CN102550293A - Method for liquid fermentation cultivation of Agaricus bisporus strain - Google Patents
Method for liquid fermentation cultivation of Agaricus bisporus strain Download PDFInfo
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- CN102550293A CN102550293A CN201210023708XA CN201210023708A CN102550293A CN 102550293 A CN102550293 A CN 102550293A CN 201210023708X A CN201210023708X A CN 201210023708XA CN 201210023708 A CN201210023708 A CN 201210023708A CN 102550293 A CN102550293 A CN 102550293A
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- 241000222519 Agaricus bisporus Species 0.000 title abstract 2
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Abstract
The invention discloses a method for liquid fermentation cultivation of Agaricus bisporus strain, which includes: firstly, inoculating activated slant culture 0.5cm2 into a 250mL culture bottle containing 60-120mL of liquid medium, controlling temperature to be 21-27 DEG C, shaking at 90-180rpm, culturing for 5-7 days to obtain primary shaking strain, transferring primary seeds accounting for 5-10% of the inoculation amount to secondary culture liquid for cultivation, transferring cultured secondary shaking liquid strain to tertiary culture liquid for cultivation, and sequentially performing all levels of cultivation to obtain seed broth by the same methods; and secondly, inoculating the seed broth accounting for 5% of the inoculation amount into a 10L fermentation jar containing 6L of fermentation medium for cultivation, and culturing at 25 DEG C for 5-6 days. The method is characterized by short production cycle, uniform fungus age, low production cost and simplicity in inoculation, a test tube of strain can be multiplied by 200000 times by five levels of cultivation, the secondary liquid strain is evidently faster than traditional solid spawn in growth speed, and the speed is increased by 33%. In addition, the growth speeds of all levels of the liquid strain are nearly equal, the average fullness time is 27.5 days, and the method is absolutely applicable to practice of factory production.
Description
Technical field
The invention belongs to edible mushroom culture technology field, be specifically related to a kind of liquid fermentation culturing method of agaricus bisporus bacterial classification.
Background technology
Agaricus bisporus (Agaricus bisporus) is a kind of edible mushroom of extensive cultivation; In existing technology; Solid spawn is generally adopted in the production of agaricus bisporus; But solid spawn has obvious defects, shows that mainly growth cycle is long, cell age is inconsistent, sends out slow, the aspects such as the cultivated species expense is big, production cost height of bacterium speed, obviously can not satisfy large-scale cultivation of agaricus bisporus demand.
Summary of the invention
Goal of the invention: to the deficiency that exists in the prior art, the purpose of this invention is to provide a kind of liquid fermentation culturing method of agaricus bisporus bacterial classification, to satisfy efficient user demand of cultivating agaricus bisporus.
Technical scheme: in order to realize the foregoing invention purpose, the technical scheme that the present invention adopts is following:
A kind of liquid fermentation culturing method of agaricus bisporus bacterial classification may further comprise the steps:
(1) gets the 0.5cm of activation
2The female kind in inclined-plane is inoculated in the 250mL blake bottle that 60～120mL liquid nutrient medium is housed; 21～27 ℃ of temperature controls; 90～180rpm shaken cultivation, 5～7d gets one-level and shakes a bottle bacterial classification; By inoculum concentration is that 5～10% switching first order seeds are cultivated to the secondary culture fluid, more cultured secondary is shaken a bottle liquid spawn and is transferred to three grades of liquid mediums and cultivates, same procedure carry out successively at different levels cultivate seed liquor; Wherein, shake-flask culture conditions at different levels are identical, and the liquid culture based formulas is: carbon source, nitrogenous source 10～20g, KH
2PO
41～3g, MgSO
40.5～1.5g, VB
110mg, water 1000mL, pH5～7; Carbon source is wheat bran, corn flour, glucose, fructose, mannose or the sucrose of 20～30g, or 20～30mL brewer's wort; Nitrogenous source is peptone, dusty yeast, corn flour, ammonium sulfate or potassium nitrate;
(2) seed liquor of step (1) gained is seeded to the 10L fermentation tank that the 6L fermentation medium is housed by 5% inoculum concentration and carries out fermented and cultured, cultivate 5～6d, get final product for 25 ℃; Wherein, fermentative medium formula is: wheat bran 30g, corn flour 10g, peptone 1g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 1g, VB
110mg/L, water 1000mL, pH6.5.
In the step (1), C/N ratio (g/g or mL/g) is preferably 3: 1 or 2.5: 1.5;
In the step (1), carbon source is preferably wheat bran, corn flour or brewer's wort; One-level is cultivated and is most preferably used brewer's wort, and secondary is cultivated and most preferably used corn flour.
In the step (1), nitrogenous source is preferably peptone, dusty yeast or corn flour, most preferably is corn flour.
In the step (1), when one-level was cultivated, the optimization formula of liquid nutrient medium was: brewer's wort 30mL, corn flour 10g, KH
2PO
41g, MgSO
40.5g, VB
110mg, water 1000mL, pH6.5.
In the step (1), when secondary was cultivated, the optimization formula of liquid nutrient medium was: corn flour 10g, peptone 20g, KH
2PO
41g, MgSO
40.5g, VB
110mg, water 1000mL, pH6.5.
In the step (1), condition of culture is preferably: liquid amount is 90mL, 25 ℃ of temperature controls, and 150rpm shaken cultivation 5～6d, inoculum concentration is 5%.
Beneficial effect: compare with existing solid culture agaricus bisporus bacterial classification method, the clear superiority that the liquid fermentation culturing method of agaricus bisporus bacterial classification of the present invention has comprises: with short production cycle, cell age is consistent, production cost is low, inoculation is easy, a test tube kind is cultivated through the Pyatyi kind and just can be amplified 200000 times; Liquid second class inoculum growth rate obviously is superior to solid traditional cultivation kind; Speed has improved 33%, and simultaneously, the growth rate between liquid strain is at different levels does not have too big-difference; The average full bottle time is 27.5d; Fully can practical application in plant produced, have good practicability, can produce good economic benefits and social effect.
Description of drawings
Fig. 1 is a carbon source to mycelium pellet amount of growth figure as a result;
Fig. 2 is a carbon source to mycelium pellet number affects figure as a result;
Fig. 3 is a carbon source to mycelium pellet diameter influences figure as a result;
Fig. 4 is a nitrogenous source to mycelium pellet amount of growth figure as a result;
Fig. 5 is different C/N ratios to mycelium pellet amount of growth figure as a result;
Fig. 6 is influence the as a result figure of pH value to biomass;
Fig. 7 is a pH value change curve in the incubation;
Fig. 8 is inoculation liquid spawn and solid spawn growth rate comparison diagram;
Fig. 9 is the mineral salt cultivation results figure of variable concentrations.
Embodiment
Below in conjunction with specific embodiment the present invention is done further explanation.
Employed material of following examples and method are:
Test material: bacterial classification is an agaricus bisporus.
NY525-2002 measures various raw material phosphorus content and nitrogen content according to organic manure The People's Republic of China agricultural industry criteria.
Mycelial biomass is measured: with well-grown, free of contamination shake-flask culture zymotic fluid, through 4 layers of filtered through gauze, mycelium pellet biomass weight in wet base is weighed on electronic balance for getting solids after draining, and mycelium pellet biomass dry weight then is that the Direct Filtration oven dry is weighed.
The bacterium bulb diameter is measured: get 10 times of 1mL zymotic fluid dilute with waters, get 20 bacterium balls at random and in culture dish, be in line along fixing straight line, measure total length with slide calliper rule, repeat 3 times, ask its mean value.
Bacterium ball density measurement: get 10 times of the uniform zymotic fluid dilute with waters of 1mL, put black box paper counting in the culture dish underlay.
PH pH-value determination pH: adopt acidometer to measure the pH value.
Reducing sugar test: get fermented liquid supernatant liquid by 3,5-dinitrosalicylic acid colorimetric method for determining reducing sugar.
Mycelial growth rate: after the inoculation of the identical inoculum concentration of solid state cultivation kind or liquid spawn, observe mycelia and in seed bottle, sprout, growth downwards, the time at the bottom of growing to bottle, i.e. a full bottle time.
The fermentation tank parametric measurement: 10L full-automatic gas lift-type stirred glass fermentation tank liquid amount is 6L; The optimal liquid culture medium prescription that draws with shake flask test disposes liquid fermentation medium; 121 ℃ of 20min that sterilize down of process empty slake fermentation tank self steam in fermentation tank; Treat that a jar interior medium temperature drops to about 25 ℃,, feed filtrated air and begin to control cultivation and fermentation according to the activated good liquid spawn seed liquor of 5% inoculum concentration inoculation.Press 12h and be interval timing sterile sampling, its mycelium pellet biomass, mycelium pellet density, mycelium pellet diameter, oxyty and pH value are with electrode measurement.
Embodiment 1
Get the female about 0.5cm of kind in inclined-plane of activation
2Fritter be inoculated in the liquid nutrient medium, get one-level in 25 ℃ of constant-temperature shaking culture and shake a bottle bacterial classification, more cultured one-level is shaken bottle liquid spawn and is transferred to each second-class liquid isolate of handling and shakes in the bottle; Carry out cultivations at different levels successively, if no specified otherwise, shake-flask culture conditions at different levels are: 250mL triangular flask liquid amount 100mL; Inoculum concentration 5% (V/V), shaking speed 150r/min, 25 ℃ of shaken cultivation 5～7d; Equally, all the other liquid spawns at different levels also are identical training methods.The liquid culture based formulas of agaricus bisporus liquid spawn is: nitrogenous source, carbon source, KH
2PO
43g, MgSO
41.5g, VB
110mg, water 1000mL, pH nature, 121 ℃ of sterilization 30min.PDA collective media prescription as the agaricus bisporus slant culture: potato 200g, sucrose 20g, water 1000mL, the pH nature, 121 ℃ of sterilization 20min.
(1) in the liquid medium within; Nitrogenous source is the 20g peptone, and carbon source selects brewer's wort, wheat bran, corn starch, fructose, glucose, mannose and sucrose to cultivate respectively, wherein; Brewer's wort is 30mL, and wheat bran, corn starch, fructose, glucose, mannose and sucrose are 30g.Agaricus bisporus liquid spawn mycelium pellet biomass is analyzed, and the result is as shown in Figure 1, in institute carbon determination source; Agaricus bisporus utilizes effect better to brewer's wort; Agaricus bisporus mycelium pellet biomass reaches 15.25g/100mL, secondly is wheat bran, corn starch, and utilizes effect the poorest to fructose; Biomass is 3.96g/100mL, and availability is merely 1/4 of brewer's wort.Obviously, no matter be from theoretical foundation or produce reality that brewer's wort wide material sources, cost are well below chemical reagent carbohydrate price, thus with the Financial cost of compounded carbons brewer's wort well below carbohydrate.Different carbon source medium have material impact to mycelium pellet quantity, bacterium bulb diameter; As shown in Figures 2 and 3; What the bacterium bulb diameter was minimum is malt extract medium, and bacterium bulb diameter average out to 1.7mm contains 465/mL of bacterium nodule number order in the unit volume zymotic fluid; Next is wheat bran bacterium bulb diameter 2.1mm, 365/mL, corn starch bacterium bulb diameter 2.2mm, 345/mL.The medium of compounded carbons can be turned out a large amount of mycelium pellets, and bacterium bulb diameter homogeneous comparatively, therefore, and compounded carbons brewer's wort best results aborning.
The compounded carbons abundant nutrients; More solid content matter is contained in the inside; Making viscosity increase thereby when mycelia utilizes, produce multiple material, must be in the viscosity scope, and viscosity increases the formation that helps mycelia; Help new mycelia fragment and grow into new mycelium pellet and sprout point, too high or too low viscosity is all to the growth of bacterium ball with breed unhelpful.And low molecule carbon source because it can directly be absorbed by mycelia, provides prescribing adequate nutrition in the mycelial growth process, makes mycelia branch, extension; Mycelial growth becomes greatly characteristic with chap, thereby oligotrophy occurs in the later stage, and causes the inner anoxic of mycelia; Form bigger mycelia fragment, so that influenced the formation of bacterium ball, cause bacterium nodule number order sharply to reduce; The mycelia fragment is many, and mycelium pellet is less, and the diameter disunity.
(2) in the liquid medium within, carbon source is the 30mL brewer's wort, and nitrogenous source selects peptone, dusty yeast, corn flour, beef extract, urea, ammonium sulfate and potassium nitrate to cultivate respectively, and wherein, nitrogenous source respectively is 20g.
Inoculate agaricus bisporus respectively,, measure agaricus bisporus liquid mycelium pellet biomass at 25 ℃ of following shake-flask culture; The result is as shown in Figure 4, and organic nitrogen is compared with inorganic nitrogen, and agaricus bisporus is higher to the utilization ratio of most of organic nitrogens; Biomass is all higher, and corn flour and peptone all are good nitrogenous sources.Utilization ratio to part inorganic nitrogen urea etc. is the poorest, and biomass is zero.In above nitrogenous source, corn flour is compared with other nitrogenous sources, and the foam of generation is minimum; Foam increases can influence dissolved oxygen coefficient; Cause when serious and escape liquid in a large number, increase and pollute probability, so optimum nitrogen source is corn flour or peptone; Because corn flour is in the advantage that increases on viscosity and the cost, the selection corn flour is a nitrogenous source.
(3) in the liquid medium within, carbon source is the 30mL brewer's wort, and nitrogenous source is the 20g peptone; And add corn flour and cultivate; Observe the influence of corn flour to agaricus bisporus liquid spawn mycelial growth, the result is as shown in table 1, when not adding corn flour; Agaricus bisporus mycelia biomass is minimum, and the bacterium bulb diameter is big, quantity is few; Along with the increase of corn flour addition, hypha biomass also increases thereupon, when addition reaches 2% (mass ratio; In the time of down together); Bacterium ball biomass diminishes on the contrary, and possible liquid stickiness is excessive, and liquid shakes oscillation rate to be reduced; Influence dissolved oxygen property in the liquid, slow at late growing stage during the mycelia raised growth owing to do not obtain sufficient oxygen.And the corn flour addition is at 1%, 1.5% o'clock, and the liquid stickiness is comparatively suitable, and oxygen supply is sufficient; And C/N ratio all relatively is fit to the growth of agaricus bisporus mycelia, and mycelia adapts to strong, induces the mycelium germination growth very soon; Obtain the mycelium pellet of a large amount of diameter homogeneous; And biomass also is at the highest notch, and therefore, the corn flour addition is preferably 1%.Corn flour not only can be used as nitrogenous source, and also as tackifier, corn flour can also provide certain organic nutrition simultaneously; Corn flour can satisfy several growth factors simultaneously like this; But this is most economical relatively material benefit, and corn flour is making full use of resource therein by thorough utilization.
Table 1 corn flour content influences agaricus bisporus mycelia biomass
| Addition (%) | 0 | 0.5 | 1.0 | 1.5 | 2.0 |
| Biomass (g/100mL) | 3.12 | 9.95 | 15.12 | 13.96 | 9.16 |
| Bacterium bulb diameter (mm) | 2.9 | 1.6 | 1.2 | 1.1 | 1.9 |
| Bacterium nodule number amount (individual/mL) | 53 | 268 | 569 | 514 | 351 |
(4) in the liquid medium within, carbon source is a brewer's wort, and nitrogenous source is a corn flour, carries out seven different mix proportion schemes and cultivates, and observes its influence to the agaricus bisporus liquid culture.Compared respectively under the different proportionings,, thereby tentatively confirmed optimum carbon nitrogen ratio the influence of hypha biomass; The result is as shown in Figure 5, under the situation of carbon source abundance, along with the increase of nitrogenous source; Biomass also increases thereupon gradually; When the carbon source ratio is 3: 1 (brewer's wort 30mL, corn flour 10g), biomass reaches maximum; And when the nitrogenous source abundance, when carbon source reduced gradually, biomass reduced along with the reduction of carbon source.Simultaneously the pH value of culture fluid is discovered that when nitrogenous source was too much, sterilization back pH value can be higher; When carbon source was enriched, the pH value can occur on the low side, and too high or low excessively pH value all is unfavorable for the growth of Dual Mushroom mushroom mycelia.Therefore, carbon source, nitrogenous source are not The more the better, but will have a proper proportion to be only the most suitable agaricus bisporus liquid spawn mycelial growth, and preferred proportion is 3: 1 or 2.5: 2.5.
(5) in the liquid medium within, carbon source is the 30mL brewer's wort, and nitrogenous source is the 10g corn flour, respectively magnesium sulfate and potassium dihydrogen phosphate has been done the cultivation of ten variable concentrations.The result is as shown in Figure 9, and the magnesium sulfate and the potassium dihydrogen phosphate that add different content have certain facilitation to mycelial growth, when the magnesium sulfate addition less than 0.05% the time; Increase along with its content; The increase also in direct ratio of agaricus bisporus biomass when content is 0.05, reaches maximum; Then, biomass is along with the increase of magnesium sulfate content becomes downward trend.When the biphosphate potassium content before 0.10%, biomass increases along with the increase of content, when content was 0.10%, biomass reached maximum, mineral salt of this explanation low concentration have facilitation to agaricus bisporus, and high concentration is unfavorable for its growth on the contrary.So, in the agaricus bisporus liquid nutrient medium, should add magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.10%.
(6) in the liquid medium within, carbon source is the 30mL brewer's wort, and nitrogenous source is the 10g corn flour; Magnesium sulfate is 0.5g, and potassium dihydrogen phosphate is 1g, changes initial pH value of medium to confirm the only initial pH value of its liquid culture; Experimental result is by shown in Fig. 6 and 7, and the pH value presents the curve of bell jar type, the different differences that cause cultivating agaricus bisporus mycelia biomass when finishing of initial pH value to the influence of liquid spawn biomass; And observe the last lower change point of agaricus bisporus growth pH value, when the pH value less than 4 perhaps greater than 8.5 the time, the agaricus bisporus mycelia can not grow; Before 6.5, along with the rising of pH value, hypha biomass also increases thereupon in the pH value; When the pH value reached 6.5, hypha biomass reached maximum; Along with the progressively increase of pH value, hypha biomass also progressively reduces.Between pH6-7; PH value situation of change is particularly remarkable to the influence of hypha biomass; Because initial pH value provides the environment of a suitable growth to microorganism, biology enzyme is in the righttest state, and mycelia need not pass through the long-term procedure of adaptation and just directly begin metabolism.The liquid nutrient medium that initial pH value 6.5 is cultivated, in the incubation, the pH value has the trend that necessarily diminishes gradually, and the starting stage changes not too obvious, and after cultivating 3d, the beginning of pH value obviously descends; Cultivate latter stage, the pH value approximately reduces about 1 than initial pH value 6.5, the reduction of pH value, and culture fluid is acid to be strengthened, and can produce some feedback inhibition to metabolic process, reduces the activity of biological various enzymes, influences mycelial growth; Make liquid nutrient medium more easily by other germ contamination simultaneously, influence whole fermentation process,, reduce pollution rate, therefore select the initial pH value of pH value 6.5 as liquid nutrient medium so should suitably shorten the liquid culture time.
So far, the preferred culture medium prescription that draws the agaricus bisporus liquid spawn is: brewer's wort 30mL, corn flour 10g, KH
2PO
43g MgSO
41.5g, VB
110mg, water 1000mL, pH6.5.
(7) at preferred culture medium (brewer's wort 30mL, corn flour 10g, the KH of agaricus bisporus liquid spawn
2PO
43g MgSO
41.5g, VB
110mg, water 1000mL, pH6.5) in; Select the different culture temperature to cultivate the agaricus bisporus liquid spawn, experimental result is as shown in table 3, and data can be found out from table; In the time of 25 ℃, except the bacterium bulb diameter is not the optimum, all the other standards all are in optimum level mycelium pellet biomass 9.26g/100mL; 421/mL of mycelium pellet density can also find simultaneously, within fluctuation range about in the of 25 ℃; Hypha biomass is not had direct remarkable influence, therefore, select 25 ℃ of the righttest cultivation temperature of conduct.
Table 2 cultivation temperature is to the influence of agaricus bisporus mycelia biomass
(8) at preferred culture medium (brewer's wort 30mL, corn flour 10g, the KH2PO43g MgSO41.5g of agaricus bisporus liquid spawn; VB110mg, water 1000mL, pH6.5) middle inoculated and cultured agaricus bisporus; Through liquid amount and the shaking speed that bottle is shaken in change, investigate dissolved oxygen level to agaricus bisporus mycelium pellet biomass, mycelium pellet density, the isoparametric influence of mycelium pellet diameter, the result is as shown in table 3; When shaking speed was 150r/min, hypha biomass was in maximum, and liquid amount is respectively when 90mL, 120mL, 250mL; Difference is not obvious especially, thereby considers that liquid amount too much can cause discharge to cause the pollution of culture fluid easily, therefore; Best liquid amount is 90mL/250mL, and shaking speed is 150r/min.
Table 3 liquid amount and shaking speed are to the influence of agaricus bisporus biomass
Embodiment 2
The test tube kind is transferred in the liquid medium; Need in time from culture fluid, to obtain abundant nutrition; Therefore the solid slant medium environment that could adapt to liquid rapidly but not before grow, needs can provide in the level liquid culture fluid sufficient nutrition; Shorten the mycelial growth procedure of adaptation, obtain more standard compliant liquid spawn.When continuing enlarged culture as seed with the level liquid kind, need to consider the change of its growing environment, this moment, different carbon was the most obvious to the influence of its growth.By certain inoculum concentration the liquid strain that is cultured to exponential phase under embodiment 1 optimal culture condition is inoculated in the secondary liquid medium; In the secondary liquid medium, cultivate with the different carbon sources of selection; Its result is as shown in table 4; Only carbon source brewer's wort is very not remarkable to the effect of secondary kind in the level liquid kind, mutually though wheat bran and glucose are obvious to the hypha biomass influence.When above various different carbon sources were cultivated, the form of bacterium ball growth all had characteristics separately, when wherein sucrose, mannitol, brewer's wort are as carbon source; The mycelium pellet size is uneven; And wheat bran is big or small consistent with the mycelium pellet in the glucose, and diameter approximately is about 1mm, is typical circular bacterium ball.Can know that the mycelium pellet of cultivating as carbon source with wheat bran is that biomass or mycelium pellet density, mycelium pellet diameter various aspects all are fit to the requirement that liquid spawn produces, so select the carbon source of wheat bran as the secondary liquid strain through comparative analysis.Remaining nutrient component of secondary liquid culture and condition of culture all with each term harmonization of level liquid bacterial classification, promptly corn flour is a small amount of as nitrogenous source (addition is 1%), potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.10%, VB1.Condition of culture: 25 ℃ of cultivation temperature, initial pH value 6.5, liquid amount 90mL/250mL, shaking speed 150r/min.
The different carbon sources of table 4 are to the influence of agaricus bisporus second-class liquid isolate
| Carbon source | Biomass (g/100mL) | Mycelium pellet density (individual/mL) | Mycelium pellet diameter (mm) |
| Glucose | 11.23 | 395 | 1.00 |
| Brewer's wort | 3.43 | 294 | 0.83 |
| Mannitol | 7.11 | 256 | 0.72 |
| Wheat bran | 10.09 | 403 | 1.10 |
| Soluble starch | 7.95 | 361 | 0.73 |
| Sucrose | 5.48 | 375 | 0.68 |
| Lactose | 5.21 | 363 | 0.82 |
By above-mentioned optimal medium prescription (wheat bran 30g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 1g, VB
110mg/L; Water 1000mL; PH value 6.5) carry out the test of the righttest inoculum concentration, respectively in certain liquid nutrient medium with the level liquid bacterial classification of different vaccination amount (v/v) inoculation 2.5,5,7.5,10,12.5%, under optimum conditions such as 25 ℃ of cultivation temperature, liquid amount 90mL/250mL, shaking speed 150r/min, cultivate; Measure mycelium pellet biomass, mycelium pellet density, mycelium pellet diameter respectively; Experimental result is as shown in table 5: when inoculum concentration is 5% o'clock hypha biomass and inoculum concentration 2.5%, be significantly improved, but surpass 5% later not too big difference of each inoculum concentration, biomass and bacterium bulb diameter, quantity all are in a metastable level; And inoculum concentration is excessive to having brought certain degree of difficulty in the operation, also therefore brings some pollution hidden troubles.Therefore, the difficulty in consideration Financial cost and the real work selects 5% as the righttest inoculum concentration.
Table 5 different vaccination amount is to the influence of secondary liquid strain biomass
| Inoculum concentration (%) | 2.5 | 5 | 7.5 | 10 | 12.5 |
| Biomass (g/100mL) | 6.46 | 10.89 | 11.02 | 10.93 | 10.76 |
| Bacterium bulb diameter (mm) | 1.6 | 1.8 | 1.8 | 1.7 | 1.8 |
| Bacterium nodule number amount (individual/mL) | 393 | 431 | 427 | 412 | 422 |
At present, for the judgement of fermentation termination is commonly used six kinds of methods are arranged, they are bacterium nodule numbers of unit of account volume, and the mycelia dry weight of unit of account volume is observed bacterium ball proterties, and the test tube cultivation of sampling is back measured reducing sugar and total reducing sugar and microscopic examination method.The present invention judges fermentation termination to measure reducing sugar; Because edible mushroom is a macro fungi; Belong to eukaryotic microorganisms,, draw logarithmic phase and stationary phase in the agaricus bisporus liquid culture process through measuring reducing sugar in the liquid nutrient medium; Can obtain best culture effect as fermentation termination this moment.Test begins from cultivating back 24h, and every separated 24h adopts once appearance, measures the content of its reducing sugar and measures its biomass simultaneously; Result of the test is: the comparative analysis through content of reducing sugar learns that agaricus bisporus one arrives the terminal point of Pyatyi liquid fungus seed all at the 6d of fermentation, and be that bacterium nodule number amount or bacterium bulb diameter all are in the best period as bacterial classification this moment; Therefore; The 6d that cultivates promptly can be used as fermentation termination, and can reach best effect, and obtains the highest economic benefit.
In cultivating 2d; Reducing sugar is that the increase with incubation time increases; This possibly be because of starch contained therein class material in corn flour in the medium and the wheat bran, the amylase degraded nutriment wherein of agaricus bisporus mycelia generation, thus increased the content of reducing sugar in the medium.This stage can be thought the lag phase cultivated, in the meantime, is the procedure of adaptation of bacterial classification in fresh medium, is used for adjustment to shake down; And somatic cells shows as own vol and increases, the division of minority thalline.The adjustment of the synthetic and secretion that shows as the outer hydrolase of born of the same parents in this stage, effective mineral matter and trace element etc.In general, the environment before and after the length in this stage and the hereditary feature of bacterial classification, cell age and the inoculation has substantial connection, for the shortening time, and take the to transfer bacterial classification of logarithmic phase or the medium that employing coincide as far as possible.
After cultivating 6～7d, thalline is in the more stable stage, can be called stationary phase; Because the vigorous growth of thalline in earlier stage, very big variation, the shortage of some nutriment have taken place in culture environment; The organic acid that produces in the accumulation of metabolite and the incubation causes the variation of pH value, has limited the quick growth and the division of thalline, and mycelia begins Growth and Differentiation by the top and becomes some characteristic organs of multiplication; Thereby the continuation that has stopped cell being extended; Even the part mycelia is dead in the mycelium pellet, makes the increased numbers of old cell death toll and new cell near poised state, and biomass also reduces.Be the deadline date of fermentation termination at this moment; Otherwise will cause getting into next stage---decline phase; This stage environment condition more is not suitable for the growth and the division of thalline, and the lethality of cell is higher, and cell activity reduces greatly; This is because the exhausting and the release of some metabolites of nutriment, thereby has changed mycelial normal growth condition.Vacuole increases in the mycelium, the beginning self-dissolving, and the mycelium pellet form changes, because self-dissolving causes the reducing sugar in this stage to be in a temporary transient stationary stage.
Embodiment 3
Adopt the full-automatic gas lift stirred fermentor of 10L, liquid amount is 6L, by 5% inoculum concentration; Under 25 ℃ of condition of culture in the liquid culture optimum medium (wheat bran 30g; Corn flour 10g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 1g, VB110mg/L, water 1000mL pH value 6.5) carry out liquid fermentation and culture; Through sample analysis and record fermenter system Monitoring Data; Agaricus bisporus liquid mycelia each growth indexes result of variations in airlift fermentor is: under the fermentation tank condition, agaricus bisporus has kept growth rate preferably, and the mycelium pellet biomass increases very obvious.Earlier fermentation, mycelial density rises rapidly, and maintains a stationary value, and the mycelium pellet diameter also has certain growth along with the propelling of fermentation process, but the mycelium pellet diameter accretion is still within zone of reasonableness.When fermentation during to 5d mycelia ask biomass to reach peaked 90%, the mycelium pellet biomass is stable gradually and begin to descend subsequently, considers from the biomass angle, has reached the hypha fermentation terminal point this moment.Through the content of reducing sugar analysis, agaricus bisporus originally content of reducing sugar does not have a vertiginous process, possibly be because agaricus bisporus begins to grow slower; Do not utilize a large amount of glucides in the medium rapidly, the pH value changes then apparent in view, along with the extension of fermentation time; The pH value demonstrates downward trend gradually, and pH influences the regulatory mechanism of a series of physiological reactions such as Apoptosis self-dissolving, through pH value and mycelium pellet biomass, mycelium pellet density and the isoparametric relationship analysis of diameter; Can select to confirm agaricus bisporus liquid fermentation tank fermentation termination according to the variation of pH value; The 6d of ferment tank is a fermentation termination, and as in the production application, the 5-6d that often should select to ferment is as the fermentation termination in the actual production; The mycelium pellet of this moment is active high, still the decline phase of no show growth of microorganism.
Select different vaccination amount inoculation wheat pedigree seed culture medium in this test, inoculate by 1%, 2%, 3%, 4% different inoculum concentration respectively, relatively each different vaccination amount is at wheat medium (wheat 90%, cotton seed hulls 8.5%, gypsum 1%, lime 0.5%; Mass percent) pollution condition of mycelial growth rate and bacterial classification on, relatively each inoculum concentration is to the influence of mycelial growth, and the result is as shown in table 6; Seed bottle is in the different vaccination amount; Mycelial growth obviously receives the wherein influence of moisture, under the identical condition of wheat medium, because the difference of inoculum concentration; Cause moisture difference in the seed bottle; The water content of agaricus bisporus mycelia growth medium is about 60-64%, and under the certain situation of wheat composts or fertilisers of cultivating water content, different liquids bacterial classification inoculation amount all is kept in the seed bottle for the moisture with medium; Therefore, its main influence factor is moisture.Can find out by last table, when inoculum concentration is 4%, because the moisture in the seed bottle is excessive; Make mycelia can not grow to the bottom from top to bottom, and when inoculum concentration was 1%, moisture was less; Mycelium pellet is evenly distributed on the top of wheat composts or fertilisers of cultivating, sprouts together from difference, and with the fast speeds growth downwards that advances side by side; In the shortest time, arrive the seed bottle bottom, and mycelia is pure white, is obviously radial downward growth.Compare with CK (being the solid state cultivation kind), mycelial growth time obviously shortens, and mycelial growth is rapid, probably can carry fast more than 35%.Therefore, when 1% and 2% inoculum concentration does not have obvious difference, select 1% (7.5mL/ bottle) inoculum concentration to get final product as optimum inoculation amount.
Table 6 inoculum concentration is to the influence of mycelial growth
| Inoculum concentration % | 1 | 2 | 3 | 4 | CK |
| Full bottle time d | 26 | 26 | 41 | / | 40 |
| Directviewing description | Mycelial growth is fast | Bottom ponding | Bottom ponding is many | Moisture is excessive | / |
Embodiment 5
Liquid spawn is convenient for production; Cell age is consistent, is convenient to inoculation, and its growth rate is to embody a big standard of its advantage; Relatively solid slant tube bacterial classification and the growth time of liquid spawn on same medium can be found out the advantage of liquid spawn for the growth of solid original seed comparatively intuitively.Inoculum concentration by 1% prepares solid wheat original seed by conventional method simultaneously to the wheat culture medium inoculated, observes the full bottle time of its mycelia respectively; The result is as shown in Figure 8, and on identical wheat medium, liquid spawns at different levels and traditional test tube slant bacterial classification are relatively; Growth rate is significantly improved, and on average about 27d, is less than the 36d of cultivated species; Still less plant the required 42d time in test tube is female, and sprout time is short, mycelium pellet is evenly distributed on the top of wheat medium after inoculation; Mycelia sprouts simultaneously, does not have the difference of each different parts.In addition, because mycelium pellet has flowability, the very little mycelium pellet of a part of diameter makes it rest on each position of seed bottle along with liquid flows to the different parts of seed bottle, after conforming, and the beginning germination and growth.Therefore, sprout the major reason mostly point is its fast growth.And in test tube slant when inoculation, be transferred on the wheat medium from agar medium, and the solid environment changes, and mycelia needs certain adaptation time.According to observations, generally speaking, test tube kind and solid state cultivation kind could obviously be observed mycelium germination in the time about one week of needs after the inoculation and go out white hypha and diffusion downwards, and liquid spawn can observe the phenomenon of downward growth in 3～4d after inoculation; Secondly because test tube slant bacterial classification and cultivated species just are distributed in the top of wheat medium, the general direction of its growth is exactly a progradation from top to bottom, and growth time is long will to cause that cell age is inconsistent up and down, influences the total quality of bacterial classification.In production of hybrid seeds process, a test tube 6 bottles of original seeds of can transferring, the every bottle of original seed 40 bottles of left and right sides cultivated speciess of can transferring, therefore, cultivated species is to be exaggerated to expand to have grown 240 times; The a same test tube 20 bottles of liquid culture one-level kinds of can transferring; And the level liquid kind goes down to posterity to cultivate by 10% inoculum concentration and amplifies 10 times at every turn, so the secondary kind is equivalent to amplify 200 times, and 2000 times of three grades of kinds amplifications; The level Four kind is amplified 20000 times, and the Pyatyi kind is amplified 200000 times; Relatively secondary kind and cultivated species are in close amplification level basically, and liquid second class inoculum growth rate obviously is superior to solid traditional cultivation kind, and speed has improved 33%, and this is to change very significantly.Simultaneously; Growth rate between liquid strain is at different levels does not have too big-difference, and the average full bottle time is 27.5d, except that 5 grades of kinds; Basically all be in one up and down between 5% wave zone within; Thereby find out that liquid spawn one is very stable to going down to posterity between the level Four kind, and amplification coefficient reaches 20000 times, fully can practical application in plant produced.
Produce liquid spawn (embodiment 1 and 2) by simple process; The cultivation of agaricus bisporus kind is produced in switching; On growth rate, the cultivated species of liquid spawn switching is obviously faster than the cultivated species that enlarges with the wheat original seed, in order further to compare the quality of cultivated species on producing of two kinds of different modes; Selection is with traditional cultivating bisporous mushroom of cow dung straw composts or fertilisers of cultivating, the independent one deck 15.5m of liquid strain cultivation
2, its " material feeding " growing state is observed in unified management; With final fruiting output is both good and bad standards of comparison, and result of the test is: through the observation in the whole process of cultivation, the cultivated species mycelial concentration of agaricus bisporus liquid switching is bigger than traditional wheat kind; Mycelia is energetic; Material feeding is than very fast on the cultivate material bedstead, and mycelia ductility is stronger, strong adhesion.Two kinds of cultivated speciess do not have too big lead time in whole material feeding process.But obviously observe the agaricus bisporus that two kinds of cultivated speciess produce behind the fruiting certain difference is arranged on individuality, unit are is buddingged manyly on the cultivate material place bedstead of liquid spawn switching, and the body size is more even; One-level mushroom ratio is big; And on the bedstead of traditional cultivation kind place, the fruiting time is also inconsistent, wayward mushroom type size; At autumn mushroom production period, the cultivated species of liquid spawn preparation average yield per unit area in the cultivation of mushroom canopy is 5.98KG/m
2, and conventional solid strain preparation cultivated species is 5.72KG/m at the average yield per unit area of cultivated species
2Volume variance between finding two kinds is also not obvious, is in peer-level, an explanation thus basically; Use liquid spawn to prepare the extensive cultivation that cultivated species is used further to agaricus bisporus and still fail directly to use the liquid strain cultivation agaricus bisporus stage at present, this is fully feasible.
Above-mentioned cultivation of agaricus bisporus material cultural method is: solid state cultivation material pack back is at 121 ℃ of 120min that sterilize down; Inoculation when being cooled to 28 ℃; The inoculation standard is that solid spawn covers the strain bag top basically, and liquid spawn then is mycelium pellet evenly to be covered go up the composts or fertilisers of cultivating top.After the inoculation cultivation bag is concentrated on through 25 ℃ of constant temperature culture in the culturing room that disinfected, regularly stir cultivation and get final product.
Claims (10)
1. the liquid fermentation culturing method of an agaricus bisporus bacterial classification is characterized in that, may further comprise the steps:
(1) gets the 0.5cm of activation
2The female kind in inclined-plane is inoculated in the 250mL blake bottle that 60 ~ 120mL liquid nutrient medium is housed; 21 ~ 27 ℃ of temperature controls; 90 ~ 180rpm shaken cultivation, 5 ~ 7d gets one-level and shakes a bottle bacterial classification; By inoculum concentration is that 5 ~ 10% switching first order seeds are cultivated to the secondary culture fluid, more cultured secondary is shaken a bottle liquid spawn and is transferred to three grades of liquid mediums and cultivates, same procedure carry out successively at different levels cultivate seed liquor; Wherein, shake-flask culture conditions at different levels are identical, and the liquid culture based formulas is: carbon source, nitrogenous source 10 ~ 20g, KH
2PO
41 ~ 3g, MgSO
40.5 ~ 1.5g, VB
110mg, water 1000mL, pH5 ~ 7; Carbon source is wheat bran, corn flour, glucose, fructose, mannose or the sucrose of 20 ~ 30g, or 20 ~ 30mL brewer's wort; Nitrogenous source is peptone, dusty yeast, corn flour, ammonium sulfate or potassium nitrate;
(2) seed liquor of step (1) gained is seeded to the 10L fermentation tank that the 6L fermentation medium is housed by 5% inoculum concentration and carries out fermented and cultured, cultivate 5 ~ 6d, get final product for 25 ℃; Wherein, fermentative medium formula is: wheat bran 30g, corn flour 10g, peptone 1g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 1g, VB
110mg/L, water 1000mL, pH6.5.
2. the liquid fermentation culturing method of agaricus bisporus bacterial classification according to claim 1 is characterized in that: in the step (1), C/N ratio is 3:1 or 2.5:1.5.
3. the liquid fermentation culturing method of agaricus bisporus bacterial classification according to claim 1 is characterized in that: in the step (1), carbon source is wheat bran, corn flour or brewer's wort.
4. the liquid fermentation culturing method of agaricus bisporus bacterial classification according to claim 1 is characterized in that: in the step (1), carbon source is a brewer's wort.
5. the liquid fermentation culturing method of agaricus bisporus bacterial classification according to claim 1 is characterized in that: in the step (1), nitrogenous source is peptone, dusty yeast or corn flour.
6. the liquid fermentation culturing method of agaricus bisporus bacterial classification according to claim 1 is characterized in that: in the step (1), nitrogenous source is a corn flour.
7. the liquid fermentation culturing method of agaricus bisporus bacterial classification according to claim 1 is characterized in that: in the step (1), carbon source was a brewer's wort when one-level was cultivated, and carbon source was a corn flour when secondary was cultivated.
8. the liquid fermentation culturing method of agaricus bisporus bacterial classification according to claim 1 is characterized in that: in the step (1), when one-level was cultivated, the liquid culture based formulas was: brewer's wort 30mL, corn flour 10g, KH
2PO
41g, MgSO
40.5g, VB
110mg, water 1000mL, pH6.5.
9. the liquid fermentation culturing method of agaricus bisporus bacterial classification according to claim 1 is characterized in that: in the step (1), when secondary was cultivated, the liquid culture based formulas was: corn flour 10g, peptone 20g, KH
2PO
41g, MgSO
40.5g, VB
110mg, water 1000mL, pH6.5.
10. the liquid fermentation culturing method of agaricus bisporus bacterial classification according to claim 1 is characterized in that: in the step (1), condition of culture: liquid amount is 90mL, 25 ℃ of temperature controls, and 150rpm shaken cultivation 5 ~ 6d, inoculum concentration is 5%.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0549346A (en) * | 1991-08-23 | 1993-03-02 | Akita:Kk | Culture of mushroom |
| CN1724634A (en) * | 2004-07-19 | 2006-01-25 | 中国食品发酵工业研究院 | 10 ton liquid submerged femrentation culturing method of Gleoesterum incarnatum |
| CN1969604A (en) * | 2005-11-24 | 2007-05-30 | 徐宝明 | Method for producing double-head mushroom and raw material thereof |
| CN101157578A (en) * | 2007-09-25 | 2008-04-09 | 黄建春 | White mushroom compost storage and deacidizing utilization method after fermentation |
| CN101617611A (en) * | 2009-07-13 | 2010-01-06 | 王春宝 | Energy-saving and environment-friendly industrialized production equipment and facility of button mushrooms |
| CN101627698A (en) * | 2009-07-13 | 2010-01-20 | 王春宝 | III area system three-fermentation button mushroom production process |
| CN101637101A (en) * | 2009-08-19 | 2010-02-03 | 蛟河市黑土白云食用菌有限公司 | Industrial cultivation method of selenium-enriched agaricus bisporus |
| CN102047812A (en) * | 2009-10-30 | 2011-05-11 | 李守民 | High-yield cultivation technique of white mushrooms |
-
2012
- 2012-02-03 CN CN201210023708.XA patent/CN102550293B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0549346A (en) * | 1991-08-23 | 1993-03-02 | Akita:Kk | Culture of mushroom |
| CN1724634A (en) * | 2004-07-19 | 2006-01-25 | 中国食品发酵工业研究院 | 10 ton liquid submerged femrentation culturing method of Gleoesterum incarnatum |
| CN1969604A (en) * | 2005-11-24 | 2007-05-30 | 徐宝明 | Method for producing double-head mushroom and raw material thereof |
| CN101157578A (en) * | 2007-09-25 | 2008-04-09 | 黄建春 | White mushroom compost storage and deacidizing utilization method after fermentation |
| CN101617611A (en) * | 2009-07-13 | 2010-01-06 | 王春宝 | Energy-saving and environment-friendly industrialized production equipment and facility of button mushrooms |
| CN101627698A (en) * | 2009-07-13 | 2010-01-20 | 王春宝 | III area system three-fermentation button mushroom production process |
| CN101637101A (en) * | 2009-08-19 | 2010-02-03 | 蛟河市黑土白云食用菌有限公司 | Industrial cultivation method of selenium-enriched agaricus bisporus |
| CN102047812A (en) * | 2009-10-30 | 2011-05-11 | 李守民 | High-yield cultivation technique of white mushrooms |
Cited By (10)
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|---|---|---|---|---|
| CN104710206A (en) * | 2013-07-12 | 2015-06-17 | 鲁东大学 | Preparation method for volvariella volvacea liquid strain |
| CN103875446A (en) * | 2013-10-24 | 2014-06-25 | 江西省农业科学院农业应用微生物研究所 | Method of producing Agrocybe aegerita strains by liquid culture and preservation method of Agrocybe aegerita strain |
| CN103782801A (en) * | 2013-12-26 | 2014-05-14 | 朝阳鑫源农副产品开发有限公司 | Agaricus Bisporus liquid spawn and preparation method thereof |
| CN105340580A (en) * | 2015-12-01 | 2016-02-24 | 仇颖超 | Method for cultivating agaricus bisporus through liquid strains |
| CN106146179A (en) * | 2016-08-12 | 2016-11-23 | 河南农业大学 | A kind of method utilizing Agaricus bisporus windrow water extract to cultivate Agaricus bisporus strain |
| CN106146179B (en) * | 2016-08-12 | 2019-11-15 | 河南农业大学 | A method of utilizing agaricus bisporus windrow water extract culture agaricus bisporus strain |
| CN106148201A (en) * | 2016-08-22 | 2016-11-23 | 山东省科创食用菌产业技术研究院 | A kind of preparation method of Agaricus Bisporus liquid spawn |
| CN106947800A (en) * | 2017-01-13 | 2017-07-14 | 山东瑞蕈天库菌种开发有限公司 | The special cultivation base of White mushroom identification |
| CN107950288A (en) * | 2017-11-20 | 2018-04-24 | 山东省农业科学院农业资源与环境研究所 | A kind of planting technique of straw mushroom |
| CN107950288B (en) * | 2017-11-20 | 2020-08-21 | 山东省农业科学院农业资源与环境研究所 | Straw mushroom cultivation process |
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