CN102550293B - 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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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 domestic fungus cultivating technical 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, the production of agaricus bisporus generally adopts solid spawn, but solid spawn has obvious defect, be mainly manifested in the aspects such as growth cycle is long, cell age is inconsistent, a bacterium speed is slow, cultivated species expense is large, production cost is high, obviously can not meet large-scale cultivation of agaricus bisporus demand.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, the object of this invention is to provide a kind of liquid fermentation culturing method of agaricus bisporus bacterial classification, to meet the user demand of high effect culture agaricus bisporus.
Technical scheme: in order to realize foregoing invention object, the technical solution used in the present invention is as follows:
A liquid fermentation culturing method for agaricus bisporus bacterial classification, comprises the following steps:
(1) get 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 obtains one-level shaking flask bacterial classification, be that 5～10% switching first order seeds are cultivated to secondary culture fluid by inoculum concentration, again cultured secondary shaking flask liquid spawn is transferred to three grades of liquid mediums and cultivates, same procedure is carried out the seed liquor of cultivating to obtain at different levels successively; Wherein, shaking flask condition of culture at different levels are identical, and 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 6L fermentation medium is housed by 5% inoculum concentration and carries out fermented and cultured, cultivate 5～6d 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 step (1), C/N ratio (g/g or mL/g) is preferably 3: 1 or 2.5: 1.5;
In 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 step (1), nitrogenous source is preferably peptone, dusty yeast or corn flour, most preferably is corn flour.
In step (1), when one-level is cultivated, the optimization formula of liquid nutrient medium is: brewer's wort 30mL, corn flour 10g, KH
2pO
41g, MgSO
40.5g, VB
110mg, water 1000mL, pH6.5.
In step (1), when secondary is cultivated, the optimization formula of liquid nutrient medium is: corn flour 10g, peptone 20g, KH
2pO
41g, MgSO
40.5g, VB
110mg, water 1000mL, pH6.5.
In 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: compared 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, inoculate easy, a test tube kind is cultivated and just can be amplified 200000 times by Pyatyi kind, liquid two stage growth speed is obviously better than solid traditional cultivation kind, speed has improved 33%, simultaneously, growth rate between liquid strain is at different levels does not have too big-difference, the average full bottle time is 27.5d, completely can practical application in plant produced, there is good practicality, can produce good economic benefit and social effect.
Accompanying drawing explanation
Fig. 1 is that carbon source is to mycelium pellet amount of growth result figure;
Fig. 2 is that carbon source is to mycelium pellet number affects result figure;
Fig. 3 is that carbon source affects result figure to mycelium pellet diameter;
Fig. 4 is that nitrogenous source is to mycelium pellet amount of growth result figure;
Fig. 5 is that different C/N ratios are to mycelium pellet amount of growth result figure;
What Fig. 6 was pH value on biomass affects result figure;
Fig. 7 is pH value change curve in cultivation process;
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 described further.
The materials and methods that following examples are used is:
Test material: bacterial classification is agaricus bisporus.
According to organic manure the People's Republic of China agricultural industry criteria, NY525-2002 measures various raw material phosphorus content and nitrogen content.
Mycelial biomass is measured: by well-grown, free of contamination shaking flask cultivation and fermentation liquid, 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 is that direct filtering drying is weighed.
Bacterium bulb diameter is measured: get 10 times of 1mL zymotic fluid dilute with waters, get at random 20 bacterium balls and be in line along fixing straight line in culture dish, measure total length with slide calliper rule, repeat 3 times, ask its mean value.
Peloton density is measured: get 10 times of the uniform zymotic fluid dilute with waters of 1mL, put black box paper counting in culture dish underlay.
PH pH-value determination pH: adopt acidometer to measure pH value.
Reducing sugar test: get fermented liquid supernatant liquid and measure reducing sugar by 3,5-dinitrosalicylic Acid Colorimetry.
Mycelial growth rate: after the inoculation of the identical inoculum concentration of solid state cultivation kind or liquid spawn, observe mycelia and sprout in seed bottle, growth downwards, until the time at the bottom of growing to bottle, i.e. the full bottle time.
Fermentation tank parametric measurement: 10L full-automatic gas lift-type stirred glass fermentation tank liquid amount is 6L, the optimal liquid culture medium prescription configuration liquid fermentation medium drawing with shake flask test, in fermentation tank through sterilizing 20min at 121 ℃ of empty slake fermentation tank self steam, treat that in tank, medium temperature drops to 25 ℃ of left and right, according to the activated good liquid spawn seed liquor of 5% inoculum concentration inoculation, pass into filtrated air and start to control cultivation and fermentation.Be interval timing sterile sampling by 12h, 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 liquid nutrient medium, obtain one-level shaking flask bacterial classification in 25 ℃ of constant-temperature shaking culture, again cultured one-level shaking flask liquid spawn is transferred in the second-class liquid isolate shaking flask of each processing, carries out successively cultivations at different levels, if without specified otherwise, shaking flask condition of culture 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 are also 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 sterilizing 30min.The PDA collective media formula of cultivating as agaricus bisporus inclined-plane: potato 200g, sucrose 20g, water 1000mL, pH nature, 121 ℃ of sterilizing 20min.
(1) in liquid medium within, nitrogenous source is 20g peptone, 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, result as shown in Figure 1, in institute's carbon determination source, agaricus bisporus utilizes effect better to brewer's wort, agaricus bisporus mycelium pellet biomass reaches 15.25g/100mL, is secondly wheat bran, corn starch, and utilizes effect the poorest to fructose, biomass is 3.96g/100mL, and availability is only 1/4 of brewer's wort.Obviously, no matter be from theoretical foundation or produce reality, brewer's wort wide material sources, cost is well below chemical reagent carbohydrate price, thus by 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, bacterium bulb diameter minimum be malt extract medium, bacterium bulb diameter average out to 1.7mm, in unit volume zymotic fluid, contain 465/mL of bacterium nodule number order, 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 comparatively homogeneous of bacterium bulb diameter, therefore, and compounded carbons brewer's wort best results aborning.
Compounded carbons nutrient component is abundant, more solid material is contained in the inside, thereby in the time that mycelia utilizes, producing many kinds of substance increases viscosity, must be within the scope of viscosity, viscosity increases the formation that is conducive to mycelia, be conducive to new mycelia fragment and grow into new mycelium pellet germination point, too high or too low viscosity is all unhelpful to the Growth and reproduction of bacterium ball.And low molecule carbon source, because it can directly be absorbed by mycelia, provides sufficient nutrition in mycelial growth process, make mycelia branch, extension, mycelial growth becomes greatly feature with chap, thereby occurs oligotrophy in the later stage, and causes the inner anoxic of mycelia, form larger mycelia fragment, so that affected the formation of bacterium ball, caused bacterium nodule number order sharply to reduce, mycelia fragment is many, mycelium pellet is less, and diameter disunity.
(2) in liquid medium within, carbon source is 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 is respectively 20g.
Inoculate respectively agaricus bisporus, at 25 ℃, shaking flask is cultivated, and measures agaricus bisporus liquid bacteria pompon biomass, as shown in Figure 4, organic nitrogen is compared with inorganic nitrogen for result, and agaricus bisporus is higher to the utilization ratio of most of organic nitrogens, biomass is all higher, and corn flour and peptone are all 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, the foam producing is minimum, foam increases can affect dissolved oxygen coefficient, when serious, cause and escape in a large number liquid, increase and pollute probability, so optimum nitrogen source is corn flour or peptone, because corn flour is in the advantage increasing on viscosity and cost, selection corn flour is nitrogenous source.
(3) in liquid medium within, carbon source is 30mL brewer's wort, nitrogenous source is 20g peptone, and add corn flour and cultivate, observe the impact of corn flour on agaricus bisporus liquid spawn mycelial growth, result is as shown in table 1, in the time not adding corn flour, agaricus bisporus mycelia biomass minimum, bacterium bulb diameter is large, quantity is few; Along with the increase of corn flour addition, hypha biomass also increases thereupon, when addition reaches 2% (mass ratio, lower same) time, bacterium ball biomass diminishes on the contrary, and possible Liquid Viscous is excessive, and liquid shake oscillation rate reduces, affect dissolved oxygen in liquid, slow at late growing stage owing to not obtaining sufficient oxygen when mycelia raised growth.And corn flour addition is 1%, 1.5% time, Liquid Viscous is comparatively suitable, oxygen supply abundance, and C/N ratio is all relatively applicable to the growth of agaricus bisporus mycelia, and mycelia adapts to strong, induces very soon mycelium germination growth, obtain the mycelium pellet of a large amount of diameter homogeneous, and biomass is also at the highest notch, therefore, 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 meet several growth factors simultaneously like this, but this is relatively most economical material benefit, and corn flour is thoroughly utilized therein, be making full use of resource.
Table 1 corn flour content affects 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 liquid medium within, carbon source is brewer's wort, and nitrogenous source is corn flour, carries out seven different mix proportion schemes and cultivates, and observes its impact on agaricus bisporus liquid culture.Compare respectively under different proportionings, on the impact of hypha biomass, thereby tentatively confirm optimum carbon nitrogen ratio, result as shown in Figure 5, the in the situation that of carbon source abundance, along with the increase of nitrogenous source, biomass also increases thereupon gradually, in the time that carbon source ratio is 3: 1 (brewer's wort 30mL, corn flour 10g), biomass reaches maximum; And when nitrogenous source abundance, when carbon source reduces gradually, biomass reduces along with the reduction of carbon source.The pH value research of culture fluid is found, when nitrogenous source is too much, after sterilizing, pH value can be higher simultaneously; When carbon source is enriched, pH value there will be pH value on the low side, too high or too low to be all unfavorable for the growth of Dual Mushroom mushroom mycelia.Therefore, carbon source, nitrogenous source are not The more the better, but will have a suitable ratio to be just best suited for agaricus bisporus liquid spawn mycelial growth, and preferred proportion is 3: 1 or 2.5: 2.5.
(5) in liquid medium within, carbon source is 30mL brewer's wort, and nitrogenous source is 10g corn flour, respectively magnesium sulfate and potassium dihydrogen phosphate has been done the cultivation of ten variable concentrations.As shown in Figure 9, the magnesium sulfate and the potassium dihydrogen phosphate that add different content have certain facilitation to mycelial growth to result, in the time that magnesium sulfate addition is less than 0.05%, along with the increase of its content, agaricus bisporus biomass is increase in direct ratio also, in the time that content is 0.05, reaches maximum; Then, biomass is along with the increase of magnesium sulfate content becomes downward trend.When biphosphate potassium content is before 0.10%, biomass increases along with the increase of content, and in the time that content is 0.10%, biomass reaches maximum, and the 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 agaricus bisporus liquid nutrient medium, should add magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.10%.
(6) in liquid medium within, carbon source is 30mL brewer's wort, nitrogenous source is 10g corn flour, magnesium sulfate is 0.5g, potassium dihydrogen phosphate is 1g, change initial pH value of medium to determine the most suitable original ph of its liquid culture, experimental result is by shown in Fig. 6 and 7, pH value presents the curve of bell-shaped on the impact of liquid spawn biomass, Initial pH difference causes cultivating the difference of agaricus bisporus mycelia biomass while end, and observe the upper lower change point of agaricus bisporus growth pH value, when pH value is less than 4 or when being greater than 8.5, agaricus bisporus mycelia can not grow, in pH value before 6.5, along with the rising of pH value, hypha biomass also increases thereupon, in the time that pH value reaches 6.5, hypha biomass reaches 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 on the impact of hypha biomass, because original ph provides the environment of a suitable growth to microorganism, biology enzyme is in the suitableeest state, and mycelia does not need just directly to start metabolism through the long-term procedure of adaptation.The liquid nutrient medium that original ph 6.5 is cultivated, in cultivation process, pH value has the trend necessarily diminishing gradually, and the starting stage changes not too obvious, and to cultivating after 3d, pH value starts obviously to decline; Cultivate latter stage, pH value approximately reduces by 1 left and right than original ph 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, affects mycelial growth; Make liquid nutrient medium more easily by other germ contamination simultaneously, affect whole fermentation process, so should suitably shorten the liquid culture time, reduce pollution rate, therefore select the original ph of pH value 6.5 as liquid nutrient medium.
So far, show that the preferred culture medium formula of 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 different cultivation temperature to cultivate agaricus bisporus liquid spawn, experimental result is as shown in table 3, as can be seen from the table, 25 ℃ time, except bacterium bulb diameter is not optimum, all the other standards are all in optimum level mycelium pellet biomass 9.26g/100mL, 421/mL of mycelium pellet density, can also find, simultaneously within 25 ℃ of upper and lower fluctuation ranges, on not directly significant impact of hypha biomass, therefore, select 25 ℃ of the suitableeest cultivation temperature of conduct.
The impact of table 2 cultivation temperature on agaricus bisporus mycelia biomass
(8) at preferred culture medium (the brewer's wort 30mL of agaricus bisporus liquid spawn, corn flour 10g, KH2PO43g MgSO41.5g, VB110mg, water 1000mL, pH6.5) inoculated and cultured agaricus bisporus in, by changing liquid amount and the shaking speed of shaking flask, investigate dissolved oxygen level to agaricus bisporus mycelium pellet biomass, mycelium pellet density, the isoparametric impact of mycelium pellet diameter, result is as shown in table 3, when shaking speed is 150r/min, hypha biomass is in maximum, and liquid amount is respectively at 90mL, 120mL, when 250mL, difference is not obvious especially, consider that thereby liquid amount too much can cause discharge easily to cause the pollution of culture fluid, therefore, best liquid amount is 90mL/250mL, shaking speed is 150r/min.
The impact on agaricus bisporus biomass of table 3 liquid amount and shaking speed
Test tube kind is transferred in liquid medium, need from culture fluid, to obtain in time abundant nutrition, the solid slant medium environment that could adapt to liquid rapidly but not previously grown, therefore, needing can provide sufficient nutrition in level liquid culture fluid, shorten the mycelial growth procedure of adaptation, obtain more standard compliant liquid spawn.In the time continuing to expand cultivation using level liquid kind as seed, need to consider the change of its growing environment, now different carbon sources is the most obvious on the impact 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 secondary liquid medium, in secondary liquid medium, cultivate with the different carbon sources of selection, its result is as shown in table 4, in level liquid kind, most suitable carbon source brewer's wort is very not remarkable to the effect of secondary kind, mutually though wheat bran and glucose are obvious on hypha biomass impact.In the time that above various different carbon sources are cultivated, the form of bacterium ball growth has feature separately, when wherein sucrose, mannitol, brewer's wort are as carbon source, mycelium pellet size is uneven, and mycelium pellet in wheat bran and glucose is in the same size, diameter is approximately 1mm left and right, is typical circular bacterium ball.Known through comparative analysis, the mycelium pellet of cultivating using wheat bran as carbon source is that biomass or mycelium pellet density, mycelium pellet diameter various aspects are all applicable to the requirement that liquid spawn produces, so select the carbon source of wheat bran as secondary liquid strain.Remaining nutrient component of secondary liquid culture and condition of culture all with each term harmonization of level liquid bacterial classification, 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, original ph 6.5, liquid amount 90mL/250mL, shaking speed 150r/min.
The impact of the different carbon sources of table 4 on 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 formula (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 suitableeest inoculum concentration, in certain liquid nutrient medium, inoculate 2.5 with different vaccination amount (v/v) respectively, 5, 7.5, 10, 12.5% level liquid bacterial classification, 25 ℃ of cultivation temperature, liquid amount 90mL/250mL, under the optimum conditions such as shaking speed 150r/min, cultivate, measure respectively mycelium pellet biomass, mycelium pellet density, mycelium pellet diameter, experimental result is as shown in table 5: in the time that inoculum concentration is 5%, be significantly improved when hypha biomass and inoculum concentration 2.5%, but exceed 5% later not too large difference of each inoculum concentration, biomass and bacterium bulb diameter, quantity is all in a metastable level, and excessive the giving in operation of inoculum concentration brought certain difficulty, also therefore band is served pollution hidden trouble.Therefore, consider the suitableeest inoculum concentration of difficulty selection 5% conduct in Financial cost and real work.
The impact of table 5 different vaccination amount on 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, commonly use and have six kinds of methods for the judgement of fermentation termination, 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 back, measures reducing sugar and total reducing sugar and microscopic examination method.The present invention judges fermentation termination to measure reducing sugar, because edible mushroom is macro fungi, belong to eukaryotic microorganisms, by measuring reducing sugar in liquid nutrient medium, draw logarithmic phase and stationary phase in agaricus bisporus liquid culture process, now, as fermentation termination, can obtain best culture effect.Test 24h from cultivating starts, adopt sample one time every 24h, measure the content of its reducing sugar and measure its biomass simultaneously, result of the test is: the comparative analysis by content of reducing sugar learns, agaricus bisporus one all at the 6d of fermentation, is now that bacterium nodule number amount or bacterium bulb diameter are all in the best period as bacterial classification to the terminal of Pyatyi liquid fungus seed, therefore, the 6d cultivating can be used as fermentation termination, and can reach best effect, and obtains the highest economic benefit.
In cultivation 2d, reducing sugar is to increase with the increase of incubation time, this may be because of starch contained therein class material in the corn flour in medium and wheat bran, the nutriment that the amylase that agaricus bisporus mycelia produces is degraded wherein, thus increase the content of reducing sugar in medium.This one-phase can be thought the lag phase of cultivating, and in the meantime, is the procedure of adaptation of bacterial classification in fresh medium, is used for adjusting to shake down; And somatic cells shows as own vol and increases, the division of minority thalline.The adjustment of the synthetic and secretion, effectively mineral matter and the trace element that show as the outer hydrolase of born of the same parents of this one-phase etc.In general, the environment before and after the length in this stage and the hereditary feature of bacterial classification, cell age and inoculation has substantial connection, for the shortening time, and the bacterial classification of the logarithmic phase of taking to transfer or the employing identical medium of trying one's best.
Cultivating after 6～7d, thalline is in the more stable stage, can be called stationary phase, due to the vigorous growth of thalline in early stage, culture environment has a very large change, the shortage of some nutriment, the organic acid producing in the accumulation of metabolite and cultivation process causes the variation of pH value, Fast Growth and the division of thalline are limited, mycelia starts Growth and Differentiation by top and becomes some characteristic organs of multiplication, thereby the continuation that has stopped cell being extended, even part mycelia death in mycelium pellet, make old cell death toll and the increased numbers of new cell approach poised state, biomass also reduces.It is now the deadline date of fermentation termination, otherwise will cause entering next stage---decline phase, this stage environment condition is more not suitable for growth and the division of thalline, the lethality of cell is higher, the activity of cell reduces greatly, this is because the exhausting and the release of some metabolites of nutriment, thereby has changed mycelial normal growth condition.In mycelium, vacuole increases, and starts self-dissolving, and mycelium pellet form changes, because self-dissolving causes the reducing sugar in this stage in a temporary transient stationary stage.
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 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, by sample analysis with record fermenter system Monitoring Data, agaricus bisporus liquid mycelia each growth indexes result of variations in airlift fermentor is: under fermentation tank condition, agaricus bisporus has kept good growth rate, mycelium pellet biomass increases very obvious.Earlier fermentation, mycelial density rises rapidly, and maintains a stationary value, and mycelium pellet diameter, along with the propelling of fermentation process, also has certain growth, but mycelium pellet diameter accretion is still within zone of reasonableness.When fermentation during to 5d mycelia ask biomass to reach peaked 90%, mycelium pellet biomass stablize gradually and is started and declines subsequently, from the consideration of biomass angle, has now reached hypha fermentation terminal.Analyze by content of reducing sugar, agaricus bisporus is a not vertiginous process of content of reducing sugar originally, may be because agaricus bisporus starts to grow slower, do not utilize rapidly a large amount of glucides in medium, pH value changes obvious, along with the extension of fermentation time, pH value presents downward trend gradually, pH affects the regulatory mechanism of a series of physiological reactions such as Apoptosis self-dissolving, by pH value and mycelium pellet biomass, the isoparametric relationship analysis of mycelium pellet density and diameter, can select to determine agaricus bisporus liquid fermentation tank fermentation termination according to the variation of pH value, the 6d of ferment tank is fermentation termination, and as in production application, often should select to ferment 5-6d as the fermentation termination in actual production, mycelium pellet activity is now high, not yet arrive the decline phase of growth of microorganism.
In this test, select different vaccination amount inoculation wheat pedigree seed culture medium, respectively by 1%, 2%, 3%, 4% different inoculum concentration inoculation, 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 upper mycelial growth rate and bacterial classification, the relatively impact of each inoculum concentration on mycelial growth, result is as shown in table 6, seed bottle is in different vaccination amount, mycelial growth is obviously subject to the wherein impact of moisture, under the identical condition of wheat medium, due to the difference of inoculum concentration, cause moisture difference in seed bottle, the water content of agaricus bisporus mycelia growth medium is in 60-64% left and right, in the certain situation of wheat composts or fertilisers of cultivating water content, different liquids bacterial classification inoculum concentration is all kept in seed bottle with the moisture of medium, therefore, its main influence factor is moisture.As can be seen from the above table, in the time that inoculum concentration is 4%, because the moisture in seed bottle is excessive, make mycelia can not grow to from top to bottom bottom, and in the time that inoculum concentration is 1%, moisture is less, and mycelium pellet is evenly distributed on the top of wheat composts or fertilisers of cultivating, sprouts together from difference, and with the growth downwards that advances side by side of speed faster, within the shortest time, arrive seed bottle bottom, and mycelia is pure white, is obviously radial downward growth.Compare with CK (being solid state cultivation kind), mycelial growth time obviously shortens, and mycelial growth is rapid, probably can carry fast more than 35%.Therefore, without in obviously distinguishing, select 1% (7.5mL/ bottle) inoculum concentration as optimum inoculation amount in 1% and 2% inoculum concentration.
The impact of table 6 inoculum concentration on 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 | / |
Liquid spawn is convenient for production, cell age is consistent, is convenient to inoculation, and its growth rate is to embody a large standard of its advantage, relatively solid slant tube bacterial classification and the growth time of liquid spawn in same medium, can find out the advantage of liquid spawn for the growth of solid original seed comparatively intuitively.Inoculum concentration by 1% is to wheat culture medium inoculated, prepare solid wheat original seed by conventional method simultaneously, observe respectively the full bottle time of its mycelia, result as shown in Figure 8, on identical wheat medium, liquid spawns at different levels and the bacterial classification comparison of traditional test tube slant, growth rate is significantly improved, on average, in 27d left and right, be less than the 36d of cultivated species, still less plant the required 42d time in test tube is female, and sprout time is short, after inoculation, mycelium pellet is evenly distributed on the top of wheat medium, and mycelia sprouts simultaneously, does not have the difference of each different parts.In addition, because mycelium pellet has mobility, the very little mycelium pellet of a part of diameter, along with liquid flows to the different parts of seed bottle, makes its each position that rests on seed bottle, after conforming, starts germination and growth.Therefore, mostly germination point is a major reason of its fast growth.And test tube slant inoculation when is transferred to wheat medium from agar medium, solid environment changes, and mycelia needs certain adaptation time.According to observations, generally, test tube kind and solid state cultivation kind need the time of about one week could obviously observe mycelium germination to go out white hypha diffusion downwards after inoculation, and liquid spawn can observe the phenomenon of downward growth after inoculation in 3～4d; Secondly because test tube slant bacterial classification and cultivated species are just 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 cause that upper and lower cell age is inconsistent, affects the total quality of bacterial classification.In production of hybrid seeds process, a test tube 6 bottles of original seeds of can transferring, every bottle of original seed 40 bottles of left and right 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 level liquid kind goes down to posterity to cultivate by 10% inoculum concentration and each amplifies 10 times, therefore secondary kind is equivalent to amplify 200 times, three grades of kinds are amplified 2000 times, level Four kind is amplified 20000 times, and Pyatyi kind is amplified 200000 times; Relatively secondary kind and cultivated species are substantially in close amplification level, and liquid two stage growth speed is obviously better than 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, the average full bottle time is 27.5d, except 5 grades of kinds, substantially within being all between upper and lower 5% wave zone, thereby find out that liquid spawn one is very stable to going down to posterity between level Four kind, and amplification coefficient reaches 20000 times, completely can practical application in plant produced.
Produce liquid spawn (embodiment 1 and 2) by simple process, cultivation of agaricus bisporus kind is produced in switching, in growth rate, the cultivated species of liquid spawn switching is obviously faster than the cultivated species expanding with wheat original seed, for the cultivated species of the further relatively two kinds of different modes quality on producing, select with traditional cultivating bisporous mushroom of cow dung Straw substrate the independent one deck 15.5m of liquid strain cultivation
2unified management, observe its " material feeding " growing state, standard take final fruiting output as both qualities of comparison, result of the test is: by the observation in whole cultivation process, the cultivated species mycelial concentration of agaricus bisporus liquid switching is larger than traditional wheat kind, mycelia is energetic, on planting material bedstead, material feeding is than very fast, and mycelia ductility is stronger, strong adhesion.Two kinds of cultivated speciess, in whole material feeding process, do not have too large lead time.But after fruiting, obviously observe the agaricus bisporus that two kinds of cultivated speciess produce and on individuality, have certain difference, on the planting material place bedstead of liquid spawn switching, unit are is buddingged many, body size is more even, one-level mushroom ratio is large, and on the bedstead of traditional cultivation kind place, the fruiting time is also inconsistent, wayward mushroom type size, at autumn mushroom production period, cultivated species prepared by liquid spawn per unit output in the cultivation of mushroom canopy is 5.98KG/m
2, and conventional solid bacterial classification to prepare cultivated species be 5.72KG/m in cultivated species per unit output
2volume variance between finding two kinds is also not obvious, substantially in peer-level, thus an explanation, use liquid spawn to prepare the extensive cultivation that cultivated species is used further to agaricus bisporus and not yet can directly use the liquid strain cultivation agaricus bisporus stage at present, this is feasible.
Above-mentioned cultivation of agaricus bisporus material cultural method is: after solid state cultivation material pack at 121 ℃ sterilizing 120min, inoculation while being cooled to 28 ℃, inoculation standard is that solid spawn covers strain bag top substantially, and liquid spawn is by composts or fertilisers of cultivating top on mycelium pellet uniform fold.After inoculation, cultivation bag is concentrated on to 25 ℃ of constant temperature culture in the culturing room through disinfecting, regularly stir cultivation.
Claims (1)
1. a liquid fermentation culturing method for agaricus bisporus bacterial classification, is characterized in that, comprises the following steps:
(1) get 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 obtains one-level shaking flask bacterial classification, be that 5～10% switching first order seeds are cultivated to secondary culture fluid by inoculum concentration, again cultured secondary shaking flask liquid spawn is transferred to three grades of liquid mediums and cultivates, same procedure is carried out the seed liquor of cultivating to obtain at different levels successively; Wherein, shaking flask condition of culture at different levels are identical: liquid amount is 90mL, 25 ℃ of temperature controls, and 150rpm shaken cultivation 5～6d, inoculum concentration is 5%; 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 20～30mL brewer's wort; Nitrogenous source is corn flour; C/N ratio is 3:1 or 2.5:1.5;
Wherein, when one-level is cultivated, liquid culture based formulas is: brewer's wort 30mL, corn flour 10g, KH
2pO
41g, MgSO
40.5g, VB
110mg, water 1000mL, pH6.5;
When secondary is cultivated, liquid culture based formulas is: corn flour 10g, peptone 20g, KH
2pO
41g, MgSO
40.5g, VB
110mg, water 1000mL, pH6.5;
(2) seed liquor of step (1) gained is seeded to the 10L fermentation tank that 6L fermentation medium is housed by 5% inoculum concentration and carries out fermented and cultured, cultivate 5～6d 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.
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