CN103404363A - Coprinus comatus liquid culture preparing and high-quality high-yield innovating method - Google Patents
Coprinus comatus liquid culture preparing and high-quality high-yield innovating method Download PDFInfo
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Abstract
The invention discloses a coprinus comatus liquid culture preparing and high-quality high-yield innovating method, and belongs to the field of edible mushroom cultivation. The coprinus comatus is a main production variety of China, but the overall level of the present production technology of China falls behind. Therefore, the technological improvement is carried out. In the production of the coprinus comatus, liquid cultures are directly connected to cultivation bags to cultivate the coprinus comatus to obtain the coprinus comatus, and the traditional method that solid cultures are used for production is changed. In addition, the technological means such as twice earthing and great watering are implemented according to the characteristics of the coprinus comatus, and therefore the yield and the quality are greatly improved. The new liquid large-scale production technology is finally researched successfully after repeated tests. According to the new liquid large-scale production technology, the culture preparing and cultivation time of edible mushroom cultures can be greatly shortened, the culture quality can be improved, the culture production cost can be reduced, the sporocarp quality, the product quality, the product value and the like can be improved, the investment is little, and the new liquid large-scale production technology is easy to master and suitable for the situation of China, and has the high promotional value.
Description
Technical field
The innovative approach of a kind of coprinus comatus liquid spawn preparation and good quality and high output.
Background technology
In China, be Edible Fungi big country, produce 6500000 tons, bright mushroom per year, account for the 65-70% of Gross World Product, mushroom industry has become the main source that China's Agricultural Export is earned foreign exchange.Coprinus comatus is as one of mainly production kind of China (at present output in domestic maximum be respectively two, fragrant, flat, chicken), 600,000 tons of annual productions.It not only for the sustainable development in rural area, transform agricultural production that it is positive to play a part; and take full advantage of the refuses such as a large amount of agricultural crop straws and offcuts; with these raw materials, produce coprinus comatus and make the mushroom industry not only obtain good economic benefit, and for protection of the environment, immeasurable effect has developed eco-agriculture.But the current production technology aggregate level of China is also very backward, most low-level, low-producing states of family workshop type on a small scale that also are in.Along with integrating with of China's accession to the WTO and international economy, thisly yield poorly, production model of poor quality, small scale can not adapt to fierce market competition far away, can't be with international same industry competition (aboundresources and labour cheap advantage more and more weak).In the face of opportunities and challenges and present situation backward in technique that entry to WTO brings to the edible mushroom industry, we must have breakthrough technically and innovate.We take as the leading factor with " development bacterium industry, science and technology is dedicated one's life to the cause of the country ", at first, with liquid spawn, directly are connected on bacteria fruiting on cultivation bag in the production of coprinus comatus, have changed traditional method with solid spawn production.Solved like this that the production cycle is long, opportunities for contamination is many, turned for often, the bacterial classification problems such as variation of easily degenerating.In addition, we have implemented secondary soil-covering and have watered the technological means such as flood according to the characteristic of coprinus comatus self, make Yield and quality greatly improve.We are through in a few years research, liquid large-scale production new technology has been studied successfully in experiment finally repeatedly; this technology not only can shorten the production of hybrid seeds and the planting time of edible fungus species greatly; improve strain quality; reduce the bacterial classification production cost; improve Fruitbody, product quality and commodity value etc., and small investment, technology is easily grasped; be applicable to China's national situation, have very high promotional value.
Summary of the invention
(1) research of coprinus comatus liquid spawn culture medium screening
1, material
1.1 bacterial strain: coprinus comatus Cc100, edible fungus research institute of Yantai Normal College provides
1.2 medium
1.2.1 slant medium: PDA
1.2.2 liquid nutrient medium: potato (peeling) 20%, glucose 1%, peptone 0.25%, MgSO
40.1%, KH
2PO
40.2%, VB
110mg/L.
1.3 culture device: rotary shaking table, electronic analytical balance, accurate PH meter
2, method:
2.1 process route: coprinus comatus preservation of bacteria strain → inclined-plane enlarges cultivation → liquid spawn preparation → liquid culture
2.2 the preparation of liquid spawn: it is 70ml that slant strains is inoculated in to the 250ml(liquid amount) in conical flask, put into 25 ℃ of insulating boxs standing 24 hours, then at 25 ℃, the rotary shaking table shaken cultivation of 180r/min 4d, make liquid spawn standby.
2.3 shaking flask culture technology: the tested medium of 70ml of packing in the 250ml conical flask, the liquid spawn prepared is inoculated according to 10% inoculum concentration, establish 8 repetitions for every, at 25 ℃, the rotary shaking table shaken cultivation of 180r/min 4d, the pH nature, observed and recorded bacterium ball growing state, measure mycelia dry weight, Peloton density and bacterium bulb diameter, and mycelium dry weight is carried out to variance analysis.
2.4 mycelium dry weight is measured: after fermentation stops, the culture fluid in shaking flask is filtered with dry absorbent cotton, then, after with distilled water, repeatedly rinsing, be put in 60 ℃ of drying boxes and dry to constant weight, measure dry weight.
2.5 the measurement of Peloton density: bacterium liquid is placed in to the standing 5min of 100ml graduated cylinder, surveys the bacterium ball and account for the long-pending percentage of bacteria liquid.
2.6 the measurement of bacterium bulb diameter:, get at random 10 bacterium balls and arrange in a line, measure its total length again divided by 10, obtain the average diameter of bacterium ball.
3, results and analysis
3.1 the screening of suitable carbon source
Use respectively 2% glucose, brown sugar, maltose, lactose, fructose, sucrose as carbon source in liquid nutrient medium, observe the mycelial growth state, various carbon sources on the impact of liquid culture shaggy mane mycelium bulk-growth in Table 1:
The impact of the different carbon sources of table 1 on the shaggy mane mycelium bulk-growth
Variance analysis shows, the mycelium mean difference between each group has significant (p<0.05).Trial various carbon source is comparatively obvious on the difference of shaggy mane mycelium bulk-growth impact, under this experimental condition, glucose, brown sugar and sucrose during as carbon source mycelium production the highest, maltose during as carbon source output minimum.By the observation to the Mycelium culture shape, find, glucose and brown sugar bacterium nodule number amount are more, and the burr shape branch on dextrose plus saccharose bacterium ball surface is more, and vigor is better, comprehensive above various factors, select glucose, brown sugar and sucrose comparatively desirable as the nitrogenous source of coprinus comatus liquid nutrient medium.
3.2 the screening of suitable nitrogenous source
Respectively by 0.2% peptone, yeast extract, beef extract, NH
4Cl, (NH
4)
2SO
4, (NH
4)
2CO
3As the nitrogenous source in liquid nutrient medium, various nitrogenous sources on the heavy impact of liquid culture shaggy mane mycelium soma in Table 2:
The impact of table 2 different nitrogen sources on the shaggy mane mycelium bulk-growth
Variance analysis shows that the difference of respectively organizing mycelium dry weight has utmost point significant (p<0.01), illustrates that there is obvious difference in different nitrogen sources to the impact of the growth of shaggy mane mycelium body.Mycelium is better than inorganic nitrogen to the utilization of organic nitrogen, with bibliographical information is consistent mostly.And in three kinds of organic nitrogens, best with the yeast extract effect, be followed successively by peptone, beef extract.
3.3 the screening of suitable mineral salt
By 5 kinds of concentration, be 0.1% mineral salt---KH
2PO
4, MgSO
4, CaSO
4, ZnSO
4, FeSO
4Add respectively in liquid nutrient medium, observe the impact that various ion pair liquid culture shaggy mane mycelium somas are heavy, in Table 3:
The impact of table 3 mineral salt on the shaggy mane mycelium bulk-growth
Analysis of variance, the difference between each group have significant (p<0.05), and the mineral salt that are applicable to the shaggy mane mycelium bulk-growth are followed successively by: MgSO
4>KH2PO
4>CaSO
4>ZnSO
4>FeSO
4.Peloton density has also reflected same trend.
3.4 orthogonal experiment screening optimum medium
Consider the interaction of Carbon and nitrogen sources, select respectively the Carbon and nitrogen sources of three kinds of applicable liquid culture shaggy mane mycelium bulk-growths, design four factors, the orthogonal experiment of three levels, 9 combinations, 5 repetitions, filter out applicable single carbon source and single nitrogenous source and content separately, result is as follows:
Table 4 L
9(3
4) Orthogonal Experiment and Design
Table 5 L
9(3
4) intuitive analysis of orthogonal table coprinus comatus strain cultivation
Table 6 L
9(3
4) variance analysis of orthogonal table coprinus comatus strain cultivation
| The difference source | SS | df | MS | F | F crit |
| Between group | 67820 | 8 | 8477.50 | 6.02 | 3.05 |
| In group | 50700 | 36 | 1408.33 | ||
| Amount to | 118520 | 44 |
Variance analysis shows, each difference of organizing average mycelium dry weight has utmost point significant (p<0.01).As can be known by the intuitive analysis of table 5 data: best of breed is A
1B
3C
3D
3.A factor R value is maximum, and B factor and D factor R value are less, and four big or small primary and secondarys of experimental factor impact sequentially are followed successively by A>C>D>B.Explanation is under this condition of culture, and the variation of carbon source has the greatest impact to result, and optimal carbon source is glucose, and best sugared concentration is 3.5%.Three kinds of difference less that nitrogenous source affects mycelium production, yeast extract is the most applicable nitrogenous source, optium concentration is 1.0%.In the selected concentration range of test, the variable effect of the concentration of Carbon and nitrogen sources is less, especially b
2And b
3, c
2And c
3The difference of two levels is minimum, considers from economic angle, can select 3% carbon source and 0.5% nitrogenous source.Finally filter out the most applicable A that is combined as
1B
2C
3D
2, i.e. 3% glucose and 0.5% yeast extract.
5, the agricultural byproducts hydrolyzate is made the research of liquid nutrient medium
Much results of study show, the agricultural byproducts such as corn flour, wheat bran, bean powder are nutritious, as Carbon and nitrogen sources, prepare the liquid nutrient medium cultured mycelia with its hydrolyzate, satisfactory for result.For this reason, after filtering out coprinus comatus liquid optimum formula, consider that the price of sucrose, peptone is higher, in order to reduce costs, we have done again the formula screening that replaces sucrose that price is higher and peptone, yeast extract etc. with the lower agricultural and sideline product of price.Having designed 3 kinds, to take agricultural byproducts be main liquid culture based formulas, formula A: corn flour 5%, bean powder 2%, glucose 0.5%, KH
2PO
40.1%, MgSO
40.1 %, VB
110mg/L; Formula B: corn flour 4%, wheat bran 3%, sucrose 0.5%, KH
2PO
40.1%, MgSO
40.05%; Formula C: potato 15%, wheat bran 5%, beancake powder 2%, brown sugar 0.5%, MgSO
40.1%, KH
2PO
40.2%, VB
110mg/L.With the optimal medium filtered out above (potato 20%, glucose 3%, yeast extract 0.5%, KH as a control group
2PO
40.2%, MgSO
40.1%, CaSO
40.1%, VB
110mg/L), establish 8 repetitions for every group, shaken cultivation 4 days, observe shaggy mane mycelium bulk-growth situation, the results are shown in Table 7:
The impact of the liquid nutrient medium that table 7 agricultural byproducts hydrolyzate is made on the shaggy mane mycelium bulk-growth
* with control group, compare p<0.05
With agricultural byproducts hydrolyzate formula with agent prescription, compare, bacterium ball size, bacterium ball vigor are all without significant difference, and the mycelium dry weight mean value of formula A and formula C is all higher than control group, and the t check shows, with control group, compare formula C average mycelium dry weight there was a significant difference meaning.Therefore select formula C to do and prepare liquid nutrient medium, not only cost is low, and effective.
(2) coprinus comatus liquid spawn shaking flask is cultivated the research of optimal culture conditions
1, material
1.1 bacterial strain: coprinus comatus Cc100, edible fungus research institute of Yantai Normal College provides
1.2 medium
1.2.1 slant medium: PDA
1.2.2 liquid nutrient medium: potato 20%, wheat bran 3%, beancake powder 2%, brown sugar 0.5%, MgSO
40.1%, KH
2PO
40.2%, CaSO
40.1%, VB
110mg/L
1.2.3 culture device: rotary shaking table, electronic analytical balance, accurate PH meter, fermentation tank
2, method
2.1 process route: coprinus comatus preservation of bacteria strain → inclined-plane enlarges cultivation → liquid spawn preparation → liquid culture
2.2 the preparation of liquid spawn: it is 70ml that slant strains is inoculated in to the 250ml(liquid amount) in conical flask, put into 25 ℃ of insulating boxs standing 24 hours, then at 25 ℃, the rotary shaking table shaken cultivation of 180r/min 4d, make liquid spawn standby.
2.3 shaking flask culture technology: the tested medium of 70ml of packing in the 250ml conical flask, the liquid spawn prepared is inoculated according to 10% inoculum concentration, establish 8 repetitions for every, at 25 ℃, the rotary shaking table shaken cultivation of 180r/min 4d, the pH nature, observed and recorded bacterium ball growing state, measure mycelia dry weight, Peloton density and bacterium bulb diameter, and mycelium dry weight is carried out to one-way analysis of variance.
2.4 mycelium dry weight is measured: after fermentation stops, the whole culture fluids in each shaking flask are filtered with dry absorbent cotton, after with distilled water, repeatedly rinsing, be put in 60 ℃ of drying boxes and dry to constant weight, measure dry weight.
2.5 the measurement of Peloton density: bacterium liquid is placed in to the standing 5min of 100ml graduated cylinder, surveys the bacterium ball and account for the long-pending percentage of bacteria liquid.
2.6 the measurement of bacterium bulb diameter:, get at random 10 bacterium balls and arrange in a line, measure its total length again divided by 10, obtain the average diameter of bacterium ball.
3, results and analysis
3.1 the screening of the suitableeest initial p H:
By after the medium wiring solution-forming, with diluted alkaline and diluted acid, adjust PH5～8, result (Fig. 1) shows: cultivating initial p H is 6 ~ 7 o'clock, the shaggy mane mycelium bulk-growth is comparatively desirable.
3.2 the screening of suitable cultivation temperature:
Under 22 ℃, 25 ℃, 28 ℃ and 31 ℃ of conditions, cultivate respectively, stopped afterwards fermentation in 5 days, survey mycelium dry weight and Peloton density, the results are shown in Figure 2:
Before 25 ℃, with the rising of temperature, the mycelia amount constantly increases, but continues to heat up, and the mycelia amount starts to descend, and temperature surpasses 30 ℃, and mycelia quantity obviously reduces, thereby definite 25 ℃ are the suitableeest fermentation temperature.
3.3 optimum inoculation amount
In order to select optimum inoculation amount, by 5%, 10 %, tetra-inoculum concentrations of 1 5%, 20 %, test, observe the impact of different vaccination amount on mycelium dry weight and Peloton density, the results are shown in Figure 3:
As can be seen from Figure 3, along with inoculum concentration increases, biomass also improves gradually.When inoculum concentration was increased to 10 % by 5%, mycelium dry weight had improved 27 %, and inoculum concentration is while being increased to 20 % from 10 %, and biomass only improves 9%.Peloton density liquid has also reflected this variation tendency.Consider economic benefit, 10 % are desirable inoculum concentration.
3.4 the research of fermentation termination
(3) Primary Study of liquid spawn fermentation tank cultivation and fermentation condition
1,, with reference to the shake flask test result, carry out the test of 5L fermentation tank, the 4L medium of packing into, regulating initial p H is 6.3, with the inoculum concentration access fermentation tank of 10 %, under 180r/min, 25 ℃ of conditions, cultivate, every 12 hours sampling 100ml, detect mycelium content (as shown in Figure 5A).
2, carry out 60L and produce the tank scale-up, the 50L medium of packing into, regulate the fermentation tank internal pressure and maintain 0.02-0.03 Mpa, and temperature is 23-25 ℃, and every 12 hours sampling 100ml, detect mycelial growth situation (seeing Fig. 5 B).
In 96h, along with the prolongation of fermentation time, mycelial biomass increases gradually, after 60h, mycelial growth tends to be steady gradually, finds by the morphologic observation to the bacterium ball, and after 72h, the bacterium ball increases, polish, vigor starts to descend, and therefore, it is more suitable after cultivating 48-72h, to inoculate.
(4), the Research on Technological Innovation of cultivating chicken leg mushroom and management
1. take full advantage of fresh fruit slag and other pollution waste material
When making cultivation bag, break the normal procedure some are discarded and the more serious pollution material of environmental hazard added as batching, not only improve to a certain extent output, and promote to harmful substance degraded, purified natural environment.Really embodied the characteristic of the ecological agriculture, the protection of whole human environment has been played to positive role.
2. adopt de-bag " vertical " soil fruiting
After coprinus comatus bacterium bag covers with, digging after a swing bag earthing can fruiting, but conventional way swing bag, adopt disconnected bag upright and two kinds of modes of sleeping pendulum, and the mode fruiting of the direct vertical swing bag of our experimental employings in actual production (constantly bag), its effect all is better than traditional method.Repeatedly adopted in practice afterwards this kind Ways of fruiting not only greatly to improve feed stock conversion (cotton seed hull of take is major ingredient) and can reach 216%, and traditional way is only 150-180% with its conversion ratio of same raw material.This is because adopt vertical mushroom springing method to make on fruiting bed unit are nutrition more concentrated, and the mycelium on surface is in case after kink formed the mushroom flower bud, the nutrients help of deep layer is continual to be carried to the fruit body place, thereby the quality of fruit body also improves greatly.In addition such Ways of fruiting has been saved to a certain extent again the labour, has reduced labour intensity, has been taken full advantage of space, and more product can be produced in same place, and therefore good promotional value is arranged.
3. the fruiting phase carries out earthing for the second time after buddingging and processes
Coprinus comatus is " without the long mushroom of soil " thereby the soil that must be covered with 2-3cm after on management of producing mushroom, the bacterium bag covers with swing-bed, and water spray keeps moistening, and kink forms the mushroom flower bud after mycelia " climbs soil ", grows to maturation and does not pluck in time during parachute-opening.And our way is: at bacterium bag earthing for the first time, fruiting immediately not when the mushroom flower bud has just formed, but the nutrition sandy soil of earthing 2-3cm again, and keep the soil moisture of 65% left and right, after processing like this, not only improve the ecological conditions such as edible mushroom humidity, temperature, air, pH value, increase simultaneously the mycelial gravity in composts or fertilisers of cultivating surface, produce the formation that physical stimulation is beneficial to fruit body.To mycelium, created in addition the chance of " savings nutrition ", make nutrition more concentrated, no longer include phenomenon (mushroom body root and the medium natural separation of " the dead mushroom of ghost ", finally cause the prematurity mushroom body withered), thereby not only output increases to some extent, also obviously changed quality and the outward appearance of fruit body, make the mushroom body increase, plump, stem is short and real, misshapen mushroom and withered mushroom are few, have improved product quality and value.
4. the first damp mushroom is gathered rear filling flood once
The damp mushroom of coprinus comatus Routine Management first just keeps moistening with a small amount of water of the instruments such as sprayer spray after gathering, and follows out the second damp mushroom.We are after the first damp mushroom is gathered, in the fruiting bed, fill with flood once, water can slowly soak into the bacterium bag like this, supplement in time mycelium because of the moisture that fruiting loses, improved the mycelia vigor, improved conveying nutrition and the fruiting ability of mycelia, also guaranteed the conveying of bacterium bag degree of depth nutrition, make dark mushroom body large, the pure white delicacy of color and luster, be difficult for parachute-opening, be easy to management.
Four, the main innovation of this research and characteristic
1, determined first the optimal medium of coprinus comatus liquid spawn: nitrogenous source, carbon source and mineral salt are screened, understand to be applicable to the medium of coprinus comatus liquid spawn growth, the method by orthogonal experiment filters out optimal medium.Determined with the agricultural byproducts culture fluid of less expensive and prepared medium, not only reduced cost, and effect has been ideal
2, more comprehensively optimize first the Liquid Spawn Culture Conditions of coprinus comatus, obtained the coprinus comatus liquid spawn that incubation time is short, vigor good, output is high.
3, first the carrying out of success the large-scale production of coprinus comatus liquid spawn: with the success of 60L fermentation tank culture coprinus comatus liquid spawn; the coprinus comatus liquid spawn that is applied to of modern biotechnology deep layer culture process success is produced; determined coprinus comatus liquid spawn producting rule, for theoretical foundation has been established in the large-scale production of coprinus comatus.
4, adopt first vertical fruiting, secondary soil-covering and adopt after water the measures such as flood, improved coprinus comatus output, and realized the efficient utilization of pomace, bacterium chaff waste material and contaminated feedstock, obtained economic benefit and optimized again environment.
The accompanying drawing explanation
Fig. 1 is the impact of initial p H on shaggy mane mycelium soma weight and Peloton density; Fig. 2 is the affect figure of cultivation temperature on shaggy mane mycelium soma weight and Peloton density; Fig. 3 is the affect figure of different vaccination amount on shaggy mane mycelium soma weight and Peloton density; Fig. 4 is the affect figure of incubation time on mycelium dry weight and Peloton density; Fig. 5 is the dynamic change figure that fermentation tank culture shaggy mane mycelium soma is heavy.
Claims (3)
1. the innovative approach of coprinus comatus liquid spawn preparation and good quality and high output, is characterized in that the optimal liquid medium is potato 15%, wheat bran 5%, beancake powder 2%, brown sugar 0.5%, MgSO40.1%, KH2PO40.2%, VB110mg/L.
2. the innovative approach of a kind of coprinus comatus liquid spawn preparation as claimed in claim 1 and good quality and high output, it is characterized in that there is obvious difference in different nitrogen sources to the impact of the growth of shaggy mane mycelium body, in three kinds of organic nitrogens, best with the yeast extract effect, be followed successively by peptone, beef extract; Select glucose, brown sugar and sucrose comparatively desirable as the nitrogenous source of coprinus comatus liquid nutrient medium; The suitableeest fermentation temperature is 25 ℃; Desirable inoculum concentration is 10%.
3. the method for coprinus comatus liquid spawn preparation and good quality and high output innovation research, is characterized in that, adopts de-bag " vertical " soil fruiting, and the fruiting phase carries out earthing for the second time after buddingging and processes, and the first damp mushroom is gathered rear filling flood once.
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| CN112956372A (en) * | 2021-03-11 | 2021-06-15 | 齐齐哈尔大学 | Method for cultivating lucid ganoderma |
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| CN112956372A (en) * | 2021-03-11 | 2021-06-15 | 齐齐哈尔大学 | Method for cultivating lucid ganoderma |
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