CN103404366A - Black abalone mushroom liquid culture preparing and high-yield cultivation method - Google Patents
Black abalone mushroom liquid culture preparing and high-yield cultivation method Download PDFInfo
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Abstract
The invention discloses a black abalone mushroom liquid culture preparing and high-yield cultivation method, and belongs to the field of edible mushroom cultivation. The black abalone mushroom belongs to the oyster mushroom category of the oyster mushroom family of the Agaricales of the Hymenomycetes of the Basidiomycotina in the biology classification. The total sugar content of the black abalone mushroom is up to 44.4%. The black abalone mushroom contains various vitamins, amino acid essential for human bodies and microelements essential for the human bodies. When people eat the black abalone mushroom frequently, the blood pressure and the cholesterol can be reduced, and the arteriosclerosis and the like can be prevented. At present, the study about liquid culture production is mainly focused on the formulation selection technology, the culture screening technology, the large-scale submerged fermentation technology and the like of edible mushrooms, such as the needle mushroom and the toadstool, which are difficult to cultivate by manpower or are low in yield. In order to sufficiently develop the advantages of the liquid culture in black abalone mushroom production, the systematic study is carried out on black abalone mushroom culture liquid cultivation in the scheme. According to the black abalone mushroom liquid culture preparing and high-yield cultivation method, the cultivation manner and the management measures are improved and innovated, and the new path for production application of liquid shake-flask cultivation and black abalone mushroom culture fermentation cylinder cultivation is finally studied out after exploration for several years and repeated tests.
Description
Technical field
A kind of method of black abalone mushroom liquid spawn preparation and high-yield culturing, belong to edible fungus culturing and learn field.
Background technology
At black abalone mushroom [Pleurotus ostreatus (Jacg.exFr.)], claim again black flat mushroom, grey flat mushroom, Fungus Pleurotus ostreatus, first mushroom, limit pin mushroom, grey mushroom (abalonelike) etc., belong to Basidiomycotina, Hymenomycetes, Agaricales, Pleurotaceae, Pleurotus in the biology classification.Its fruit body cap is shell-like or fan-shaped etc., the greyish black light in cap surface, and the fertile matter of meat is tender, delicious flavour, nutritious.Its total sugar content is up to 44.4%, and contains amino acid and the trace element of various vitamin and needed by human, frequent edible can reducing blood pressure and cholesterol, and the prevention of arterial sclerosis, treatment autonomic nerve disorders etc., be called functional food, health food both at home and abroad.The abalone flavor that dense aquatic foods are arranged because of its fruit body, therefore be referred to as " grey mushroom (abalonelike) ".The research of producing about liquid spawn at present mainly concentrates on the formula screening, bacterial screening, scale deep layer fermenting process of the edible mushroom that the artificial cultivations such as Asparagus, dictyophora phalloidea, hickory chick are more difficult or output is lower etc.; and only make some progress, and the production-scale liquid shaking bottle of current strain plant is cultivated the physiology of bacterial classification and the research of application technology is reported seldom to relatively being applicable to.In order to give full play to the advantage of liquid spawn in black abalone mushroom is produced, this problem compares systematic research to the black abalone mushroom kind of liquid culture.In addition, our improvement and bring new ideas in addition on planting type and control measures, through in a few years exploring and repetition test has worked out finally that liquid shaking bottle is cultivated and fermentation tank culture is deceived the new way of the production application of abalone mushroom bacterial classification.
Summary of the invention
(1) research of liquid strain nutriment and Screening of Media
1 material
11 for the black abalone mushroom PL-AD of examination bacterial classification
28, provided by edible fungus research institute of Yantai Normal College.
1.2 basal medium sucrose 1%, peptone 0.5%, KH
2PO
40.05%, MgSO
40.05%, VB
110mg/L.
1.3 shaking flask medium soluble starch 5%, sucrose 1%, MgSO
40.15%, KH
2PO
40.3%, yeast extract (powder) 0.1%, pH value nature.
2 methods
2.1 the preparation of one-level shaking flask kind accesses bacterial classification picking soybean grain size 4-6 piece under aseptic condition in firm cultured test tube slant in 500ml (liquid amount 200ml) conical flask, allow mycelia face up and float on liquid level, be placed in the 25 ℃ of static cultivation of insulating box 48h, then in 25 ℃, 5～6d. is cultivated in the rotary shaking table concussion of 180r/min
2.2 the preparation of secondary shaking flask kind accesses one-level shaking flask kind in 500ml (liquid amount 200ml) conical flask, inoculum concentration is 10%, is placed in 25 ℃, the rotary shaking table shaken cultivation of 180r/min 5d, plant liquid clarification, yellowish, take out kind of quiet the putting of bottle, be evenly distributed, stop fermentation.Then survey its biomass, Peloton density and bacterium bulb diameter, carry out variance analysis.
2.3 after biomass estimation fermentation stops, the culture fluid in the secondary shaking flask by 15 * 15cm3～4 layer filtered through gauze, then, after water flushing several times, is dried to constant weight in 60 ℃ of drying bakers, surveys its biomass.
2.4 the mensuration of Peloton density is placed in the static 5min of 200ml graduated cylinder by bacterium liquid, surveys the bacterium ball and accounts for the long-pending percentage of bacteria liquid.
2.5 the mensuration of bacterium bulb diameter is got at random the bacterium ball and lined up a 5cm straight line, number goes out the bacterium nodule number in the 5cm straight line, then uses the bacterium nodule number divided by 5cm, obtains the average diameter of bacterium ball.
2.6 the screening experiment of suitable carbon source uses respectively 1% sucrose, glucose, brown sugar, maltose, fructose, white sugar as carbon source the carbon source in basal medium, with without the carbon source basal medium as a control group, each carbon source repeats 3 times, and condition of culture is with the shaking flask kind.Fermentation stops, and measures the impact of various carbon sources on its biomass.
2.7 the screening experiment of suitable nitrogenous source is used the nitrogenous source in basal medium respectively 0.1% yeast extract (powder), peptone, beef extract, NH
4CL, NaNO
3, (NH
4)
2CO
3As nitrogenous source, take and be contrast without the nitrogenous source basal medium, each repeats 3 times, and condition of culture is with the shaking flask kind, and fermentation stops, and measures the impact of various nitrogenous sources on its biomass.
2.8 the screening experiment of suitable mineral salt, by the mineral salt in basal medium, is used respectively 0.2% KH
2PO
4, K
2HPO
4, MgSO
4, ZnSO
4, FeSO
4, CaCO
3As mineral salt, the mineral salt basal medium of take is contrast, and each repeats 3 times, and condition of culture is the same, and fermentation stops, and measures the impact of various mineral salt on its biomass.
2.9 the agricultural byproducts hydrolyzate is made the screening experiment of black abalone mushroom liquid nutrient medium in order to reduce costs, we are after the black abalone mushroom liquid strain optimum formula of screening, studied again the test of the agricultural byproducts such as corn flour, wheat bran, bean powder, groundnut meal, rice bran as Carbon and nitrogen sources, and respectively add 1% the red pool, set four kinds of culture medium prescriptions.Formula A: corn flour 5%, brown sugar 1%, wheat bran 3～5%, KH
2PO
40.05%, MgSO
40.05%, pH value 6～7.Formula B: corn flour 6%, brown sugar 1%, bean powder 2%, KH
2PO
40.05%, MgSO
40.05%, pH value 6.0～7.0.Formula C: corn flour 5%, brown sugar 0.5%, groundnut meal 2%, KH
2PO
40.05%, MgSO
40.05%, VB
110mg, pH value 6.0～7.0.Formula D: corn flour 5%, brown sugar 0.5%, rice bran 4%, KH
2PO
40.05%, MgSO
40.05%, pH value 6.0～7.0.Control group: soluble starch 5%, sucrose 1%, KH
2PO
40.3%, MgSO
40.15%, yeast extract (powder) 0.1%.Establish 8 repetitions for every group, 5d is cultivated in concussion, detects black abalone mushroom mycelial growth situation.
3. results and analysis
3.1 the selection result Analysis of variance of suitable carbon source shows, the average dry weight difference of the mycelium between each group has significant (P<0.05=.Illustrate that different carbon sources have obvious difference to black abalone mushroom mycelial growth.
Table 1 is the selection result of suitable carbon source
As can be seen from Table 1, black abalone mushroom mycelium is more extensive to the utilization of carbon source, and monosaccharide and disaccharide all adapts to, but, in monose, is advisable with glucose, and in disaccharide, sucrose is advisable.In this experiment, sucrose is optimal carbon source, and under same condition of culture, bacterium nodule number amount is maximum, and biomass is the highest.It is the poorest that fructose utilizes.Therefore it is that optimum carbon source is cultivated the shaking flask kind that sucrose is selected in this experiment, is secondly glucose.
3.2 the selection result Analysis of variance of suitable nitrogenous source shows, each otherness of organizing mycelial biomass is (p<0.05) significantly, illustrate that different nitrogen sources exists obvious difference to the impact of deceiving the abalone mushroom mycelial biomass.
The impact of table 2 different nitrogen sources on black abalone mushroom mycelial growth
As can be seen from Table 2, mycelium is better than inorganic nitrogen to the utilization of organic nitrogen, and this also meets the requirement of most edible mushroom.The yeast extract (powder) of wherein take is best organic nitrogen, is secondly peptone and beef extract.Black abalone mushroom nitrate nitrogen in the utilization of inorganic nitrogen slightly is better than ammoniacal nitrogen.
3.3 the screening test Analysis of variance of suitable mineral salt, each otherness of organizing the mycelium biology is (P<0.05) significantly.In this experiment, the mineral salt of suitable black abalone mushroom mycelial growth are according to being
MgSO
4＞KH
2PO
4＞K
2HPO
4＞ZnSO
4＞CaCO
3＞FeSO
4。
The impact of the different mineral salt of table 3 on black abalone mushroom mycelial growth
As can be seen from Table 3, with MgSO
4And KH
2PO
4For the best, be secondly K
2HPO
4.Visible Mg, P, K have larger impact to the black mycelial growth of abalone mushroom.
3.4 the selection result of agricultural byproducts hydrolyzate medium
As can be seen from Table 4, with agricultural byproducts hydrolyzate formula with agent prescription, compare, bacterium ball size, bacterium ball vigor are all without significant difference, the mycelium dry weight mean value of formula A and formula B is all higher than control group, the t check shows, with control group, compare, the difference of formula A, the average mycelium dry weight of B has significant.Therefore not only cost is low to select formula A to make liquid nutrient medium, and effective.
Table 4 agricultural byproducts hydrolyzate medium culture is deceived the mycelial growing state of abalone mushroom
3.5 orthogonal experiment screening optimal medium is selected the Carbon and nitrogen sources of the black abalone mushroom mycelial growth of three kinds of applicable liquid culture, designs 4 factors, the orthogonal experiment of 3 levels, 9 combinations, 5 repetitions, filter out suitable single carbon source and single nitrogenous source and content separately, the results are shown in Table 5, table 6.
Table 5L9 (3
4) Orthogonal Experiment and Design
Each component of table 6 L9 (3
4) Orthogonal Experiment and Design
From table 6 analysis, find out, the mycelia dry weight of take is reference index, and best of breed is A
1B
3C
3D
3, namely brown sugar 1%, corn flour 5%, wheat bran 5%, peptone 0.5%.With best of breed, add trace element, i.e. KH
2PO
40.05%, MgSO
40.05%, V
B110mg/L, namely become black abalone mushroom optimal liquid medium after cooperation.
(2) research of physical and chemical factor to black abalone mushroom Mycelia growth effect
In recent years, the black abalone mushroom production development of our province is very fast, makes but the black abalone mushroom bacterial classification used at present mostly is the solid mode, and the cycle is longer, consumes energy more.With black abalone mushroom solid spawn, compare the advantage such as liquid spawn possesses with short production cycle, and cell age is neat.In order to give full play to the advantage of liquid spawn in black abalone mushroom is produced, the part physiology that this experiment is cultivated black abalone mushroom bacterial classification to liquid shaking bottle is studied.
1. materials and methods
1.1 material
1.1.1 strains tested PL-AD
28, provided by edible fungus research institute of Yantai Normal College.
1.1.2 inclined-plane mother culture media PDA medium.
1.1.3 liquid seed culture medium soluble starch 5%, sucrose 1%, dusty yeast 0.1%, KH
2PO
40.3%, MgSO
40.15%.pH be worth nature.
1.2 method
1.2.1 the preparation of liquid spawn fills the female slant strains access 500ml that plants of cultured test tube in the conical flask of 200ml culture fluid, every bottle graft enters 3～6 (soybean grain sizes), make its mycelia be suspended on liquid level upward, after being placed in the 25 ℃ of static cultivation of constant temperature 48h, be placed on 25 ℃ of lower shaken cultivation 5d on the rotary shaking table of 180r/min, as liquid spawn.
1.2.2 the mensuration of mycelia dry weight by 4 layers of filtered through gauze, then, after water rinses well, is placed in cultured bacterium liquid 60 ℃ of drying bakers and dries to constant weight, and then claims its weight.
2. result and discussion
2.1 the culture fluid of medium initial p H on the different gradient pH of impact configuration of black abalone mushroom fermentation, after packing inoculation shaking flask is cultivated routinely, measure cultivation front and back culture fluid pH, the variation of observing pH in the black abalone mushroom process of growth of contrast reaches the impact on mycelial growth, the results are shown in Table 7.
The impact of table 7 initial pH value of medium on black abalone mushroom fermentation
Result shows, cultivates initial pH very large on the fermentation impact, matches with orthogonal experiments.The situation of change of culture fluid pH before and after the contrast inoculated and cultured, find that black abalone mushroom is seeded in the culture fluid of different PH gradients and cultivate, initially all culture fluid pH is adjusted to 6～7 left and right, illustrate that the black comparatively ideal culture fluid of abalone mushroom growth fraction is at the micro-acid environment of neutrality, and can regulation of secretion in metabolic process the material of environmental PH, with the stable environment acid-base value.
2.2 the impact of shaking flask liquid amount on black abalone mushroom fermentation
With the 500ml conical flask pack 50,100,120,150,180,200 into, the culture fluid of 220mL carries out the shaking flask cultivation.The results are shown in Table 8.
The impact of table 8 shaking flask liquid amount on black abalone mushroom fermentation
Result shows, 200ml contains the highest, the too much or very few Sheng liquid measure of liquid measure mycelial yield, all may produce the bacterium ball that diameter is larger.
2.3 shaking flask rotating speed and the cultural method impact on black abalone mushroom fermentation
(1) shaking flask rotating speed: will contain the 500ml triangular flask of 200ml zymotic fluid, and be placed in respectively 160,180,200, carry out shake flask fermentation under the rotating speed of 220r/min.(2) cultural method: a. gets primary inclined plane 1cm
2, being inoculated in shaking flask, rotational oscillation is cultivated immediately.B. get primary inclined plane 1cm
2, bacterium faces up, and is inoculated in gently in shaking flask, static cultivation 48h, then rotational oscillation is cultivated.The results are shown in Table 9.
The impact on black abalone mushroom fermentation of table 9 shaking flask rotating speed and inoculation method
Result shows, within the specific limits, rotating speed increases, throughput increases, dissolved oxygen content increases, and the mycelial growth amount increases, and the mycelium pellet diameter diminishes, when rotating speed reaches 100～180r/min, the air abundance, nutrient is abundant, and mycelia is along the direction growth of inoculation piece cross section, and be staggered to form network structure, become the core of granular structure later.When rotating speed reaches 200, excessive velocities, network structure can not form, and causes the mycelia granule to reduce.In addition, static cultivation 48h after inoculation, then the mycelium production of upper shaking table shaken cultivation, after the inoculation, the mycelium production of shaken cultivation is high immediately.This may be while due to the bacterial classification piece, cultivating on surface, and the air abundance is applicable to thalline and grows, and the part mycelia, along the bacterial classification cross growth, forms network structure, carries out shaken cultivation later, and these netted dispersions become the core of mycelium pellet, are convenient to form mycelium pellet.
2.4 the impact of fermentation temperature on black abalone mushroom mycelium pellet output and size
500ml shaking flask bacterium liquid is placed in to 15 ℃, 20 ℃, 25 ℃, 30 ℃ bottom fermentations.Measure the impact of cultivation temperature on mycelium pellet dry weight and size, the results are shown in Table 10.
The impact of table 10 fermentation temperature on black abalone mushroom fermentation
As can be seen from Table 10, in certain temperature range, the mycelium pellet diameter reduces gradually along with continuous the increasing of rising mycelia amount of temperature, but, after 25 ℃, continues to heat up, and while reaching 30 ℃, the mycelia amount no longer increases.Visible 25 ℃ are black abalone mushroom preference temperature.Therefore, determine that 25 ℃ are suitable fermentation temperature.
2.5 impact in 500ml triangular flask the packing 200ml culture fluid of shaking flask culture fluid inoculum concentration on black abalone mushroom fermentation, sterilizing rear 5%, 10%, 15%, the 20% different amount one-level shaking flask bacterial classifications that access respectively are placed in 25 ℃, the rotary shaking table shaken cultivation of 180r/min 5～6d, survey the situation of its inoculum concentration to mycelial growth, measure mycelia dry weight and bacterium ball size.The results are shown in Table 11.
The impact of table 11 shaking flask culture fluid inoculum concentration on black abalone mushroom fermentation
From upper table, illustrating, from 5%～10% increase progressively, mycelial output improves gradually along with inoculum concentration, inoculum concentration with 10%～15% is advisable, and when inoculum concentration reaches 20%, the amount of growth of mycelia is without increasing significantly, consider economic benefit, determine that inoculum concentration is 10%.
2.6 the different fermentations time is on black abalone mushroom biomass and after slightly the impact of polysaccharide will fill the 500ml triangular flask inoculation of 200ml culture fluid, 25 ℃ of lower 180r/min shaken cultivation, start to measure mycelium production from 72h, every the 24h sampling, measure once, measure altogether 5 times, each 3 bottles, average, measurement result is in Table 12.
The mycelial growing state of black abalone mushroom of the different incubation times of table 12
After result is shaking table shaken cultivation 4d, it is light yellow transparent and fresh and sweet taste arranged that culture fluid also is, higher in the mycelial output of fermented and cultured 120～144h and thick polyoses content as seen from Table 12, cultivate the 120h value of peaking, bacterium liquid is fast and healthy and strong in 25 ℃ of dull and stereotyped long speed of mycelia of cultivating.After 168h, mycelial biomass sharply descends, the self-dissolving that senesces of bacterium ball, and the mycelia observed under microscope is thin and weak.
2.7 broth viscosity is on mycelium pellet quantity and big or small to affect the mycelium pellet number of liquid spawn in unit volume the more the better, diameter is healed better little.Can increase the contact-making surface of bacterial classification and composts or fertilisers of cultivating like this, be conducive to send out bacterium.It is reported, the viscosity of the big or small major decision medium of mycelium pellet.In certain scope, the viscosity of medium is larger, and the mycelium pellet number is more, and the bacterium ball is less.We adopt soluble starch to improve broth viscosity, and the research broth viscosity, on mycelium pellet number and big or small impact, the results are shown in Table 13 (cultivating 5d under 25 ℃) through repetition test.
Table 13 broth viscosity and bacterium ball amount and big or small relation (unit: individual/ml.mm)
3. brief summary
By system test, determined that the suitableeest carbon source of black abalone mushroom liquid fermentation is sucrose (or brown sugar or corn flour), the suitableeest nitrogenous source is yeast extract (or wheat bran, peptone). the suitableeest mineral salt are KH
2PO
4And MgSO
4, the suitableeest preliminary examination pH value 6.0～7.0, the suitableeest liquid amount 500ml triangular flask is contained 200ml, and the suitableeest cultural method is shaken cultivation after the rear 48h of cultivation of inoculation.The suitableeest shaking speed is 180r/min, and the suitableeest cultivation temperature is 25 ℃, and the suitableeest inoculum concentration is 10%, and the suitableeest incubation time is 5d.If in medium, add appropriate stickum, the size of hypha biomass and bacterium ball can be better.
(3) Primary Study of liquid fermentation tank kind deep layer cultivation.
1. materials and methods
1.1 bacterial classification: PL-AD
28, provided by edible fungus research institute of Yantai Normal College.
1.2 medium:
1.2.1 slant medium: PDA medium.
1.2.2 shaking flask medium: soluble starch 5%, sucrose 1%, yeast extract 1%, MgSO
40.15%, KH
2PO
40.3%, PH nature.
1.2.3 fermentation tank culture medium: corn flour 5～6%, brown sugar 1%, wheat bran 5%, peptone 0.5%, KH
2PO
40.05%.MgSO
40.05%, PH6～7.
1.3 method:
1.3.1 actication of culture: will be stored in the PL-AD in refrigerating box
28Bacterial strain is received on the PDA medium, cultivates 6～7d in 25 ℃.
1.3.2 the one-level shaking flask is cultivated: in 500ml triangular pyramidal bottle, be loaded on 200ml shaking flask medium, inoculate 2～3 slant strains that soya bean is large, static cultivation 24h, in 25 ℃, 180r/min shaking table shaken cultivation 5～6d.
1.3.3 the secondary shaking flask is cultivated: the 200ml shaking flask of packing in 500ml triangular pyramidal bottle medium, the one-level shake-flask seed liquid of access 10% amount, in 25 ℃, 180r/ml shaking table shaken cultivation 5d.
1.3.4 submerged fermentation is cultivated:
1.3.4.15L fermentation tank culture: the 5L fermentation tank 4L fermentation tank culture medium of packing into, by 10% inoculum concentration, connect the secondary shake-flask seed liquid, in 25 ℃, the 180r/min cultivation of ventilating.Every 24h sampling detects the mycelial growth situation.
1.3.4.2100L fermentation tank culture: the 100L canned people 70L medium that ferments, fermentation tank internal pressure maintain and carry a 0.03M
Pa, temperature is 25～26 ℃, every 24h sampling detects the mycelial growth situation.
2. result and discussion
2.1 ferment tank growth curve: from Fig. 1 and Fig. 2, can find out, 5L, 100L fermentation tank mycelia yield all are the double logarithmic curve relation with incubation time, fermentation tank culture 3～4d, mycelia yield maximum.
In cultivation process, black abalone mushroom mycelium is constantly grown, and forms a large amount of mycelium pellets, constantly consumes nutrient component in medium, and mycelium dry weight constantly increases, and reaches maximum at 96-120h, and now biomass is the highest.Continue fermented and cultured, nutriment is poor, and mycelium falls into a decline, and nidus is molten, and biomass starts to descend.According to tracing analysis, fermentation termination should be determined at 84-100h.Although now the mycelia dry weight is not maximum, is conducive to mycelial growth.
2.2 the relation of fermentation time and pH value
The 5L fermented and cultured, self registering PH change curve such as Fig. 3.
As can be known by shake flask test, the suitableeest preliminary examination pH value is 6～7, and medium makes approximately 0.5 position of PH decline at sterilization process, therefore before sterilizing, the PH of medium is adjusted to 6.5～7.5.As can be seen from Figure 3, under the environment of suitable mycelial growth, mycelia itself has the ability of certain adjusting pH value, and makes PH be in plateau.But the C/N ratio in medium, the addition of anti-crawl agentfoam oil etc. are not suitable for, the thalline cocktail party loses the ability of regulating PH, and the PH of medium will fluctuate.Therefore by the observation of fermentation process PH change curve, judge whether mycelium is in the growing environment of adaptation.
(4) comparative study of the result of use of liquid strain and solid kind.
1, materials and methods
1.1 material
1.1.1 strains tested PL-AD
28, provided by edible fungus research institute of Yantai Normal College.Make routinely the female kind of test tube and liquid strain.
11.2 culture medium A. the wheat pedigree seed culture medium; B. cultivation bag medium: cotton seed hulls 50%, corncob 30%, pomace 20%, quicklime 5%.(additional)
1.2 method
1.2.1 the preparation of wheat medium is soaked 24h by wheat, then adds poach to thoroughly well cooked but not mushy, namely the white heart of broken nothing is advisable, and the packing Cans are to bottleneck, conventional sterilizing rear standby.
1.2.2 the preparation of cultivation material bag is by the formulated normal fermentation, by the cultivation material bag the fermented polyethylene plastic bag of packing into, and every sacked material heavy 1.2Kg that gives money as a gift, the long 28cm of the clean charging of bag.
1.2.3 1. the mycelia vigour accesses respectively the wheat medium by the female kind of test tube, liquid strain, inoculum concentration is 10%.Observe mycelia and cover with a bottle time, to determine the difference in quality of liquid strain and solid kind.Each processes 30 bottles of various combination inoculations, is divided into 3 groups, cultivates under 25 ℃.The cotton seed hulls that 2. will prepare is cultivated pocket and is accessed respectively immediately wheat kind and liquid strain, and inoculum concentration is 10%.Observe mycelium germination time, growth rate, purseful number of days, pollution rate etc., to determine the vigor difference of liquid strain and solid kind.60 bags of the various combination inoculations of each processing, be divided into three groups, 25 ℃ of cultivations.
1.2.4 fruiting relatively adopts wall formula stereoscopic bag to plant the above-mentioned cultivation bag of sending out bacterium good, 60 bags of each combinations, be divided into three groups, and Routine Management, using first three damp mushroom as measurement index.
2, results and analysis
2.1 mycelia vitality test result shows, liquid strain, female access wheat medium and the cotton seed hulls medium of planting of test tube, and mycelia covers with Cans and the required time difference of cultivation bag is remarkable.The wheat medium is respectively 16d and 30d, differs 14d; The cotton seed hulls medium is respectively 21d and 34d, differs 13d, and liquid strain is than test tube kind tool have great advantage (in Table 14, table 15) all when breeding original seed and the cultivation bag no matter illustrate.
The comparison of table 14 liquid strain and solid kind growing state on the wheat medium
The main cause of analytical table 14 has: one, and liquid strain is comprised of mycelium pellet, and inoculation shakes up rear bacterium ball and can be evenly dispersed in medium, carries out the Multipoint synchronous growth; Its two, the new fresh oxygen that liquid strain constantly provides during the fermentation, can make hyphal cell breathe vigorous, cell division is quick, has shortened a bacterium time.
Table 15 liquid strain and solid kind growing state on the cotton seed hulls medium compares
The result of table 15 is similar to table 14, and the day long speed of liquid strain is 1.07cm, and the solid kind is 0.76cm, and its difference is 0.31, purseful time advance 13d, and the mycelia vigor of visible liquid strain is apparently higher than the solid kind.
2.2 the experiment in cultivation output of two kinds of bacterial classifications of output comparison has obvious difference, in Table 16.
Table 16 liquid strain and solid kind fresh mushroom production be (Kg) relatively
As can be seen from Table 16, bag output differs 0.18Kg, and biological efficiency improves 15%.
3, discuss
(1) liquid strain has shortened the production of hybrid seeds cycle, cultivates original seed and than solid spawn, can shift to an earlier date 14d and cover with the bacterium bottle, and cultivate cultivation bag and can shift to an earlier date 13d and send out the bacterium bag full, and mycelial growth is vigorous, the bacterial classification stalwartness, cell age is consistent, and survival rate is high, and pollution rate is low.
(2) liquid spawn is in composts or fertilisers of cultivating, and same time intramatrical mycelium kink is many, and early, change of tide is fast for fruiting, can shift to an earlier date the 11d fruiting than the composts or fertilisers of cultivating of solid spawn cultivation, and whole cultivation cycle can shorten 35～40d, and output is high, and fruiting is neat, is convenient to management.
(3) the liquid spawn feeding is convenient, and cost is low, saves the energy, as long as strictly observe the rules of sterile working, is to be worth scale, batch production to produce a kind of method of upper application.
(5) experimental study of liquid strain storage temperature and the term of validity
With tank fermentation method, produce liquid spawn, have that output is high, the cycle is short, cell age is neat, pollute less, low cost and other advantages, but storage life is short, after inoculated and cultured is good, must use in a short time, having lost the solid kind has the advantage of certain storage life, incompatible with the production and selling of strain plant, seriously restrict the black abalone mushroom liquid strain of strain plant development.In order to explore black abalone mushroom liquid strain storage temperature and the term of validity, we have carried out following experimental study.
1 materials and methods
1.1 material
1.1.1 strains tested PL-AD
28, provided by edible fungus research institute of Yantai Normal College.
1.1.2 the preparation of bacterial classification through test tube slant and liquid strain expand numerous after, access 5L stainless steel standard fermentation tank culture 120～132h, the bacterium ball is 1000～1200/ml, mycelia 800～850mg/100ml.
1.2 method
1.2.1 bringing back to life test, plate establishes 0～5 ℃, 6～10 ℃, and 11～15 ℃, 16～20 ℃, 21～25 ℃ of five tests.Each processes bacterial classification with the encapsulation of 500ml triangular flask, puts under different temperatures and preserves.During resurrection, the large ball of picking, bead, sheet respectively connect respectively three points on plating medium, put under 25 ℃ and to cultivate, and record the bacterium ball situation of sprouting.
1.2.2 the test of bacterial classification survival rate is established 5 temperature ranges and is preserved, and every 3d, gets at random 10 bacterium balls and is seeded on Agar Plating, puts 25 ℃ of cultivations, is as the criterion to inoculate sprouting of rear 2d bacterial classification, is calculated to be motility rate, observes altogether 16d.
2 results and analysis
2.1 plate brings back to life test: by five kinds of humid tests, each form bacterium ball all carries 24h and sprouts, and along with the prolongation of preserving number of days and the raising of storage temperature, bacterium ball sprout time has notable difference, in Table 17.
Table 17 plate brings back to life test (getting the mean of three some start-up times)
2.2 bacterial classification survival rate test: black abalone mushroom liquid strain carries within 0～10 ℃ to be preserved the 18d survival rate and is more than 100%, 10 ℃ along with the prolongation of holding time, and survival rate obviously reduces, in Table 18.
Under table 18 different temperatures, preserve the survival rate (%) of bacterial classification
From above-mentioned two tables, finding out 1. at identical temperature, with the reduction of storage life prolongation bacterial classification survival rate; 2. storage life is identical, and the bacterial classification survival rate raises and weakens with temperature; 3. in this test, the survival rate of preserving bacterial classification does not show downward trend within 0～10 ℃, within 11～15 ℃, start to descend after 15d, but fall is little, within 16～20 ℃, 9d starts to descend, and after 6d, is sharply downward trend within 21～25 ℃; 4. survival rate drops to 95% and makes the black abalone mushroom liquid strain term of validity of summation balance: 0～10 ℃ is 18d, and 10～15 ℃ is 15～18d, and 16～20 ℃ is 9～12d, and 21～25 ℃ is 3d.
3 discuss
3.1 at plate, bring back to life in test, we find that the size of bacterium ball is also a direct factor that affects the term of validity.Large ball, bead and the sheet of institute's picking are within the same holding time, and its speed of sprouting is followed successively by large ball>bead>sheet mycelia; Under same temperature, preserving number of days is also large ball>bead>plates.It seems, thalline is large, and nutritional amt is many, and adaptability early, sprout fast, and the bacterium ball is less, sheet mycelia especially, and its adaptability is poor, very easily dead.At this, be noted that large ball requirement is advisable in about 0.2cm.
3.2 we have only made the bottled liquid strain term of validity of 500ml, 300ml triangle and observe in test, relevant other packing method, on the impact of the term of validity, need further research.
3.3 in view of above characteristics, we think that the term of validity preservation of liquid strain is subjected to the multiple-factor restriction, not only is subjected to the impact of temperature, different time, and is subjected to the impact of packing container, bacterium ball size and cell age.
(6) experimental study of liquid strain fruiting biological transformation ratio of different storage period
1 materials and methods
1.1 material
1.1.1 strains tested PL-AD
28, provided by edible fungus research institute of Yantai Normal College.
1.1.2 medium is female PDA medium of planting 1..2. liquid nutrient medium: corn flour 5%, sucrose 1%, yeast extract (powder) 0.5%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.05% is solid original seed and the foster base of bag cultivating 3.: all employing modulation water content is 65% pure cotton seed hulls, cultivation bag is the Polythene Bag of 17cm * 33cm * 0.04, and every sacked material 250g that gives money as a gift is conventional sterilizing.
1.2 method
1.2.1 the preparation of liquid strain is at the 500ml triangular flask that fills 15 beades liquid nutrient medium 150ml that packs into, in 1.1kg/cm
2Under went out equal 30 minutes, cooling rear every bottle graft enters female 3～4 of the soybean grain sizes of planting in inclined-plane, puts 25 ℃ of concussions on 180r/min rotation mode bottle swingging machine and cultivates 5d, and start to calculate and deposit the effect time.
1.2.2 shelf life test different under different temperatures are the liquid strain after shaking table shaken cultivation 5d, by the length production of hybrid seeds successively of holding time, under 25～30 ℃ of room temperatures, deposit respectively 0,1,5,7,15,25,95d, and under 5 ℃, deposit 95d in refrigerator.The solid kind is after cultivation 35d, to cover with the original seed of bottle, compares to connect the solid kind.Then by the liquid strain of different storage lives access cultivation bag.3 repetitions of every processing, each 20 bags.Culture farm ground temperature is 25～26 ℃, and air humidity remains on 65～70%, manages under the same conditions.After mycelia was covered with bag, in 10～22 ℃, air humidity was 90～95% fruiting place fruitings.
Result
2.1 the fresh mushroom production comparative result is because liquid strain is in better growing conditions, mycelial growth is vigorous, and day growth speed is fast, so in cell, the nutriment accumulation is many, and output is generally higher than the solid kind.In 1～5d, liquid strain is than solid kind increase yield significantly when the holding time, and biologicak efficiency can improve more than 10%.In Table 19, table 20.
The impact on fresh mushroom production of table 19 liquid strain different storage periods
From explanation table 19, the liquid strain output of depositing 1d under room temperature (25～30 ℃) is the highest, gross yield 8028g, and biologicak efficiency reaches 160.6%; Deposit 15d with interior liquid strain fresh mushroom production higher than solid spawn; Deposit the liquid strain output of 25d a little less than solid spawn; Deposited the liquid strain of 95d, although mycelia day, long speed also was not less than 6.4mm, mycelia is sparse, and a little less than vitality, the microscopy mycelia is aging, thin and weak.Many cavitys in cell.Through multiple range test, its significance of difference is in Table 20.
The SSR multiple ratio of table 20 liquid strain bag output of different storage period
Result of the test shows, black abalone mushroom liquid strain is at 25～30 ℃, deposits between 25d mushroom output without significant difference, deposits liquid strain output in 15d higher than the solid kind, if (5 ℃) are preserved in refrigerator, can also extend storage life (3 months) and little to the mushroom yield effect.
2.2 fruiting time and mushroom matter relatively, the results are shown in Table 21.
The impact of table 21 storage life on fruiting time and mushroom matter
Result of the test shows, liquid strain is deposited 7d under room temperature (25～30 ℃), little to the fruiting time effects.Within certain resting period, one resting period is longer, and the first damp mushroom goes out more early, and the growth and development stage of this and black abalone mushroom has substantial connection, namely black abalone mushroom by the hyphal development stage to differentiation fruiting, the physiological maturity that needs are certain.In addition, the bacterial classification resting period is shorter, and the fruiting time is overstepping the bounds of propriety loose, irregular.The long-time bacterial classification of preserving of low temperature, its distinguishing feature is that fruiting is neat, this is mainly synchronously to cultivate effect, makes mycelia be in same vegetative stage.
Discuss
By this test, show: in temperature higher period, under room temperature, the storage life of liquid strain can not surpass 15d, and best storage life is 1～7d, and comparable like this solid kind improves 4～12% output, and the quality of mushroom body is also relatively good.If words with good conditionsi, field of bacterial and mushroom agriculture adopt cryopreservation solution body kind, so not only can guarantee the fruiting Yield and quality, extend the holding time, and fruiting is neat, be convenient to management.
Two, the research of black abalone mushroom liquid spawn good quality and high output pattern.
(1) screening of composts or fertilisers of cultivating
1 materials and methods
1.1 material
1.1.1 strains tested PL-AD
28, edible fungus research institute of Yantai Normal College provides.
1.1.2 cultivated species adopts 500ml triangular flask loading amount 200ml, shaking speed is controlled at 180r/min, 25 ℃ of cultivation temperature, and constant temperature culture 4～5d, its culture fluid terminal have special " abalone " delicious food, and bacterium ball size is even, without the secondary shaking flask kind of living contaminants.
1.2 method
1.2.1 the composts or fertilisers of cultivating processing selecting, without going mouldy, without the fresh cotton seed hulls of insect pest, corncob, pomace (water content is below 5%), after tanning by the sun processing, then is pulverized corncob, crosses diameter 0.5～1cm sieve, standby.
1.2.2 making major ingredient, pomace, wheat bran, quicklime by cotton seed hulls, corncob, culture material formula forms 11 formulas (in Table 22) as auxiliary material
Table 22 different formulations composts or fertilisers of cultivating component content (%)
1.2.3 grog wall formula stereoscopic bag hydroponics is adopted in experiment in cultivation.By different formulations furnishing water content in table 1 60～65%, in the polypropylene plastics pocket of the 17 * 33cm that packs into, the every bag of heavy 250g that gives money as a gift, 126 ℃ of sterilizing 2h, cooling after, with inoculating gun injection type access liquid strain, inoculum concentration is 10% at desinfection chamber.Each formula connects 20 bags, and 25 ℃ of constant temperature culture, after the mycelia purseful, move into the mushroom room, carry out management of producing mushroom.The fruiting temperature is controlled at 15～20 ℃, and the air relative temperature keeps 85～95%.
1.2.4 biological efficiency and yield meter algorithm are by the mean value of 20 bags of three damp mushroom fresh mushroom productions.Biological efficiency (%)=fruit body fresh weight/composts or fertilisers of cultivating dry weight.
2 results and discussion
2.1 its mycelium germination time of mycelial growth situation different formulations, day long speed, purseful number of days, and the difference of pollution rate in Table 23.
Table 23 different formulations mycelial growth situation
As can be seen from Table 23, each the formula mycelium germination time except the formula 1 and 2 a little later, other indifference.All variant on material feeding time, day long speed, purseful number of days and pollution rate, from total situation, to fill a prescription 7,8,9 for well.
2.2 the biological efficiency of different composts or fertilisers of cultivating is deceived abalone mushroom PL-AD
28Growth biological efficiency different (in Table 24) in different composts or fertilisers of cultivating
Table 24 is deceived abalone mushroom PL-AD
28Biological efficiency in different composts or fertilisers of cultivating
By table 24, illustrated: 1. with cotton seed hulls, make major ingredient higher than the biological efficiency of making major ingredient with corncob, the black abalone mushroom of this explanation is the same with other edible mushrooms, and cotton seed hulls is its good cultivation matrix.2. the collocation of two kinds of cotton seed hulls and corncobs material is used highlyer than single kind of major ingredient biological efficiency, to have good air capacity of soils relevant with the nutrition complement of two kinds of major ingredients and corncob for this.3. within the specific limits, in the height of black abalone mushroom biological efficiency and composts or fertilisers of cultivating, there is certain positive correlation in the increase of cotton seed hulls.When the cotton seed hulls recruitment surpassed 63%, biologicak efficiency started to descend.This explanation, although cotton seed hulls is a kind of good cultivation matrix.But the material in sterilizing back pkt. easily hardens, therefore its air capacity of soils is poor, affected growth and the physiological ripening of mycelia.Therefore when with pomace, wheat bran etc., making auxiliary material, cotton seed hulls and corncob should be transferred to certain ratio, could improve its biological efficiency.This test recipe 7,8,9 biological efficiencies are higher.Because 8 and 9 formula variations are little, cotton seed hulls is higher than corncob price in addition, so this research apolegamy side 8 is optimum formula.
3 brief summaries
3.1 our Shandong cotton seed hulls and corncob wide material sources are cheap, this bi-material collocation made to the major ingredient economic benefits comparison high.
3.2 this research adds appropriate pomace in each formula, help the growth of black abalone mushroom mycelia and the raising of output.Analyzing its reason may contain the small-molecule substance simply such as ready-made carbohydrate and amino acid in pomace, and these materials can directly be absorbed, and are conducive to the g and D of mycelia.
3.3 in composts or fertilisers of cultivating, add appropriate pomace, both reduced cost, turn waste into wealth, fruit juice factory has solved difficulty entirely again, has also reduced environmental pollution simultaneously.
(2) research of liquid strain cultivation in raw material
According to document, black abalone mushroom liquid strain cultivation in raw material only has the lab scale report, up to now, does not also have one to use black abalone mushroom liquid spawn for raw material production fully.For this reason, we,, on the basis of black abalone mushroom liquid strain grog experiment in cultivation, have carried out again the cultivation in raw material exploration, have obtained good effect.Now be reported as follows:
1 materials and methods
1.1 material
1.1.1 strains tested PL-AD
28, provided by edible fungus research institute of Yantai Normal College.
1.1.2 culture fluid is ordinary maize powder sucrose culture fluid.
1.1.3 plant formulation cotton seed hulls 50%, corncob 30%, fruit juice slag 20%, additional 5% quicklime, water is appropriate.
1.2 method
5～6d 4～5d
4～5d 4d
25℃ 25℃
Quality control standard mycelium pellet diameter 0.5～1.5mm, bacteria containing amount (weight in wet base) 50% left and right, without living contaminants.
1.2.2 planting material process choose fresh, without damage by disease and insect, after tanning by the sun in a few days, soaked 15～20 minutes with 0.1% liquor potassic permanganate, pull rear adjusting material water content 60% left and right cotton seed hulls out.
1.2.3 the bed hydroponics is adopted in experiment in cultivation, the She Sange community, and each community bed surface area is 1m
2, the 15Kg that feeds intake, use liquid spawn 250ml, in the mode of sprinkle, it mixed with planting material.With solid state cultivation kind routinely, compare, the sowing quantity of solid kind is 3 bottles of Cans bacterial classifications, adopts the layering vaccination ways.After inoculation, all want compacting and cover film, take off film after mycelia is covered with the composts or fertilisers of cultivating surface, routine manages.The project that will observe during this period: observe the mycelial growth situation, 10d, 15d survey the growth rate of its mycelia; The bright mushroom amount of three damp mushrooms is received in the fruiting time Ji Ge community of a record damp mushroom.
3 results and analysis
3.1 the average daily long speed of mycelial growth in mycelial growth situation 15 days, liquid spawn increases fast 0.33cm than solid spawn mycelia, and liquid spawn is covered with Zao 7 days of composts or fertilisers of cultivating (in Table 25) than solid spawn mycelia
Mycelial growth situation in table 2515 day
3.2 a damp mushroom fruiting phase is because the liquid spawn mycelia is covered with composts or fertilisers of cultivating than solid spawn 7 days ahead of time, so also corresponding fruiting ahead of time 7～8 days has shortened the production cycle.
3.3 output is experiment in cultivation output such as the table 26 of two types of spawn relatively
Table 26 liquid strain raw material bed is planted test output (Kg)
As can be known from Table 26, the average 13.73Kg/m of liquid spawn output
2, the average 11.33Kg/m of solid spawn output
2, liquid strain can increase production 2.40Kg/m
2.
Discuss and brief summary
4.1 liquid spawn, because the mycelia fragment is many, germination point is many, is the liquid condition dispersity large, cultivating the neutral body growth, mycelia mostly, for the growth animated period, grows stalwartness, in case ripe cell age is consistent, in the same period intramatrical mycelium kink is many, therefore fruiting early, neat, change of tide is fast, is convenient to management, and output is high.
4.2 by this research, the black abalone mushroom of liquid spawn cultivation in raw material is feasible, not only can shorten the production cycle, if composts or fertilisers of cultivating is handled well, can also avoid living contaminants, cost is low, saving of work and time.
4.3 the composts or fertilisers of cultivating water content of liquid strain cultivation in raw material should be lower than the water content of solid kind cultivation in raw material, one is 50%～60% to be advisable.
(3) research of liquid strain Different sowing ways
At present, for black abalone mushroom class, adopt raw material or the packed solid kind of fermentation material layer to broadcast the cultivation of earthing formula more.This research liquid strain, adopt that the raw material layer is broadcast, drilling, three kinds of Different sowing ways of bunch planting carry out the ridge cultivation test relatively.Now result of the test is reported as follows:
1 materials and methods
1.1 material
1.1.1 strains tested PL-AD
28, provided by edible fungus research institute of Yantai Normal College.
1.1.2 main composts or fertilisers of cultivating murphy juice, corn flour, cotton seed hulls, corncob, wheat bran etc.
1.2 method
1.2.1 the production technology of liquid spawn is with above-mentioned (two)
1.2.2 experiment in cultivation
1.2.2.1 formula cotton seed hulls 50%, corncob 30%, fruit juice slag 20%, additional 5% quicklime, 40% carbendazim glue suspension 0.2%, material-water ratio 1:1, inventory 25Kg/m
2.
1.2.2.2 seeding quantity is 100ml/m 1.
22. 200ml/m
23. 300ml/m
2.Above-mentioned three kinds of bacterium liquid are all with 3 times of water dilutions.
1.2.2.3 seeding method (1) layer is broadcast, composts or fertilisers of cultivating divides 4 layers to enter furrow, and lower floor is uniform broadcasting 1/3 bacterium liquid respectively; (2) drilling, composts or fertilisers of cultivating are drawn ditch on top layer after all entering furrow, ditch depth 3～4cm, and ditch, apart from 6～7cm, evenly is sprinkling upon bacterium liquid in ditch; (3) after bunch planting, composts or fertilisers of cultivating all enter furrow, at Wa Xue， cave, top layer distance 6～7cm, the dark 3～4cm in cave, bacterium liquid quantitatively is sown in cave with little spoon.After above-mentioned 3 kinds of modes are sowed, all will be by surface evening, compacting, covering newspaper and plastic film.
1.2.2.4 output is the yield per unit area of a weighing statistics damp mushroom relatively.
Results and analysis
It the results are shown in Table 27 2.1 the black abalone mushroom of the cultivation of Different sowing ways liquid strain cultivation as a result adopts Different sowing ways
The cultivation result of table 27 Different sowing ways
From table 27, can find out, seeding method is with bunch planting the best, is mainly because after bacterium liquid enters cave, more concentrated, has community superiority, and radiation growth is fast, and a part of bacterium grain penetrates into certain depth along cave, enlarged a bacterium area.
2.2 abalone mushroom bunch planting way different seeding quantity is deceived in the cultivation of different seeding quantity liquid strain bedding cultivation as a result, its result has notable difference (in Table 28)
The bunch planting cultivation result of table 28 different vaccination amount
As can be seen from Table 28, strengthening inoculum concentration is to improve the effective ways of black abalone mushroom output.Seeding quantity 300ml/m
2, cultivating 27d composts or fertilisers of cultivating mycelia and all cover with, 37d adopts a damp mushroom in left and right, and only the biological efficiency of a damp mushroom can reach 76.8%.
3 discuss and brief summary
3.1 this test is to adopt the research on Different sowing ways and different seeding quantity method liquid spawn, its advantage be inoculation speed fast, can be quantitatively, location inoculation, effect better, a bacterium is neat.But the quality requirement to liquid strain is high, the processing requirements of composts or fertilisers of cultivating is strict.
3.2 during the black abalone mushroom of liquid strain cultivation, in this research, take bunch planting as good, inoculum concentration is 300ml/m
2.
(4) experimental study of liquid strain different planting.
1 materials and methods
1.1 material
1.1.1 strains tested PL-AD
28, provided by edible fungus research institute of Yantai Normal College.
1.1.2 cultivating, liquid spawn fermentation tank plants.
1.1.3 culturing raw material and formula cotton seed hulls 75Kg, corncob 45Kg, pomace 10Kg, additional quicklime 8Kg.
1.2 method
1.2.1 the heap fermentation of raw material weighs up composts or fertilisers of cultivating by the formula requirement, adds suitable quantity of water, drops in agitator and mixes thoroughly.During spice, main is to grasp water content, adheres to peaceful little not large principle, and the moisture while finally packing is agglomerating to hold wet feed, i.e. loose being advisable of abandoning, and this is the crucial water content of normally sending out bacterium.
The raw material of mixing thoroughly are piled wide 1.0～1.5 meters, high 0.5～1.0 meter, the stockpile that length is not limit (stockpile is changeable according to the height of temperature).After building up stockpile, on stockpile, get through pore with the waddy of diameter 5cm left and right, every span 40～50cm, then cover straw screen or mat.When expecting that interior temperature rises to more than 50 ℃, carry out turning for the first time, keep every 24h turning once later, so carry out 3～4 times and get final product.Fermentation produces a large amount of thermophilic aerobic microorganisms such as actinomycetes through windrow, these microorganisms can be improved the constituents such as its material inner cellulose, hemicellulose, lignin and crude protein, be conducive to directly absorbing of mycelia, induce simultaneously the sprouting of miscellaneous bacteria spore in stockpile, accomplished with the bacterium bacteria, with bacterium gram bacterium, induce sterilizing, the standard of successful base-material of fermenting is: evenly, material has special wine flavour for well to the inside and outside thermophilic aerobic microbiological actinomycetes growth of material.
1.2.2 the base-material pack, can pack and sowing with the sowing raw material after heap fermentation is processed.The sack specification is in the Polythene Bag of 17cm * 33cm * 0.02cm, the every bag of heavy 300g that gives money as a gift.By the inoculating gun gunite, sow at the bag two ends, seeding quantity is 10%.
1.2.3 send out bacterium, the bacterium bag of having inoculated is placed in culturing room, temperature is controlled at 25 ℃ ± 1.Approximately through 20～22d, can send out the bacterium bag full.
1.2.4 the bacterium bag of the screening of high-yield culturing mode will be sent out full bacterium is moved in canopy and is carried out fruiting experiment, adopts following three kinds of modes to cultivate: the vertical covering method of (1) de-bag section; (2) the horizontal covering method of de-bag; (3) de-bag upright covering method.Process for every kind and repeat 3 times, take out at random 145 bags at every turn and arrange in furrow.The long 6.5m of furrow, wide 0.9m, dark 0.15～0.28m, row spacing 0.3m, bag and bag interval 0.02m, fill with soil, and then the tank flood, cover 0.02～0.03 meter of humus soil after the water infiltration.Fruiting situation and the output of every kind of planting type of observed and recorded.
The measure fruiting phase of high yield management of producing mushroom is controlled temperature at 10～25 ℃ as far as possible, keeps temperature of shed near the sporophore growth optimum temperature, and humidity remains at 85～95%.The measure of taking in the meantime is: (1) every damp mushroom is filled with a flood after gathering and finishing, and the fruiting phase only gently sprays water to the furrow face, mainly to aerial, ground water spray; (2) every damp mushroom tank flood not after gathering and finishing, and in every day fruiting phase to the furrow face fog of respraying, keep its furrow face moistening.Observe the fruiting situation of above each processing, the quality of fruit body and biological efficiency etc.
Results and analysis
2.1 the screening of high-yield culturing mode the results are shown in Table 29 by the black three kinds of modes of abalone mushroom of liquid strain cultivation.
The impact of table 29 different planting on black abalone mushroom fruit body
From the result of table 29, can find out: biological efficiency is buried the highest to stand, and can reach 196.33%, and mushroom body hypertrophy, and sleeping buried poor.Analyze its reason and may be and adopt vertical mushroom springing method to make on fruiting bed unit are nutrition more concentrated, after in a single day the mycelium on surface formed the mushroom flower bud, the nutrients help of deep layer is continual to be carried to the fruit body place, thereby fruit body is also loose.In addition may be relevant with the direction of growth of mycelia.
2.2 the improvement Routine Management of control measures adopts repeatedly to spray water often every day, keep moistening this of relative air humidity and furrow face not only to increase labour intensity, also be difficult to guarantee the water content of thalline.We are under the prerequisite that guarantees relative air humidity, mainly grabbed the measure of carrying out the flood full irrigation of adopting after mushroom, at first guaranteed bacterium bag internal cause fruiting and dried out, mycelia can be reached maturity under normal environment, also fundamentally having met fruit body absorbing moisture simultaneously.This not only is convenient to management, the more important thing is and has improved output and quality.It the results are shown in Table 30
Table 30 is filled with the comparative trial of flood and spray water
By table 30, learnt: fill with flood after adopting mushroom, the needs of mycelial growth have not only been guaranteed, be conducive to physiological ripening, also met simultaneously the requirement of fruit body development phase to moisture, make fruit body plentiful big and fleshy, rotten mushroom or dead mushroom phenomenon, do not appear in glossy black light, biological transformation ratio can reach more than 196%, and this technology has also reduced labour intensity.
3 discuss
3.1, from our cultivation practices, adopt vertical earthing obviously to be better than sleeping burying with section is vertical and bury.Not only there were significant differences for biological efficiency, and take full advantage of space, and same site area can be produced more product, is best planting type.
3.2 carry out first, fill with the flood control measures after mushroom, improved the aspects such as Yield and quality, color and luster and mushroom shape, but suitable water spray keeps air humidity or necessity.
Five. the characteristic of this project and main innovation
Determined the optimum medium of black abalone mushroom liquid strain: on the basis of understanding black abalone mushroom solid culture medium, at first carbon source, nitrogenous source and the mineral salt of black abalone mushroom liquid strain are screened, method by orthogonal experiment filters out optimum medium, determined that black abalone mushroom liquid strain can produce by industrial three grade fermemtation, the culture fluid formula of comparatively ideal fermentation tank: corn flour 5～6%, brown sugar 1%, wheat bran 0.05%, peptone 0.05% (or dusty yeast 0.5%), KH
2PO
40.05%, MgSO
40.05%, VB
110mg/L, pH value 6.0～7.0.
Determined the optimum condition of culture of black abalone mushroom liquid strain: different condition of culture have obvious difference to black abalone mushroom liquid strain mycelial growth, this test is on the basis of determining optimal medium, condition of culture is carried out to comparative study, determined that finally best PH is 6.0～7.0, best liquid amount is 180～200ml, after inoculation, first static cultivation 48h is 170～180r/min in shaking speed again, and cultivation temperature is that under 25～26 ℃, 4～5d the best is cultivated in concussion.
Determined black abalone mushroom liquid strain storage temperature and the term of validity: the abalone mushroom liquid strain is kept at temperature and the term of validity is studied to deceiving, determined that black abalone mushroom liquid strain is deposited the 1d biological efficiency under room temperature (26～30 ℃) the highest, deposit 15d with the bright mushroom of interior output higher than the solid kind.In refrigerator, preserve and also be unlikely to affect fresh mushroom production in 3 months under 5 ℃.
Carried out first black abalone mushroom liquid strain fermentation pocket and planted research, and succeeded.Determined that with the inoculation of inoculating gun calibrated shot formula be the first-selected inoculation method of this research.
While having determined black abalone mushroom liquid strain raw material ridge cultivation, take bunch planting as good, inoculum concentration is 300ml/m
2.
Successfully carried out first the large-scale production of black abalone mushroom liquid strain: with the black abalone mushroom liquid strain success of 100L fermentation tank culture; modernization biotechnology deep layer culture process successfully is applied to black abalone mushroom liquid spawn to be produced; determined black abalone mushroom liquid strain producting rule, for the large-scale production of black abalone mushroom is laid a good foundation.
The accompanying drawing explanation
Fig. 1 5L fermentation tank mycelial growth curve
Fig. 2 100L fermentation tank mycelial growth curve
Fig. 3 5L fermentation tank PH change curve.
Claims (4)
1. a kind of method prepared black abalone mushroom liquid spawn, the optimum medium that it is characterized in that determining black abalone mushroom liquid strain is corn flour 5～6%, brown sugar 1%, wheat bran 0.05%, peptone 0.05%(or dusty yeast 0.5%), KH2PO40.05%, MgSO40.05%, VB110mg/L, pH value 6.0～7.0.
2. the method for preparing of a kind of black abalone mushroom liquid spawn as claimed in claim 1, it is characterized in that having determined the optimum condition of culture of black abalone mushroom liquid strain: best PH is 6.0～7.0, best liquid amount is 180～200ml, after inoculation, first static cultivation 48h is 170～180r/min in shaking speed again, and cultivation temperature is that under 25～26 ℃, 4～5d the best is cultivated in concussion.
3. the method for preparing of a kind of black abalone mushroom liquid spawn as claimed in claim 1, is characterized in that having determined that black abalone mushroom liquid strain is deposited the 1d biological efficiency under room temperature (26～30 ℃) the highest, deposit 15d with the bright mushroom of interior output higher than the solid kind,
In refrigerator, preserve and also be unlikely to affect fresh mushroom production in 3 months under 5 ℃.
4. the method for a black abalone mushroom cultivation, it is characterized in that selecting the best three kinds of formulas of the raw material that is applicable to black abalone mushroom cultivation: 1. cotton seed hulls 90%, wheat bran (rice bran) 10%, quicklime 2%(is additional), water content is adjusted to 60～65%, pH value 2. corncob 30% naturally, cotton seed hulls 50%, fruit juice slag 20%, quicklime 5%(is additional), water content is adjusted to 3. cotton seed hulls (or corncob) 58% of 60～65%, pH value 6.5～7.0, wood chip 40%, sucrose 1%, land plaster 1%, quicklime 1%(is additional), water content 60～65%, pH value nature.
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| CN104904498A (en) * | 2015-06-30 | 2015-09-16 | 兴安县铭晖食用菌种植有限公司 | Cultivation method of black fungi |
| CN107889687A (en) * | 2017-11-22 | 2018-04-10 | 湖南轻工研究院有限责任公司 | A kind of culture medium of edible fungus and preparation method thereof |
| CN107955802A (en) * | 2017-12-05 | 2018-04-24 | 福建农林大学 | A kind of method of thickness wall spore Pu Keniya bacteria solid fermentations production spore |
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