ml, agar
The mixture of kind or the two, using stirring, shaking table shake culture, agar addition is more than in the above-mentioned published patent culture medium
0.4g/100ml solidifies, and can not carry out Liquid Culture, is difficult to control, and there is fermentation failure hidden danger, and shaking table culture obtains
Liquid spawn amount is few, is not suitable for scale, mechanization production, while strain quality is not fine.Application No. is
201410244283.4 Chinese patent discloses a kind of production method of edible fungi liquid strain, is the edible fungi that will be preserved
It in solid medium culture after strain is activated, then crushes, continues sealing culture 2-3 days on solid medium, with 0.15-
0.2% agar solution contact is uniformly mixed so as to obtain liquid spawn, and the above-mentioned published patent substantially still has lacking for solid fermentation
It falls into, and crushing therein is further cultured for technique and allocating technology miscellaneous bacteria easy to pollute, the high shear force of high-speed stirred mashing and crushing
Strain is injured quite big.A northern gardening edible mushrooms piece discloses the preparation research for the pleurotus eryngii liquid strain that Liu Guanhui etc. is delivered
【2009(11)230-232】, fermentation medium and fermentation condition are optimized, but still not comprehensively, it cannot be suitble to well
Growth, breeding, the physical characteristics such as size, density, the uniformity of zymophyte pompon of obtained liquid spawn are not highly desirable,
Stirring shearing force is equally existed to seriously affect mycelium pellet quality.
Air-blowing fermentation is the fermentation method for being passed through oxygen or filtrated air from airlift fermentation pot bottom, is provided simultaneously with logical
The effect of oxygen and soft stirring, logical oxygen efficiency is high, dissolved oxygen effect is good, and stirring-type fermentation shearing force can be effectively prevent to Pleurotus eryngii liquid
The influence of body strain quality, obtained liquid spawn mycelia is sturdy, modest viscosity, mycelia bulb diameter is small, density is high, distribution is equal
It is even.Application No. is 201310479951.7 Chinese patents to disclose a kind of cultural method of pleurotus eryngii liquid strain, will be traditional
Potato carbon source and wheat bran nitrogen source replace with bean cake powder, culture medium is poured into fermentation tank, 121 DEG C sterilize 60-120 minutes, go out
Water leaching is cooling after bacterium and is passed through filtrated air；The good liquid parent species of inoculated and cultured, inoculum concentration are 1-2 ‰, fermentation tank culture temperature
It it is 21-26 DEG C, venting pressure is 0.5-1.5 megapascal, and culture obtains pleurotus eryngii liquid strain, but the above-mentioned published patent in 7-10 days
Culture medium not comprehensive nutrition, zymotechnique lag, fermentation period is long, and obtained liquid spawn quality is still undesirable.
To sum up, according to the strain properties of Pleurotus eryngii, by Optimal Medium and condition of culture, being fermented using air-blowing, it is high to prepare
Quality, high yield, pleurotus eryngii liquid strain at low cost are still the unremitting pursuit of those skilled in the art.
Invention content
Technical problem solved by the invention is the defect for overcoming existing air-blowing fermentation to prepare pleurotus eryngii liquid strain, in bacterium
The Chinese herbal medicine extract that strain resists hot and cold irritability and immunity can be improved in science compounding in kind each stage culture medium of incubation
And adaptability raw material wood dust shortens strain cell age, proliferation algebraic step by the way of the additional inoculation of segmentation and temperature-variable fermentation
Away from, the viscosity of the chitosan adjustment fermentation medium after stream plus coating in due course, turn out a kind of fermented hypha it is sturdy, it is active it is high,
Mycelia bulb diameter is small, density is big, is evenly distributed, the pleurotus eryngii liquid strain that fermentation period is short.
In order to achieve the above object, the present invention uses following technical scheme：
A kind of fermentation process of high density liquid strain, includes the following steps：
1) go bail for the Pleurotus eryngii slant strains 0.2cm kept22 pieces of strain block be inoculated in plating medium and activated,
25 DEG C of constant temperature incubations 10 days, so activation 2 times, obtain tablet seed；
2) the tablet seed card punch of a diameter of 0.5cm is intercepted into 0.2cm23 pieces of strain block access 250mL triangular flasks
In, liquid seed culture medium liquid amount is 100mL, in 25 DEG C of constant temperature incubations 2 days, is then placed in constant-temperature table, 25 DEG C,
100r/min shaken cultivations obtain liquid seeds in 3 days；
3) liquid seeds are inoculated in 10% inoculum concentration in 500mL shaking flasks, Shake flask medium liquid amount 250mL, in
25 DEG C, 150r/min oscillation constant temperature incubations 4 days shake-flask seed；
4) shake-flask seed is inoculated in 10% inoculum concentration in 5L seeding tanks, seed tank culture base liquid amount 3L, in 25
DEG C, under the conditions of ventilatory capacity 4L/min constant temperature incubation 4 days seeding tank seed；
5) seeding tank seed is inoculated in 30L fermentation tanks with 5% inoculum concentration first, fermentation medium liquid amount 15L, in
30-32 DEG C, ventilatory capacity 3-5L/min ferment at constant temperature 12-24h, are then cooled to 18-22 DEG C with the rate of 0.3-0.5 DEG C/min,
8-10g coating chitosans and 900mL seeding tank seeds, ferment at constant temperature under the conditions of ventilatory capacity 6-8L/min are added into fermentation tank
10-16h is finally warming up to 24-26 DEG C with the rate of 0.2-0.4 DEG C/min, ferment at constant temperature under the conditions of ventilatory capacity 5-7L/min
45-52h is up to high density liquid strain；
The Pleurotus eryngii slant strains are that Pleurotus eryngii purchased in market purifies bacterial strain；
The plating medium group becomes：Potato 200g/L, glucose 20g/L, peptone 5g/L, potassium dihydrogen phosphate
3g/L, magnesium sulfate 1.5g/L, agar 20g/L, VB10.02g/L, surplus are water, pH value 6.0；
The liquid seed culture medium group becomes：Glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar
2g/L, Chinese herbal medicine extract 0.3-0.5g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.1-0.3g/L,
VB10.01g/L, surplus are water, pH value 6.0；
The Shake flask medium group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extraction
Object 0.5-1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.2-0.4g/L, VB10.01g/L, surplus
For water, pH value 6.0；
The seed tank culture base group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine carry
Take object 0.8-1.2g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3-0.5g/L, VB10.01g/L, it is remaining
Amount is water, pH value 6.0；
The group of the fermentation medium becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine carry
Take object 1.5-2g/L, wood dust 0.6-1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB10.01g/L, surplus
For water, pH value 6.0；
Preferably, the preparation method of the Chinese herbal medicine extract, includes the following steps：It counts in parts by weight, weighs Radix Astragali
30 parts, 30 parts of rhizoma zingiberis, 30 parts of dried orange peel, 25 parts of cow-bezoar, 25 parts of the root bark of tree peony, 20 parts of Radix Glycyrrhizae, 18 parts of rhizoma anemarrhenae, 18 parts of honeysuckle, cordate houttuynia
17 parts, 15 parts of radix paeoniae rubra, 15 parts of radix scutellariae, 10 parts of Rhizoma Chuanxiong, 8 parts of Radix Angelicae Sinensis；Set the supersonic wave cleaning machine for filling 0.2% sodium bicarbonate solution
In in 200W, 30KHz clean 12min, drain, by said herbal medicine be crushed to grain size be 2 millimeters uniformly mixed hereinafter, setting in container
The water for merging 5 times of weight of addition, obtains mixed material, and 80 DEG C of holding 3h of control temperature are then cooled to 45 DEG C, with newborn acid for adjusting pH
Value is 4, Microwave Extraction 12min is carried out under the conditions of power 200W, while being surpassed under the conditions of power 250W, frequency 35KHz
Sound wave assisted extraction；Then the mixed enzyme that mixed material total weight 1.0% is added is digested, and is 6.2 with newborn acid for adjusting pH value,
3h is digested, the mixture of mixed material 2 times of w ethanols and propyl alcohol is finally added, the mass ratio that ethyl alcohol and propyl alcohol mix is 1:2,
Temperature is controlled to 70 DEG C of holding 3.5h, filtering obtains the first filtrate；Add the water of 2 times of weight of filter residue, the 90 DEG C of holdings of control temperature
2h, is then cooled to 30 DEG C, and filtering obtains the second filtrate；By the first filtrate and the second filtrate according to mass ratio 3:2 merge, filtrate
It is freeze-dried, crushed up to Chinese herbal medicine extract after vacuum concentration；
The mixed enzyme is dextranase, zytase, pentosanase, pectase in mass ratio 3:2:2:1 uniformly mixing.
Preferably, the preparation method of the wood dust includes the following steps：Will without it is mouldy, without disease pest sawmilling end or waste wood
Remove sundries, be placed in the supersonic wave cleaning machine of the sodium bicarbonate solution equipped with mass percent concentration 0.5% in 200W,
30KHz cleans 10min, rinses, drains, and is put into steam kettle in 0.3-0.4MPa boiling 20-30min, takes out, drain, with plant
Object oil in mass ratio 100:Then mixture is set microwave dryer in 2000W, 120 DEG C of microwave irradiation 5min by 1 uniformly mixing,
It is allowed to moisture and reaches 8-10%, last ultramicro grinding is packed to grain size 0.3-0.5mm up to wood dust；
It is highly preferred that the drying process is：Microwave irradiation 10s stops 10s.
Preferably, the preparation method of the fermentation medium is：According to each raw material of culture medium prescription precise, take first
The water of 4-6 times of soybean cake powder quality is 5-6 with newborn acid for adjusting pH value, and the thermostableα-amylase of 5u/g soybean cake powders, side is added
It stirs side and soybean cake powder is added, be warming up to 90-95 DEG C, heat preservation, liquefy 10-15min, is then cooled to 50-60 DEG C, keeps the temperature, side
The protease hydrolytic 20-30min that the carbohydrase and 30u/g soybean cake powders of 40u/g soybean cake powders is added in side is stirred, is added while stirring
Enter remaining water and other raw materials, uniformly mix, adjustment pH value is 6.0,121-123 DEG C of sterilizing 30-40min up to fermented and cultured
Base；
Preferably, the preparation method of the coating chitosan, includes the following steps：Take chitosan that its quality 30- is added
50% mass percent concentration is that softwood is made in the cyclohexaamylose aqueous solution of 10-12%, is sieved and is pelletized with 10-20 mesh, obtained
Wet granular is set in 2000W, 60-80 DEG C of dry 4-6min of power in microwave dryer, sieves quick whole grain with 10-20 mesh up to wrapping
By chitosan.
The detection method of fluid present invention strain primary quality measure：
The measurement of mycelium pellet dry weight
It takes 50ml zymotic fluids to be filtered with the sieve of 80 mesh, collects bacterium ball, rinsed repeatedly with clear water, and be placed on
80 DEG C of drying are weighed after 4 hours in constant temperature drying box, and weigh every half an hour it is primary, until weighing difference twice is no more than 2mg,
As constant weight.Finally calculate the average value of dry mycelial weight.
The measurement of mycelium pellet density
It takes zymotic fluid 1ml in culture dish with pipette, bacterium ball quantity is counted, repeat numeration three times, finally count
Calculate the average value of Peloton density.
The measurement of mycelia bulb diameter
Zymotic fluid 1ml is taken with pipette, takes 10 bacterium balls to line up with tweezers a queue of, total length is measured, is then averaging
Value, duplicate measurements three times, calculate the average value of bacterium bulb diameter.
The measurement of mycelium pellet tieback tablet sprout time
A bacterium ball is taken to be placed in PDA culture medium tablet center, each fermented and cultured from the culture solution of fermentation medium
Base does three repetitions, and the PDA plate for being placed with bacterium ball is placed in incubator and is cultivated for 25 DEG C, primary every observation in 1 hour, etc.
To after having observed that one group of bacterium ball is sprouted, it is changed to observation in every 0.5 hour once, and record the sprout time of each experimental group, finally
Calculate the average value of sprout time after bacterium ball tieback tablet in each fermentation medium.
The measurement of exocellular polysaccharide content
Zymotic fluid 50ml is taken, is filtered, is taken supernatant, the industrial alcohol of two volumes is added, precipitate polysaccharides are overnight, then
3600r/min is centrifuged 10 minutes, is collected polysaccharide, is placed at 40 DEG C and dries, and one is weighed every half an hour after weighing after 2 hours
It is secondary, until weighing difference twice is no more than 2mg, as constant weight.
Another object of the present invention is to provide the high density liquid strain of above method preparation.
The fermented tank fermentation 67-92h of high density liquid strain, mycelium pellet density is 4.5-5.5 X 105A/L, bacterium
The a diameter of 0.5-0.7mm of pompon, mycelium pellet dry weight are 90-100g/L, sprout time 4-5h, exocellular polysaccharide 3.0-3.5g/
L。
It is a still further object of the present invention to provide application of the above-specified high density liquid spawn in planting almond abalone mushroom.
Advantageous effect：
The present invention is according to the characteristic of Pleurotus eryngii opportunistic pathogen strain, in liquid seeds, shake-flask seed, seeding tank seed and fermentation tank liquid
Strain can be improved from as little as more science compoundings in each stage culture medium of body strain and resist hot and cold irritability, immunity and antibiotic property
Chinese herbal medicine extract, while science has compounded not only with adaptability to raw material, but also the wood dust with antifoaming agent function, by step by step
Domestication, rejuvenation expand culture, enhance pleurotus eryngii quel strains high temperature resistance and Cold stress ability, improve the energy for producing a variety of degrading enzymes
Power excites its proliferative capacity and proliferation density, shortens sprout time, improve the yield of mycelia and exocellular polysaccharide, improves
Adaptability of the bacterial strain to yeasting and follow-up planting environment, while in such a way that additional inoculation and staged variable temperature are fermented,
Strain cell age and proliferation algebraic step are shortened away from stream, which adds after being coated with, in due course has thickening property, intermiscibility, stability, adsorptivity
The viscosity and stability that fermentation medium is adjusted with the chitosan of the specific functions such as biocidal property, improve mycelium pellet in zymotic fluid
The homogeneity and dissolved oxygen concentration of distribution, and broken the preparation method of traditional zymotic culture medium, to soybean cake powder therein into
One step biological enzymolysis, shortens fermentation period, turn out a kind of fermented hypha it is sturdy, it is active it is high, mycelia bulb diameter is small, density
Greatly, the liquid spawn being evenly distributed, mycelium pellet density are 4.5-5.5 X 105A/L, a diameter of 0.5-0.7mm of mycelium pellet, bacterium
Pompon dry weight is 90-100g/L, sprout time 4-5h, exocellular polysaccharide 3.0-3.5g/L.Improve liquid spawn comprehensively
Quality and yield, reduce production cost, and solid base has been established for high quality, high yield and the large-scale planting of Pleurotus eryngii
Plinth shows through experiment in cultivation：Liquid spawn prepared by the present invention is healthy and strong, vigor is high, and the speed of growth is fast, mycelial yield and quality
Height, anti-miscellaneous bacteria ability is strong, long shelf-life, and the bacterium germination time shortening 22.22% compared with commercial liquid strain, miscellaneous bacteria infection rate reduces
96.67%, level-one mushroom rate improves 5%, and production capacity is commercial liquid strain 2 times or more, embodies higher economic value, more
Add suitable batch production, large-scale planting.
Particular technique principle is：
1. the Chinese herbal medicine extract science compounding of the present invention significantly improves strain and resists hot and cold irritability, immunity and disease-resistant
Property Chinese herbal medicine be raw material, through be cleaned by ultrasonic, low temperature enzymolysis, water extracting alcohol carry with ultrasonic wave assisted microwave synthesis extract and prepare, carry
Take object active constituent content high, it is at low cost, can effectively by taming step by step, rejuvenation expand culture, it is anti-to enhance pleurotus eryngii quel strains
High temperature and Cold stress ability, improve the ability of the degrading enzymes such as cellulase-producing, protease, amylase, excite its proliferation
Ability and proliferation density, shorten sprout time, effectively increase the production of the density, dry mycelial weight and exocellular polysaccharide of liquid spawn
Amount, reduces mycelia bulb diameter, enhances adaptability of the bacterial strain to yeasting and follow-up planting environment, enhance comprehensively highly dense
Resisting stress, disease resistance and the immunocompetence of liquid spawn are spent, while can effectively kill the worm's ovum in raw material using ultrasonic cleaning
And miscellaneous bacteria, heavy metal ion content and persticide residue are reduced, the raw material for reducing Pleurotus eryngii enriching heavy metal ion is possible, carries
The high foodsafety of edible mushroom and the shelf-life of liquid spawn.
2. wood dust of the present invention is using the sawdust of high cellulose content and high lignin content and vegetable oil as raw material, using super
Prepared by sound cleaning, autoclaving and microwave drying process, quality is fine and soft soft, and trophic fiber cellulose content is high, is very beneficial for apricot
Abalone mushroom strain fermentation, while network is abundant, uniform, strong adsorption force, vegetable oil adhesion amount is big, may be either that Pleurotus eryngii offer is rich
Rich nutrition can also solve the problems, such as air-blowing fermentation foam easy to foaming under high ventilation quantity, also provide ring for the cultivation in later stage
Border adaptive capacity, and using worm's ovum and the miscellaneous bacteria that can effectively kill in raw material is cleaned by ultrasonic, reduce heavy metal ion content
And persticide residue, the raw material for reducing Pleurotus eryngii enriching heavy metal ion is possible, improves the foodsafety of edible mushroom.
3. the present invention overcomes the method that tradition prepares fermentation medium, to the further liquefaction of soybean cake powder therein and enzyme
Solution, small molecule is degraded to by starch therein and protein macromolecule nutriment, is played ferment and fermentation convenient for Pleurotus eryngii, is shortened
Pleurotus eryngii fermentation period.
4. the coating chitosan of the present invention, while there is the multi-functional of chitosan and cyclohexaamylose, moderately have adjusted
Viscosity, intermiscibility, stability, adsorptivity and the biocidal property of Pleurotus eryngii zymotic fluid, improve what mycelium pellet was distributed in zymotic fluid
Homogeneity and dissolved oxygen concentration.
5. the fermentation process of high density liquid strain of the present invention uses air-blowing zymotechnique, simple for process, easy to operate, hair
The ferment period is short (fermentation tank 67-92h), can scale and mechanization production, by optimize strain spread cultivation and fermentation process culture medium
And zymotechnique is shortened cell age and is proliferated algebraic step away from effectively improving by the way of the additional inoculation of segmentation and temperature-variable fermentation
The bioactivity and fermenting power of liquid spawn, significantly improve the density of mycelium pellet, the yield of dry mycelial weight and exocellular polysaccharide,
Mycelia bulb diameter is reduced, the quality and yield of liquid spawn is improved comprehensively, reduces production cost.
Specific implementation mode
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
Under the premise of invention spirit and scope, various changes or change to material component and dosage progress in these embodiments
It belongs to the scope of protection of the present invention.
It is prepared by 1 raw material of embodiment
1. the preparation of Chinese herbal medicine extract：
The preparation method of the Chinese herbal medicine extract, includes the following steps：It counts in parts by weight, weighs 30 parts of Radix Astragali, do
30 parts of ginger, 30 parts of dried orange peel, 25 parts of cow-bezoar, 25 parts of the root bark of tree peony, 20 parts of Radix Glycyrrhizae, 18 parts of rhizoma anemarrhenae, 18 parts of honeysuckle, 17 parts of cordate houttuynia are red
15 parts of Chinese herbaceous peony, 15 parts of radix scutellariae, 10 parts of Rhizoma Chuanxiong, 8 parts of Radix Angelicae Sinensis；Set in the supersonic wave cleaning machine for filling 0.2% sodium bicarbonate solution in
200W, 30KHz clean 12min, drain, and said herbal medicine, which is crushed to grain size, uniformly to be mixed simultaneously for 2 millimeters hereinafter, setting in container
The water for adding 5 times of weight, obtains mixed material, and 80 DEG C of holding 3h of control temperature are then cooled to 45 DEG C, are with newborn acid for adjusting pH value
4, Microwave Extraction 12min is carried out under the conditions of power 200W, while ultrasonic wave is carried out under the conditions of power 250W, frequency 35KHz
Assisted extraction；Then the mixed enzyme that mixed material total weight 1.0% is added is digested, and is 6.2 with newborn acid for adjusting pH value, enzymolysis
3h, finally adds the mixture of mixed material 2 times of w ethanols and propyl alcohol, and the mass ratio that ethyl alcohol and propyl alcohol mix is 1:2, control
Temperature to 70 DEG C of holding 3.5h, filtering obtains the first filtrate；The water of 2 times of weight of filter residue is added, 90 DEG C of control temperature keeps 2h, so
After be cooled to 30 DEG C, filtering obtains the second filtrate；By the first filtrate and the second filtrate according to mass ratio 3:2 merge, and filter vacuum is dense
It is freeze-dried, crushed up to Chinese herbal medicine extract after contracting；
The mixed enzyme is dextranase, zytase, pentosanase, pectase in mass ratio 3:2:2:1 uniformly mixing.
2. the preparation of wood dust：
The preparation method of the wood dust, includes the following steps：It will be without mouldy, last without disease pest sawmilling or waste wood removes impurity elimination
Object is placed in the supersonic wave cleaning machine of the sodium bicarbonate solution equipped with mass percent concentration 0.5% and is cleaned in 200W, 30KHz
10min is rinsed, is drained, and is put into steam kettle in 0.35MPa boiling 25min, is taken out, drain, with vegetable oil in mass ratio 100:
Then mixture is set microwave dryer in 2000W, 120 DEG C of microwave irradiation 5min, wherein microwave irradiation 10s by 1 uniformly mixing,
10s is stopped, last ultramicro grinding is packed to grain size 0.4mm up to wood dust.
3. being coated with the preparation of chitosan：
The preparation method of the coating chitosan, includes the following steps：Take chitosan that the quality hundred of its quality 40% is added
Divide in the cyclohexaamylose aqueous solution that specific concentration is 11%, softwood is made, is sieved and pelletized with 20 mesh, obtained wet granular, set microwave drying
In power 2000W, 70 DEG C of dry 5min in machine, sieves quick whole grain with 20 mesh and be coated with chitosan to obtain the final product.
Chinese herbal medicine extract, wood dust and coating chitosan used in following embodiment 2-6 are that embodiment 1 is made
Standby, pleurotus eryngii quel strains are " Longhai City 3 " that edible mushroom research institute of Longhai City of Zhangzhou City of Fujian Province provides, and other raw materials are purchased in market.
Embodiment 2
A kind of fermentation process of high density liquid strain, includes the following steps：
1) go bail for the Pleurotus eryngii slant strains 0.2cm kept22 pieces of strain block be inoculated in plating medium and activated,
25 DEG C of constant temperature incubations 10 days, so activation 2 times, obtain tablet seed；
2) the tablet seed card punch of a diameter of 0.5cm is intercepted into 0.2cm23 pieces of strain block access 250mL triangular flasks
In, liquid seed culture medium liquid amount is 100mL, in 25 DEG C of constant temperature incubations 2 days, is then placed in constant-temperature table, 25 DEG C,
100r/min shaken cultivations obtain liquid seeds in 3 days；
3) liquid seeds are inoculated in 10% inoculum concentration in 500mL shaking flasks, Shake flask medium liquid amount 250mL, in
25 DEG C, 150r/min oscillation constant temperature incubations 4 days shake-flask seed；
4) shake-flask seed is inoculated in 10% inoculum concentration in 5L seeding tanks, seed tank culture base liquid amount 3L, in 25
DEG C, under the conditions of ventilatory capacity 4L/min constant temperature incubation 4 days seeding tank seed；
5) seeding tank seed is inoculated in 30L fermentation tanks, fermentation medium liquid amount 15L, in 31 with 5% inoculum concentration first
DEG C, ventilatory capacity 4L/min ferment at constant temperature 18h, be then cooled to 20 DEG C with 0.4 DEG C/min rates, into fermentation tank be added 9g coating
Chitosan and 900mL seeding tank seeds, ferment at constant temperature 13h under the conditions of ventilatory capacity 7L/min, finally with 0.3 DEG C/min rate liters
Temperature is to 25 DEG C, and ferment at constant temperature 48h is up to high density liquid strain under the conditions of ventilatory capacity 6L/min；
The plating medium group becomes：Potato 200g/L, glucose 20g/L, peptone 5g/L, potassium dihydrogen phosphate
3g/L, magnesium sulfate 1.5g/L, agar 20g/L, VB10.02g/L, surplus are water, pH value 6.0；
The liquid seed culture medium group becomes：Glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar
2g/L, Chinese herbal medicine extract 0.4g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.2g/L, VB1 0.01g/
L, surplus are water, pH value 6.0；
The Shake flask medium group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extraction
Object 0.8g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3g/L, VB10.01g/L, surplus are water, pH value
6.0；
The seed tank culture base group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine carry
Take object 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.4g/L, VB10.01g/L, surplus are water, pH
Value 6.0；
The group of the fermentation medium becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine carry
Take object 1.8g/L, wood dust 0.8g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB10.01g/L, surplus are water, pH
Value 6.0；
The preparation method of the fermentation medium is：According to each raw material of culture medium prescription precise, soyabean cake is taken first
The water that 5 times of silty amount is 5.5 with newborn acid for adjusting pH value, the thermostableα-amylase of 5u/g soybean cake powders is added, adds while stirring
Enter soybean cake powder, be warming up to 93 DEG C, heat preservation, liquefy 12min, is then cooled to 55 DEG C, and it is yellow that 40u/g is added in heat preservation while stirring
Remaining water and other raw materials is added in the carbohydrase of beancake powder and the protease hydrolytic 25min of 30u/g soybean cake powders while stirring,
Uniformly mixing, adjustment pH value are 6.0,121 DEG C of sterilizing 40min up to fermentation medium.
The high density liquid strain mycelium pellet density prepared through the above method is 5.5 X 105A/L, mycelium pellet are a diameter of
0.5mm, mycelium pellet dry weight are 100g/L, exocellular polysaccharide 3.5g/L.
Embodiment 3
A kind of fermentation process of high density liquid strain, includes the following steps：
Step 1) is to 4) the same as embodiment 2；
5) seeding tank seed is inoculated in 30L fermentation tanks, fermentation medium liquid amount 15L, in 32 with 5% inoculum concentration first
DEG C, ventilatory capacity 5L/min ferment at constant temperature for 24 hours, be then cooled to 22 DEG C with the rate of 0.5 DEG C/min, 10g be added into fermentation tank
It is coated with chitosan and 900mL seeding tank seeds, ferment at constant temperature 16h under the conditions of ventilatory capacity 8L/min, finally with 0.4 DEG C/min's
Rate is warming up to 26 DEG C, and ferment at constant temperature 52h is up to high density liquid strain under the conditions of ventilatory capacity 7L/min；
The plating medium composition is the same as embodiment 2；
The liquid seed culture medium group becomes：Glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar
2g/L, Chinese herbal medicine extract 0.5g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3g/L, VB1 0.01g/
L, surplus are water, pH value 6.0；
The Shake flask medium group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extraction
Object 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.4g/L, VB10.01g/L, surplus are water, pH value
6.0；
The seed tank culture base group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine carry
Take object 1.2g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.5g/L, VB10.01g/L, surplus are water, pH
Value 6.0；
The group of the fermentation medium becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine carry
Take object 2g/L, wood dust 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB10.01g/L, surplus are water, pH value
6.0；
The preparation method of the fermentation medium is：According to each raw material of culture medium prescription precise, soyabean cake is taken first
The water that 6 times of silty amount is 6 with newborn acid for adjusting pH value, the thermostableα-amylase of 5u/g soybean cake powders is added, is added while stirring
Soybean cake powder is warming up to 95 DEG C, and heat preservation, liquefy 15min, is then cooled to 60 DEG C, and 40u/g soya beans are added in heat preservation while stirring
Remaining water and other raw materials is added, in the carbohydrase of cake powder and the protease hydrolytic 30min of 30u/g soybean cake powders while stirring
Even mixing, adjustment pH value are 6.0,121 DEG C of sterilizing 30min up to fermentation medium.
The high density liquid strain mycelium pellet density prepared through the above method is 4.8 X 105A/L, mycelium pellet are a diameter of
0.5mm, mycelium pellet dry weight are 92g/L, exocellular polysaccharide 3.1g/L.
Embodiment 4
A kind of fermentation process of high density liquid strain, includes the following steps：
Step 1) is to 4) the same as embodiment 2；
5) seeding tank seed is inoculated in 30L fermentation tanks, fermentation medium liquid amount 15L, in 32 with 5% inoculum concentration first
DEG C, ventilatory capacity 3L/min ferment at constant temperature for 24 hours, be then cooled to 22 DEG C with the rate of 0.3 DEG C/min, into fermentation tank be added 8g packets
By chitosan and 900mL seeding tank seeds, ferment at constant temperature 10h under the conditions of ventilatory capacity 8L/min, finally with the speed of 0.4 DEG C/min
Rate is warming up to 24 DEG C, and ferment at constant temperature 45h is up to high density liquid strain under the conditions of ventilatory capacity 7L/min；
The plating medium composition is the same as embodiment 2；
The liquid seed culture medium group becomes：Glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar
2g/L, Chinese herbal medicine extract 0.3g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3g/L, VB1 0.01g/
L, surplus are water, pH value 6.0；
The Shake flask medium group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extraction
Object 0.5g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.4g/L, VB10.01g/L, surplus are water, pH value
6.0；
The seed tank culture base group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine carry
Take object 0.8g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.5g/L, VB10.01g/L, surplus are water, pH
Value 6.0；
The group of the fermentation medium becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine carry
Take object 1.5g/L, wood dust 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB10.01g/L, surplus are water, pH
Value 6.0；
The preparation method of the fermentation medium is：According to each raw material of culture medium prescription precise, soyabean cake is taken first
The water that 4 times of silty amount is 6 with newborn acid for adjusting pH value, the thermostableα-amylase of 5u/g soybean cake powders is added, is added while stirring
Soybean cake powder is warming up to 90 DEG C, and heat preservation, liquefy 15min, is then cooled to 50 DEG C, and 40u/g soya beans are added in heat preservation while stirring
Remaining water and other raw materials is added, in the carbohydrase of cake powder and the protease hydrolytic 30min of 30u/g soybean cake powders while stirring
Even mixing, adjustment pH value are 6.0,121 DEG C of sterilizing 40min up to fermentation medium.
The high density liquid strain mycelium pellet density prepared through the above method is 4.6 X 105A/L, mycelium pellet are a diameter of
0.7mm, mycelium pellet dry weight are 90g/L, exocellular polysaccharide 3.1g/L.
Embodiment 5
A kind of fermentation process of high density liquid strain, includes the following steps：
Step 1) is to 4) the same as embodiment 2；
5) seeding tank seed is inoculated in 30L fermentation tanks, fermentation medium liquid amount 15L, in 30 with 5% inoculum concentration first
DEG C, ventilatory capacity 5L/min ferment at constant temperature 12h, be then cooled to 18 DEG C with the rate of 0.5 DEG C/min, 10g be added into fermentation tank
It is coated with chitosan and 900mL seeding tank seeds, ferment at constant temperature 16h under the conditions of ventilatory capacity 6L/min, finally with 0.2 DEG C/min's
Rate is warming up to 26 DEG C, and ferment at constant temperature 52h is up to high density liquid strain under the conditions of ventilatory capacity 5L/min；
The plating medium composition is the same as embodiment 2；
The liquid seed culture medium group becomes：Glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar
2g/L, Chinese herbal medicine extract 0.5g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.1g/L, VB1 0.01g/
L, surplus are water, pH value 6.0；
The Shake flask medium group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extraction
Object 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.2g/L, VB10.01g/L, surplus are water, pH value
6.0；
The seed tank culture base group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine carry
Take object 1.2g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3g/L, VB10.01g/L, surplus are water, pH
Value 6.0；
The group of the fermentation medium becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine carry
Take object 2g/L, wood dust 0.6g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB10.01g/L, surplus are water, pH value
6.0；
The preparation method of the fermentation medium is：According to each raw material of culture medium prescription precise, soyabean cake is taken first
The water that 6 times of silty amount is 5 with newborn acid for adjusting pH value, the thermostableα-amylase of 5u/g soybean cake powders is added, is added while stirring
Soybean cake powder is warming up to 95 DEG C, and heat preservation, liquefy 10min, is then cooled to 60 DEG C, and 40u/g soya beans are added in heat preservation while stirring
Remaining water and other raw materials is added, in the carbohydrase of cake powder and the protease hydrolytic 20min of 30u/g soybean cake powders while stirring
Even mixing, adjustment pH value are 6.0,123 DEG C of sterilizing 30min up to fermentation medium.
The high density liquid strain mycelium pellet density prepared through the above method is 5.2 X 105A/L, mycelium pellet are a diameter of
0.6mm, mycelium pellet dry weight are 98g/L, exocellular polysaccharide 3.4g/L.
Embodiment 6
A kind of fermentation process of high density liquid strain, includes the following steps：
Step 1) is to 4) the same as embodiment 2；
5) seeding tank seed is inoculated in 30L fermentation tanks, fermentation medium liquid amount 15L, in 31 with 5% inoculum concentration first
DEG C, ventilatory capacity 4L/min ferment at constant temperature 18h, be then cooled to 20 DEG C with 0.4 DEG C/min rates, into fermentation tank be added 9g coating
Chitosan and 900mL seeding tank seeds, ferment at constant temperature 13h under the conditions of ventilatory capacity 7L/min, finally with 0.3 DEG C/min rate liters
Temperature is to 25 DEG C, and ferment at constant temperature 48h is up to high density liquid strain under the conditions of ventilatory capacity 6L/min；
The plating medium group becomes：Potato 200g/L, glucose 20g/L, peptone 5g/L, potassium dihydrogen phosphate
3g/L, magnesium sulfate 1.5g/L, agar 20g/L, VB10.02g/L, surplus are water, pH value 6.0；
The liquid seed culture medium group becomes：Glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar
2g/L, Chinese herbal medicine extract 0.4g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.2g/L, VB1 0.01g/
L, surplus are water, pH value 6.0；
The Shake flask medium group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extraction
Object 0.8g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3g/L, VB10.01g/L, surplus are water, pH value
6.0；
The seed tank culture base group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine carry
Take object 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.4g/L, VB10.01g/L, surplus are water, pH
Value 6.0；
The group of the fermentation medium becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine carry
Take object 1.8g/L, wood dust 0.8g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB10.01g/L, surplus are water, pH
Value 6.0.
The high density liquid strain mycelium pellet density prepared through the above method is 4.5 X 105A/L, mycelium pellet are a diameter of
0.7mm, mycelium pellet dry weight are 90g/L, exocellular polysaccharide 3.0g/L.
The experiment in cultivation of 7 high density liquid strain of the present invention of embodiment
1. test strain：The liquid spawn and commercially available " Longhai City 3 " liquid spawn prepared with the embodiment of the present invention 2 is examination
Strain is tested, test group and control group are divided into；
2. inoculation method：Test group and control group are inoculated with identical inoculum concentration and inoculum density using conventional method, point
It Jie Zhong not be 2000 bottles；
3. production management：Test group and control group use same management method
3.1 bacterium germination management
Bacterium bottle is placed in constant temperature incubation room after inoculation, is protected from light culture.Temperature control at 23 DEG C, relative humidity 70%~
75%.The nutrient that this stage is used in culture material decomposes completely and accumulation.The key of bacterium germination period management is heat preservation, moisturizing, early period
If temperature is too low, the duration is longer, can reduce spawn activity, and mycelia speed of production slows down.All bacterium bottle mycelia it is dense it is white after
15d is persistently cultivated again, nutrient is made thoroughly to decompose, and mycelia reaches physiological maturity.
3.2 corkages, mycelium stimulation
It after bacterium bottle latter stage of ripening, is opened according to a conventional method, mycelium stimulation processing.During mycelium stimulation, when liquid spawn mycelium stimulation
Remove surface thin layer.Bacterium bottle is moved into mushroom room after the completion of mycelium stimulation, bacterium bottle is inverted, and mycelia restoration ecosystem is made.Wait for bacterium
After silk restores, temperature control is at 14 DEG C~16 DEG C, and relative humidity is 90~95%, illumination 500~800Lx, CO2Concentration 600~
1500ppm, fruiting to be buddingged.
3.3 sporophore growth management
Temperature is controlled at 15~18 DEG C, and air humidity is maintained at 85%~90%, CO2Concentration 1500~1800ppm it
Between, sporophore growth is rapid.When mushroom lid is unfolded substantially, spore harvests a damp mushroom when not yet launching.
4. mass and yield index measure
Observe and record mycelial concentration, mycelia color, bacterium germination time, the miscellaneous bacteria infection shape of test group and control group culture bottle
Fructification character is compared in condition, observation, measures fructification stem length, cap size and stem diameter, single bottle yield, and count meter
Miscellaneous bacteria infection rate, level-one mushroom rate and biological transformation ratio are calculated, as a result such as table 1-3：
Biological conversion rate (%)=fructification fresh goods yield (g)/compost dry weight (g) × 100%
Table 1
| Project | The bacterium germination time | Mycelia color | Mycelial concentration | Miscellaneous bacteria infection rate |
| Test group | 14 days | White | It is dense | 0.5% |
| Control group | 18 days | It is partially yellow in vain | It is thin | 15% |
The above result shows that：Fluid present invention strain is healthy and strong, vigor is high, and the speed of growth is fast, and mycelial yield and quality are high, resist
Miscellaneous bacteria ability is strong, compared with the control group bacterium germination time shortening 22.22%, and miscellaneous bacteria infection rate reduces by 96.67%.
Table 2
| Project | Stem average length (cm) | Cap average diameter (cm) | Stem average diameter (cm) | Level-one mushroom rate |
| Test group | 15.17 | 7.34 | 6.28 | 97% |
| Control group | 10.36 | 7.79 | 3.42 | 92% |
The above result shows that：Fructification cap and stem growth ratio after fluid present invention cultivating bacterial spawn is suitable, appearance
Grace, credit rating is high, and level-one mushroom rate improves 5% compared with the control group.
Table 3
| Project | Single bottle average weight (g) | Culture material dry weight (g) | Biological transformation ratio (%) |
| Test group | 123.41 | 180 | 68.56 |
| Control group | 91.73 | 180 | 50.96 |
The above result shows that：For fructification after fluid present invention cultivating bacterial spawn only according to yield height, biological transformation ratio is high, has
Preferable economic benefit is more suitable for batch production, large-scale planting.Compared with the control group, biological transformation ratio improves 34.79%.
Meanwhile a height of 5.5 X of fluid present invention strain mycelium pellet density 105A/L, commercial liquid strain mycelium pellet density
Up to 2.7 X 105A/L, compared with the control group, production capacity are commercial liquid strain 2 times or more, are embodied higher
Economic value.
It should be noted that：Abalone mushroom liquid culture prepared by 3-6 of the embodiment of the present invention equally has above-mentioned experiment effect, respectively
It is little between embodiment and with above-mentioned experiment effect otherness.
Claims (5)
1. a kind of fermentation process of high density liquid strain, which is characterized in that include the following steps：By the Pleurotus eryngii inclined-plane of preservation
Strain expands through liquid seed culture medium, shake-flask seed culture medium and seed tank culture base step by step successively after plating medium activates
Big culture obtains seeding tank seed, and seeding tank seed is inoculated in 30L fermentation tanks, fermentation medium dress with 5% inoculum concentration first
Then liquid measure 15L is cooled down in 30-32 DEG C, ventilatory capacity 3-5L/min ferment at constant temperature 12-24h with the rate of 0.3-0.5 DEG C/min
To 18-22 DEG C, 8-10g coating chitosans and 900mL seeding tank seeds are added into fermentation tank, in ventilatory capacity 6-8L/min conditions
Lower ferment at constant temperature 10-16h, is finally warming up to 24-26 DEG C with the rate of 0.2-0.4 DEG C/min, in ventilatory capacity 5-7L/min conditions
Lower ferment at constant temperature 45-52h is up to high density liquid strain；
The group of the fermentation medium becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract
1.5-2g/L, wood dust 0.6-1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB10.01g/L, surplus are water,
PH value 6.0；
The preparation method of the Chinese herbal medicine extract, includes the following steps：It counts in parts by weight, weighs 30 parts of Radix Astragali, rhizoma zingiberis 30
Part, 30 parts of dried orange peel, 25 parts of cow-bezoar, 25 parts of the root bark of tree peony, 20 parts of Radix Glycyrrhizae, 18 parts of rhizoma anemarrhenae, 18 parts of honeysuckle, 17 parts of cordate houttuynia, radix paeoniae rubra 15
Part, 15 parts of radix scutellariae, 10 parts of Rhizoma Chuanxiong, 8 parts of Radix Angelicae Sinensis；Set in the supersonic wave cleaning machine for filling 0.2% sodium bicarbonate solution in 200W,
30KHz cleans 12min, drains, and said herbal medicine, which is crushed to grain size as 2 millimeters, uniformly to be mixed in container hereinafter, setting and add 5
The water of times weight obtains mixed material, and 80 DEG C of holding 3h of control temperature are then cooled to 45 DEG C, are 4 with newborn acid for adjusting pH value,
Microwave Extraction 12min is carried out under the conditions of power 200W, while ultrasonic wave auxiliary is carried out under the conditions of power 250W, frequency 35KHz
Extraction；Then the mixed enzyme that mixed material total weight 1.0% is added is digested, and is 6.2 with newborn acid for adjusting pH value, is digested 3h,
The mixture of mixed material 2 times of w ethanols and propyl alcohol is finally added, the mass ratio that ethyl alcohol and propyl alcohol mix is 1:2, control temperature
Degree to 70 DEG C of holding 3.5h, filtering obtains the first filtrate；The water of 2 times of weight of filter residue is added, 90 DEG C of control temperature keeps 2h, then
30 DEG C are cooled to, filtering obtains the second filtrate；By the first filtrate and the second filtrate according to mass ratio 3:2 merge, filter vacuum concentration
After be freeze-dried, crush up to Chinese herbal medicine extract；
The mixed enzyme is dextranase, zytase, pentosanase, pectase in mass ratio 3:2:2:1 uniformly mixing；
The preparation method of the wood dust includes the following steps：Will without it is mouldy, without disease pest sawmilling end or waste wood remove sundries, set
10min is cleaned in 200W, 30KHz in the supersonic wave cleaning machine of the sodium bicarbonate solution equipped with mass percent concentration 0.5%,
It rinses, drain, be put into steam kettle in 0.3-0.4MPa boiling 20-30min, take out, drain, with vegetable oil in mass ratio 100:
1 uniformly mixing, then sets microwave dryer in 2000W, 120 DEG C of microwave irradiation 5min, last ultramicro grinding to grain by mixture
Diameter 0.3-0.5mm packs up to wood dust；
The preparation method of the fermentation medium is：According to each raw material of culture medium prescription precise, soyabean cake silty is taken first
The water of 4-6 times of amount, is 5-6 with newborn acid for adjusting pH value, and the thermostableα-amylase of 5u/g soybean cake powders is added, is added while stirring
Soybean cake powder is warming up to 90-95 DEG C, and heat preservation, liquefy 10-15min, is then cooled to 50-60 DEG C, and heat preservation is added while stirring
The carbohydrase of 40u/g soybean cake powders and the protease hydrolytic 20-30min of 30u/g soybean cake powders, be added while stirring remaining water and
Other raw materials uniformly mix, and adjustment pH value is 6.0,121-123 DEG C of sterilizing 30-40min up to fermentation medium；
The preparation method of the coating chitosan includes the following steps：Take chitosan that the quality percentage of its quality 30-50% is added
Specific concentration is that softwood is made in the cyclohexaamylose aqueous solution of 10-12%, is sieved and is pelletized with 10-20 mesh, obtained wet granular, set microwave
In 2000W, 60-80 DEG C of dry 4-6min of power in drying machine, sieves quick whole grain with 10-20 mesh and be coated with chitosan to obtain the final product.
2. a kind of fermentation process of high density liquid strain as described in claim 1, which is characterized in that the liquid seeds training
Supporting base group becomes：Glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar 2g/L, Chinese herbal medicine extract 0.3-
0.5g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.1-0.3g/L, VB10.01g/L, surplus are water, pH
Value 6.0.
3. a kind of fermentation process of high density liquid strain as described in claim 1, which is characterized in that the shake-flask seed training
Supporting base group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 0.5-1.0g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.2-0.4g/L, VB10.01g/L, surplus are water, pH value 6.0.
4. a kind of fermentation process of high density liquid strain as described in claim 1, which is characterized in that the seed tank culture
Base group becomes：Soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 0.8-1.2g/L, biphosphate
Potassium 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3-0.5g/L, VB10.01g/L, surplus are water, pH value 6.0.
5. a kind of fermentation process of high density liquid strain as described in claim 1, which is characterized in that microwave when microwave drying
10s is irradiated, 10s is stopped.
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| CN110819563B (en) * | 2019-11-18 | 2022-03-01 | 曲阜师范大学 | Brevibacillus brevis 3C and application thereof in promoting growth of pleurotus eryngii |
| CN111615996B (en) * | 2020-05-19 | 2021-09-21 | 江西省农业科学院农业应用微生物研究所（江西省农村能源研究中心） | Stropharia rugosoannulata strain breeding method |
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| CN104119134A (en) * | 2014-06-20 | 2014-10-29 | 东至林野生态种植中心 | Pleurotus eryngii culture medium prepared from rice bran pulp and preparation method thereof |
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| CN104119134A (en) * | 2014-06-20 | 2014-10-29 | 东至林野生态种植中心 | Pleurotus eryngii culture medium prepared from rice bran pulp and preparation method thereof |
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| CN104920069B (en) | 2018-03-30 |
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