CN103733873A - Study on liquid pholiota adiposa strains and polysaccharide - Google Patents

Study on liquid pholiota adiposa strains and polysaccharide Download PDF

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CN103733873A
CN103733873A CN201310213973.9A CN201310213973A CN103733873A CN 103733873 A CN103733873 A CN 103733873A CN 201310213973 A CN201310213973 A CN 201310213973A CN 103733873 A CN103733873 A CN 103733873A
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yellow umbrella
polysaccharide
medium
yellow
liquid
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蔡德华
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Ludong University
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Ludong University
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Abstract

The invention provides a study on liquid pholiota adiposa strains and polysaccharide and belongs to the technical field of edible mushroom cultivation. The liquid strains have the advantages of being short in production cycle, uniform in fungus age and neat in fruiting. The liquid pholiota adiposa strains are obtained through a submerged cultivation technology and are mycelia which grow in fluid nutrient media and are used for breeding next-generation strains or used for cultivation. The liquid strains have characteristics and advantages that solid strains can not compare with. In china, the report about large-scale cultivation of the pholiota adipose strains does not appear yet. Only some scattered cultivation of the solid strains exists, and the further development of the pholiota adipose strains is greatly restricted in the breeding mode, so it is urgently needed that the cultivation of the liquid strains replace the cultivation of the solid strains.

Description

The research of a kind of yellow umbrella liquid spawn and polysaccharide
Technical field
The invention belongs to the edible fungus culturing field that learns a skill, relate to the research to yellow umbrella liquid spawn and polysaccharide.
Background technology
Biotechnology, as a part for bio-science field, will have development and application prospect widely in 21st century.Along with the development of the natural science applied research in the world, the research field of China edible mushroom is constantly widened.Fermentation engineering as one of biotechnology is bionic basis, submerged fermentation technology is the application of fermentation engineering, it grows up in antibiotic fermentation technical foundation, from the forties, be progressively applied to edible mushroom latter stage, series of studies shows, edible fungi submerged fermentation technology has wide practical use.
Yellow umbrella [Pholiota adipose (Fr.) Qu é l.], have another name called willow mushroom, yellow mushroom, Pholiota adiposa, Eumycota (Eumycota), Basidiomycetes (Basidiomycetes), Agaricales (Agaricaies), a kind of edible medicinal fungi of holding concurrently of Strophariaceae (Strophariaceae).This bacterium bacterial context is plump, and taste is good, sliding tender tasty and refreshing, can match in excellence or beauty with bolete, is a kind of unique flavor, peculiar precious mushroom.Its fruit body is rich in protein, fat and carbohydrate and multivitamin and mineral salt, very useful to health, and especially one deck mucus on fruit body surface, proves a kind of nucleic acid through biochemical analysis, is of value to human body energy and mental recovery.The polysaccharide body that fruit body is extracted by organic solvent, reaches 80% ~ 90% to the inhibiting rate of sarcoma180 and EC; Also can prevent in addition the infection of staphylococcus, Escherichia coli, pneumobacillus and tubercle bacillus.Yellow umbrella is popular in Japanese market, is also the fashion delicacies of restaurant, city restaurant in China.Therefore there is vast potential for future development, the report that does not also have the extensive cultivation of yellow umbrella in China, and have some scattered cultivations also to only limit to the cultivation of solid spawn, on seed production way, just greatly restricted further developing of yellow umbrella, therefore just in the urgent need to replace the cultivation of solid spawn with liquid spawn.
" liquid spawn " is comparatively speaking with " solid spawn ", by deep layer culture technique, obtains, and it refers to and is grown in liquid nutrient medium for breeding the mycelium of bacterial classification of future generation or cultivation.Liquid spawn has the characteristics and advantages that solid spawn can not be compared:
production cycle shortens greatly
Preparation liquid spawn is general only needs 3 ~ 7 days, and the cycle is short, and speed is fast; And cultivate one bottle of solid state cultivation kind need 30 ~ 50 days.The advantages such as in addition, the liquid spawn of usining expands while cultivating cultivated species as original seed, and also than adopting fast many of solid spawn, and it is even to have each bottle of mycelial growth rate with the solid spawn that this method is produced, and extremely bacterium (mycelia does not sprout) rate is low.With this cultivated species carry out that bed is planted, bag is planted, brick is planted, plant, its Quality and yield that produces mushroom (ear) is all equal to or higher than the cultivated species that uses solid original seed.Also liquid spawn directly can be used as to cultivated species, be inoculated in composts or fertilisers of cultivating and produce, this was than solid spawn precocity 10 ~ 20 days.Because liquid spawn has mobility, each mycelium pellet and mycelia segment can be scattered and be sprouted at different positions, and growth point is many, and the pollution that has reduced inoculation (is 5% with solid kind pollution rate, and be 1% with liquid strain pollution rate), cover with the also just shortening greatly of required time of bag simultaneously.
cell age is consistent, and fruiting (ear) is neat, is convenient to management
Because solid spawn is to be abutted against mycelia on kind of piece to spread and grow up to, inoculation can only be in media surface in addition, and the speed of so not only cultivating bacterial classification is slow, and it is widely different to be in the mycelium cell age of seed bottle upper and lower, generally differs 20 ~ 30 days.In the time of often at the bottom of bottom mycelia grows to bottle, the mycelia that is in top is just aging in succession.Liquid spawn is different, their uniformities that grows, and cell age is neat, and mycelium when deep layer is cultivated 3 ~ 7 days is on the occasion of vigorous period, sprouts soon after inoculation, grows healthy and strong.With the cultivation of its spice, its Mycelium growth rate is consistent, budding and the fruiting time more consistent, be convenient to management, gather and process.To the kind of grog cultivation, especially bag cultivation is more applicable, and therefore same medium adopts liquid-spawn inoculation, and its bacteria time is only 1/2 of solid kind.So more shortening the bacteria phase provides assurance.
the cost of bacterial classification is low
Adopt this technique fermenting and producing liquid spawn, output is high, raw material is cheap, and cost is low compared with solid spawn.
inoculation is convenient
The liquid spawn that is liquid state is convenient to the mechanization of inoculating process, automation, is conducive to the raising of operating efficiency.Be convenient to carry out large-scale industrialized production, and reduced to a great extent labour intensity.
be convenient to obtain more object product
Some can not carry out tame wild mushroom can obtain object product by submerged fermentation, as polysaccharide, amino acid etc.
Summary of the invention
The optimization research of yellow umbrella deep layer culture systems
1 materials and methods
1.1 material
1.1.1 bacterial classification Huang Sanyou Shandong Province edible mushroom work station provides
1.1.2 mother culture media
Add rich PDA: potato 20%, glucose 2%, agar 2%, wheat bran filtrate 5%, peptone 0.5%, V b10.001%, KH 2pO 40.3%, MgSO 40.25%.
1.1.3 one-level shaking flask medium
Potato 20%, wheat bran filtrate 3%, glucose 1%, peptone 0.2%, KH 2pO 40.2%, MgSO 40.1%, VB 10.001%
1.1.4 carbon source is for examination medium
Potato 20%, peptone 0.2%, KH 2pO 40.2%, MgSO 40.1%, V b10.001%, carbon source.With 1% glucose, brown sugar, lactose, fructose, maltose, sucrose, starch, replace carbon source wherein respectively.
1.1.5 nitrogenous source is for examination medium
Potato 20%, glucose 1%, KH 2pO 40.2%, MgSO 40.1%, V b10.001%, nitrogenous source.Use respectively 0.2% peptone, beef extract, yeast extract, NH 4nO 3, NH 4cl, (NH 4) 2sO 4replace nitrogenous source wherein with 3% wheat bran filtrate. .
1.1.6 C/N ratio is for examination medium
Peptone 0.3%, KH 2pO 40.2%, MgSO 40.1%, VB 10.001%, glucose.Take peptone as the fixing nitrogen content of radix, and conversion glucose phosphorus content, adds respectively glucose according to C/N ratio 10:1,20:1,30:1,40:1,50:1.
1.2 method
1.2.1 medium preparation
Potato is cut into piece after peeling, and puts into water together with wheat bran, maintains 25min left and right after boiling.After eight layers of filtered through gauze, get filtrate, then add respectively each medicine, constant volume.Solid culture medium is distributed into test tube, and 1/4 ~ 1/3,121 ℃ of sterilizing 0.5h that loading amount is test tube take out pendulum inclined-plane.Liquid nutrient medium is sub-packed in 500ml conical flask, every bottled 150ml, 121 ℃ of sterilizing 0.5h..
1.2.2 actication of culture
The bacterial classification sterile working of preservation is connected to inclined-plane, cultivates about one week at 25 ℃, now mycelia is covered with inclined-plane, can use.
1.2.2 the preparation of liquid spawn
0.5 ㎝ * 0.5 ㎝ that size is close is got in sterile working mother from inclined-plane plants piece, is inoculated in (bacterium piece wants the thin bacterium piece that as far as possible makes to swim on liquid level) in liquid nutrient medium.4 ~ 5 of every bottle graft kinds.25 ℃ of standing 24h, are then placed in 25 ℃, on 160 r/min Leftward/rightward rotating shaking tables, cultivate about 6 days.
1.2.3 inoculate the sterile working of secondary shaking flask, inoculum concentration is 10%, is placed on rotary shaking table 25 ℃, and 160r/min cultivates 4 ~ 5 days.Establish 6 repetitions for every group.
1.2.4 result treatment
1. the mensuration of bacterium sphere volume ratio is poured zymotic fluid in graduated cylinder into, fully static after, measure bacterium sphere volume, calculate its volume ratio.
2. the number of bacterium ball and size measurement are drawn respectively 1ml zymotic fluid and are put into 3 culture dishes from graduated cylinder with pipette, and culture dish below lining, with squared paper, is measured bacterium bulb diameter and records bacterium nodule number order.
3. after the mensuration of mycelial growth amount is filtered zymotic fluid with absorbent cotton, then with distilled water flushing for several times, drain, then in 60 ℃ of baking ovens, dry to constant weight, with electronic analytical balance, weigh.
4. C/N ratio is tested the accurate pH meter of mensuration of zymotic fluid pH value, the pH value before mensuration fermentation and the pH value after fermentation.
2 results and analysis
The impact of 2.1 different carbon sources on yellow umbrella liquid culture
2.1.1 carbon source result of the test is in Table 1
The impact of the different carbon sources of table 1 on yellow umbrella liquid culture
From table 1, can find out that yellow umbrella take on the medium that glucose is carbon source, mycelia growing way is best, and bacterium ball growth fraction is more even.Take on the medium that lactose, fructose, brown sugar, maltose be carbon source, yellow umbrella growth is also better, but good not as good as growing way on the medium that is carbon source at glucose.Take on the medium that starch is carbon source, yellow umbrella growth is poor, and it is consistent that this reports with pertinent literature.
2.1.2 variance analysis
As shown in Table 1, yellow umbrella, take on the medium that glucose, fructose, lactose, brown sugar, maltose, sucrose is carbon source, is all grown better, but exists very large difference on the number of bacterium ball, diameter.The variance analysis of carrying out by its dry weight, filters out preferably carbon source.Mycelia dry weight in 7 kinds of medium is carried out to variance analysis, the results are shown in Table 2.
Table 2 carbon source medium mycelium dry weight variance analysis table
Difference source SS df MS F value F(0.05) F(0.01)
Between group 0.7223 6 0.0120 19.8911 2.8477 4.46
In group 0.0847 14 0.0061
Amount to 0.8071 20
SS-sum of squares of deviations, df-degree of freedom, MS-is side all, F (0.05)-F critical value (as follows)
As shown in Table 2, carbon source F value 19.8911 > F (0.05)=2.8477, and F value 19.8911 > F (0.01)=4.46, can find out that yellow umbrella supplies to utilize the ability of carbon source different in examination medium at each, difference is very remarkable.In order to further illustrate the significance of difference between medium, carried out again multiple ratio between mean, the results are shown in Table 3.
Table 3 carbon source medium mycelium dry weight SSR check table
As can be seen from Table 3,1,2 and 5,6,7 significant differences, and between 3,4 and 5, difference is not remarkable, 1 and 5,6,7 differences are extremely remarkable, 2,3,4,5 differences are very not remarkable, mycelia dry weight in known 1, apparently higher than 3,4,5,6,7, and 2 is more or less the same, therefore, glucose is the carbon source that is suitable for yellow umbrella growth most, be secondly fructose, and sugarcane sugar and starch is unsuitable for the growth of yellow umbrella.
The impact of 2.2 different nitrogen sources on yellow umbrella submerged fermentation
2.2.1 nitrogenous source result of the test is in Table 4
The impact of table 4 different nitrogen sources on yellow umbrella liquid culture
As shown in Table 4, yellow umbrella is grown better in organic nitrogen source medium, and pellet form is better, size is more suitable.In inorganic nitrogen-sourced medium, grow very fast, but mycelia is tiny, is fine hair shape, majority does not grow up to the good bacterium ball of form.Totally it seems, yellow umbrella is grown and is obviously better than inorganic nitrogen-sourced medium in organic nitrogen source medium.
2.2.2 variance analysis
As shown in Table 4, yellow umbrella all can be grown preferably in each organic nitrogen source medium, but exists difference in bacterium nodule number order, size, and its dry weight is carried out to variance analysis, filters out preferably nitrogenous source.Mycelia dry weight in 7 kinds of medium is carried out to variance analysis, the results are shown in Table 5
Table 5 nitrogenous source medium mycelium dry weight variance analysis table
Difference source SS df MS F F(0.05) F(0.01)
Between group 1.3234 6 0.2206 38.8138 2.8477 4.46
In group 0.0796 14 0.0576
Amount to 1.4030 20
As shown in Table 5, nitrogenous source F value > F(0.05)=2.8477, and F value > F(0.01)=4.46.Can find out, yellow umbrella utilizes the ability of nitrogenous source different, and difference is extremely remarkable.In order to further illustrate the significance of difference between different nitrogen sources, carried out again multiple ratio between mean, the results are shown in Table 6.
Table 6 nitrogenous source medium mycelium dry weight SSR check table
As shown in Table 6, significant difference between 1,2,3,4 and 5,6,7, between 1,2,3 and 5,6,7, difference is very remarkable, and 4 and 5 differences are not extremely remarkable.Hence one can see that, yellow umbrella take on the medium that peptone, wheat bran, beef extract be nitrogenous source growth better, and take grow on the medium that ammonium nitrate, ammonium sulfate, ammonium chloride is nitrogenous source poor, so yellow umbrella utilizes the ability of organic nitrogen more much better than than inorganic nitrogen, result on solid culture medium is consistent with it for this.
2.3 impacts of different C/N ratios on yellow umbrella liquid culture
2.3.1 yellow umbrella submerged fermentation C/N ratio experimental result is in Table 7
The impact of the different C/N ratios of table 7 on pholiota adiposa mycelium dry weight
As can be seen from Table 7, on the medium that yellow umbrella is 40:1 in C/N ratio, can obtain maximum biomass, every group of C/N ratio medium endpoint pH is on a declining curve, endpoint pH between each group declines generally with the rising of C/N ratio, but pH value exception when C/N ratio is 40:1, may be more vigorous because of mycelial growth under condition for this reason, metabolism be than very fast.
2.3.2 the mycelium dry weight that C/N ratio is respectively organized in variance analysis is difference to some extent, and its dry weight is carried out to variance analysis, filters out the suitableeest C/N ratio.In Table 8
The different C/N ratio mycelium dry weight of table 8 variance analysis table
Difference source SS df MS F value F(0.05) F(0.01)
Between group 0.5559 4 0.1390 17.0192 3.478 5.99
In group 0.08116 10 0.00817
Amount to 0.6376 14
As shown in Table 8, C/N ratio F value >F (0.05)=3.478, and F value > F (0.01)=5.99, this shows, yellow umbrella biomass on different C/N ratio medium is different, has utmost point significant difference.In order to further illustrate the significance of difference between medium, carried out again multiple ratio between mean, the results are shown in Table 9.
The different C/N ratio mycelium dry weight of table 9 SSR check table
As shown in Table 9, on the medium that yellow umbrella is 40:1 in C/N ratio, grow and be better than on the medium of 50:1,30:1,20:1,10:1, there is significant difference with 50:1,30:1,20:1 but be not extremely remarkable, and it is extremely remarkable with the difference of 10:1, this illustrates that yellow umbrella can well growth in the medium of C/N ratio in a big way, and growth is the poorest on the medium that is 10:1 in C/N ratio, therefore the suitableeest C/N ratio of yellow umbrella medium should be 40:1, and this condition can obtain maximum biomass.
3 discuss
Through above-mentioned experiment and analysis, can draw the following conclusions: yellow umbrella all can well be grown take on the medium that monose is carbon source, grows poor on polysaccharide medium, and optimum carbon source is glucose.On organic nitrogen source medium, growth is all better than inorganic nitrogen-sourced medium, and take grow on the medium that peptone, wheat bran, beef extract be nitrogenous source best.But consider from economic aspect, wheat bran is cheap, widely distributed, it is first-selected nitrogenous source.When C/N ratio changes between 20:1 ~ 50:1, yellow umbrella all can well be grown, and can obtain larger biomass.And the medium that C/N ratio is 40:1 is suitable for yellow umbrella growth most, can obtain maximum biomass.Impact as for concentration of carbon and nitrogen sources on pholiota adiposa mycelium output also needs to do further experimental study when producing on a large scale.
The research of second section, yellow umbrella liquid nutrient medium orthogonal experiment
1 materials and methods
1.1 material
1.1.1 strains tested: yellow umbrella, is provided by Shandong Province's edible mushroom work station.
1.1.2 medium:
(1) mother culture media: potato 20%, glucose 2%, peptone 0.5%, wheat bran 5%, agar 2%, MgSO 40.3%, KH 2pO 40.3%, V b10.001%.
(2) one-level shaking flask medium: potato 20%, wheat bran 3%, peptone 0.2%, glucose 1%, KH 2pO 40.2%, MgSO 40.1%, V b10.001%.
(3) nitrogenous source is for trying culture medium prescription: potato 20%, glucose 1%, KH 2pO 40.2%, MgSO 40.1%, V b10.001%, nitrogenous source.
Use respectively 0.4% beef extract, peptone, wheat bran, yeast extract, NH 4nO 3, NH 4cl, (NH 4) 2cO 3replace nitrogenous source wherein.
(4) carbon source is for trying culture medium prescription: potato 20%, peptone 0.2%, KH 2pO 40.2%, MgSO 40.1%, V b10.001%, carbon source.
With 1% brown sugar, glucose, fructose, starch, lactose, maltose, sucrose, replace carbon source wherein respectively.
(5) orthogonal experiment culture medium prescription: potato 20%, KH 2pO 40.2%, V b10.001%, carbon source 1%, nitrogenous source 0.2%, mineral salt 0.1%.Wherein carbon source, nitrogenous source, mineral salt are in Table 1 and table 3.
Table 1 factor level table
1.2 method
1.2.1 actication of culture: the bacterial classification sterile working of preservation is seeded to inclined-plane and adds rich PDA medium, cultivate 10d left and right at 25 ℃.
1.2.2 inoculate one-level shaking flask: sterile working is planted and got 0.5 ㎝ from inclined-plane mother 2the bacterial classification piece of size, is inoculated in the one-level shaking flask that sterilizing is good, and 4 ~ 5 of every bottle graft kinds make bacterial classification piece swim on liquid level as far as possible.Standing 24hr under 25 ℃ of conditions, is placed in thereafter on 25 ℃, the rotary shaking table of 160 about r/min and cultivates 6 ~ 7 days.
1.2.3 inoculating carbon and nitrogen sources shaking flask packs ready-made medium in the shaking flask of 500mL by formula (3), (4), every bottle approximately fills 150mL, sterilizing 20min at 121 ℃, cooling rear sterile working by the one-level shaking flask kind access of having grown wherein, inoculum concentration 10%, puts into thereafter on 25 ℃, the shaking table of 160r/min and cultivates 4 ~ 5d.Observe and record the upgrowth situation of bacterium ball, weigh mycelial dry weight.
1.2.4 orthogonal experiment
(1) experimental scheme, from carbon nitrogen source for choosing good four kinds of carbon and nitrogen sources examination cultivation results, is added four kinds of more common mineral salt, and design three factor four hydraulic test schemes, are shown in Table 3.
(2) result treatment
1. bacterium ball number and size measurement are poured whole zymotic fluids in culture dish into, and culture dish below lining one lattice paper, counts its bacterium ball number, and measure bacterium bulb diameter.
2. the mensuration of the shared volume ratio of bacterium ball is poured whole zymotic fluids in graduated cylinder into, fully static after, measure its volume ratio.
3. the mensuration of mycelial growth amount is after zymotic fluid suction filtration, then with distilled water flushing for several times, drains, and then in 60 ℃ of baking ovens, dries to constant weight, with electronic analytical balance, weighs.
4. the mensuration of zymotic fluid pH value adopts pH meter, surveys the pH value before fermentation, the pH value after fermentation.
2 results and analysis
2.1 carbon sources, nitrogenous source test results and analysis
As can be seen from Figure 1, it is peptone > wheat bran > beef extract > yeast extract > ammonium nitrate > ammonium sulfate > ammonium chloride that Pholiota adiposa mycelia utilizes the order of nitrogenous source, the order of utilizing carbon source is glucose > fructose > brown sugar > lactose > maltose > sucrose > starch, so the suitableeest nitrogenous source of Pholiota adiposa mycelia growth is peptone, wheat bran, beef extract, yeast extract, the suitableeest carbon source is glucose, fructose, lactose, brown sugar, visible yellow umbrella can utilize various carbon nitrogen sources more widely.Select four kinds of the suitableeest nitrogenous sources, carbon sources wherein to carry out orthogonal experiment, filter out the optimum combination of liquid culture based formulas.
2.2 orthogonal experiments and variance analysis
2.2.1 the shared volume ratio of bacterium nodule number order, bacterium ball of each group of orthogonal experiment and the variation of pH value are in Table 2
The bacterium nodule number order of table 2 orthogonal experiment, the shared volume of bacterium ball when pH value change
By table 2, we can find out, orthogonal experiment is respectively organized the indexs such as the number, size of bacterium ball obvious difference, can observe with the naked eye it significantly distinguishes, this formula obvious and medium has certain relation, group 1,4 bacterium ball sums are more and bacterium ball size is more even, and volume ratio is close to 100%.Each variation of organizing pH value is all in rising trend, but amplitude of variation differs.Their dry weight is carried out to variance analysis, filter out the optimum combination of culture medium prescription.
2.2.2 Orthogonal Experiment and Design and interpretation of result
39.1828 > F (0.01)=9.78 impacts of carbon source F value are extremely remarkable, and nitrogenous source F value 23.9354 > F (0.01)=9.78 affects extremely significantly, and in like manner known mineral salt affect not remarkable.From R value, carbon source has the greatest impact to Pholiota adiposa mycelia growth, is secondly nitrogenous source, and mineral salt impact is minimum, and this is consistent with the condition of most of hypha of edible fungus growth.Therefore the optimum combination of its culture medium prescription is: brown sugar 1%+ yeast extract 0.2%+K 2sO 40.1%.
2.2.3 verification experimental verification
Optimum combination according to culture medium prescription is tested, and weighs mycelium dry weight, repeated test three times, and recording mean value is 17.52g/L, has obtained comparatively desirable result.
3 discuss
By above test with analyze knownly, yellow umbrella can utilize various carbon nitrogen sources more widely, and the optimization formula of its liquid nutrient medium is potato 20%, brown sugar 1%, yeast extract 0.2%, K 2sO 40.1%, KH 2pO 40.2%, V b10.001%.As for the how many impacts on mycelium production of content of each composition in formula, further experimental study needs.
The 3rd joint, different concentration of carbon and nitrogen sources are cultivated the research of impact on yellow umbrella deep layer
1. materials and methods
1.1 material
1.1.1 bacterial classification Huang Sanyou Shandong Province edible mushroom work station provides.
1.1.2 mother culture media adds rich PDA: potato 20%, glucose 2%, wheat bran 5%, peptone 0.2%, Cobastab 10.001%, KH 2pO 40.3%, MgSO 40.25%, agar 2%.
1.1.3 one-level shaking flask medium potato 20%, wheat bran 3%, glucose 2%, peptone 0.2%, KH 2pO 40.25%, MgSO 40.15%, Cobastab 10.001%.
1.1.4 carbon source concentration is for examination medium potato 20%, peptone 0.5%, KH 2pO 40.25%, MgSO 40.15%, Cobastab 10.001%, glucose.Wherein the concentration of glucose is respectively 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%.
1.1.5 nitrogen concentration is for examination medium potato 20%, glucose 2%, KH 2pO 40.25%, MgSO 40.15%, Cobastab 10.001%, peptone.Wherein peptone concentration is respectively 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%.
1.1.6 orthogonal experiment medium is chosen more suitable glucose (2.0%, 2.5%, 3.0%) and peptone (0.1%, 0.2%, 0.3%), and conventional mineral salt MgSO 4+ KH 2pO 4(0.10%+0.20%, 0.15%+0.25%, 0.20%+0.30%) carries out orthogonal experiment.
1.2 method
1.2.1 medium is prepared after potato is peeled and is cut into small pieces, and puts into water together with wheat bran, maintains about 30min after boiling, and after filtering, gets filtrate and adds successively other compositions, constant volume after other compositions dissolve with double gauze.Solid culture medium is distributed into test tube, and 1/4~1/3,121 ℃ of sterilizing 30min that loading amount is test tube take out pendulum inclined-plane.Liquid nutrient medium is sub-packed in 500mL conical flask, every bottled 150mL, 121 ℃ of sterilizing 30min.
1.2.2 actication of culture is connected to inclined-plane by the bacterial classification sterile working of preservation, cultivates approximately 1 week at 25 ℃, and now mycelia is covered with inclined-plane, can use.
1.2.3 the preparation sterile working of one-level shaking flask kind is got size from inclined-plane and is about 0.5cm 2mother plant piece, be inoculated in liquid nutrient medium, make bacterium piece swim on liquid level as far as possible.4~5 of every bottle graft kinds.25 ℃ of standing cultivation 24h, are then placed in 25 ℃, cultivate approximately 7 d on the rotary shaking table of about 160r/min.
1.2.4 inoculate under secondary shaking flask aseptic technique, by the one-level shaking flask kind of having grown, with in aseptic pipette access secondary shaking flask, inoculum concentration is 10%, is placed on rotary shaking table, and 25 ℃, 160r/min cultivates 4d.Establish 3 repetitions for every group.
1.2.5 the (1) mensuration of Peloton density of result treatment.Zymotic fluid is poured in graduated cylinder, after fully static, measured bacterium sphere volume, calculate its volume ratio.(2) the mensuration of growth quantity of mycelium.Zymotic fluid is used after filter paper suction filtration, then with distilled water flushing for several times, drained, then in 60 ℃ of baking ovens, dry to constant weight, with electronic analytical balance, weigh.
2 results and analysis
2. the impact that the carbon source of 1 variable concentrations is cultivated yellow umbrella deep layer
What the carbon source of variable concentrations was cultivated yellow umbrella deep layer affects result as shown in Figure 2.
Yellow umbrella is grown take in the liquid nutrient medium that the glucose of variable concentrations is carbon source, and bacterium ball size is more consistent, and between 1 ~ 3mm, but mycelium dry weight has obvious difference; Along with the increase of carbon source concentration, mycelium dry weight increases gradually, arrived concentration 2.0% reach the highest, then along with the rising mycelium dry weight of concentration reduces, the growth of the neither suitable pholiota adiposa mycelium of carbon source concentration that this explanation is lower or higher.And the Peloton density difference of each concentration is little, just under High Concentration Situation, there is obvious minimizing.
Mycelium dry weight in 7 kinds of variable concentrations carbon source medium is carried out to variance analysis, the results are shown in Table 1.
The carbon source medium mycelium dry weight variance analysis of table 1 variable concentrations
As can be seen from Table 1: the carbon source F=3.4678 > F of variable concentrations 0.05=2.8477, but F=3.4678 < F 0.01=4.4558, illustrate that yellow umbrella utilizes the ability difference of the carbon source of variable concentrations, significant difference in each supplies to try medium.For further illustrating the significance of difference between medium, then carry out multiple ratio between mean, the results are shown in Table 2.
Table 2 variable concentrations carbon source medium mycelium dry weight SSR check
As can be seen from Table 2: the mycelium dry weight difference of carbon source concentration 2.0%, 3.0%, 2.5%, 1.0% time is not remarkable, and the mycelium dry weight difference of carbon source concentration 3.5%, 1.5%, 0.5% time is not remarkable yet, but the mycelium dry weight difference of carbon source concentration 2.0% and 3.5% time is significant, therefore the better carbon source concentration of Pholiota adiposa mycelia bulk-growth is 2.0%, 3.0% and 2.5%.
The impact that the nitrogenous source of 2.2 variable concentrations is cultivated yellow umbrella deep layer
What the nitrogenous source of variable concentrations was cultivated yellow umbrella deep layer affects result as shown in Figure 3.
As seen from Figure 3, yellow umbrella is grown take in the liquid nutrient medium that the peptone of variable concentrations is nitrogenous source, and the mycelium dry weight when low concentration 0.1%, 0.2%, 0.3% is larger, and Peloton density is larger, and volume ratio is all more than 90%, and mycelium growing way is also better; And when high concentration 0.4%, 0.5%, 0.6%, 0.7%, mycelium growing way obviously a little less than, Peloton density is obvious downward trend, mycelium dry weight also obviously diminishes.
Mycelium dry weight in 7 kinds of variable concentrations nitrogenous source medium is carried out to variance analysis, the results are shown in Table 3.
The nitrogenous source medium mycelium dry weight variance analysis of table 3 variable concentrations
As can be seen from Table 3: the nitrogenous source F=3.5350 > F of variable concentrations 0.05=2.8477, but F=3.5350 < F 0.01=4.4558, illustrate that yellow umbrella supplies to utilize the ability of variable concentrations nitrogenous source different in examination medium at each, significant difference.For further illustrating the significance of difference between each concentration, then carry out multiple ratio between mean, the results are shown in Table 4.
Table 4 variable concentrations nitrogenous source medium mycelium dry weight SSR check
As can be seen from Table 4: the concentration of nitrogen is 0.3%, 0.1%, 0.2% time, and gained mycelium dry weight difference is not remarkable; There is not significant otherness in the mycelium dry weight when concentration is 0.4%, 0.6%, 0.5%, 0.7%, but has significant otherness at concentration 0.3% and 0.4% 's mycelium dry weight yet.Therefore the better nitrogen concentration of suitable Pholiota adiposa mycelia bulk-growth is 0.3%, 0.1%, 0.2%, and now Pholiota adiposa mycelia bulk-growth is vigorous, and bacterium ball size evenly, can obtain more mycelium.
2.3 orthogonal experiment
2.3.1 experimental scheme
Foundation is the results of univariate logistic analysis above, selects 3 suitable carbon sources, nitrogen concentration and conventional mineral salt MgSO 4with KH 2pO 4variable concentrations matched combined is carried out the experimental scheme (in Table 5) of Three factors-levels.Therefrom choose the arranging scheme of optimum carbon source concentration, nitrogen concentration and inorganic salt concentration, determine the optimum medium of this bacterial classification.
Table 5 L 9(3 3) orthogonal experiment factor level table
2.3.2 orthogonal experiments
The R value of A factor (carbon source) is maximum, and the R value of B factor (nitrogenous source) is less, and the R value of C factor (mineral salt) is minimum, can judge thus, and what carbon source concentration was cultivated yellow umbrella deep layer has the greatest impact, and the impact of inorganic salt concentration is minimum.From the result of further variance analysis, can find out, carbon source concentration is cultivated impact to the deep layer of yellow umbrella and is reached significance level, so carbon source concentration should be selected optimum A 1level; But nitrogenous source and inorganic salt concentration do not reach significance level to its impact, and the difference between them is little, and from economic angle, consider the B that should select concentration low 1, C 1level, so the optimum combination of this medium is A 1b 1c 1.
3. conclusion
By above test with analyze knownly, yellow umbrella can adapt to carbon nitrogen source and the mineral salt of variable concentrations more widely, and the optimization formula of its liquid nutrient medium is potato 20%, glucose 2.0%, peptone 0.1%, MgSO 40.10%, KH 2pO 40.20%, Cobastab 10.001%.As for the optium concentration of other carbon nitrogen sources and the mineral salt later further experiment that needs.
The research of the 4th joint, yellow umbrella deep layer condition of culture
1. materials and methods
1.1 material
1.1.1 bacterial classification: yellow umbrella, is provided by Shandong Province's edible mushroom work station.
1.1.2 mother culture media adds rich PDA: potato 20%, glucose 2%, wheat bran 5%, peptone 0.5%, V b10.001%, KH 2pO 40.3%, MgSO 40.25%, agar 2%.
1.1.3 one-level shaking flask medium potato 20%, glucose 2%, peptone 0.5%, KH 2pO 40.25%, MgSO 40.15%, V b10.001%
1.1.4 secondary shaking flask medium potato 20%, glucose 2%, peptone 0.3%, KH 2pO 40.25%, MgSO 40.15%, V b10.001%
1.2 method
1.2.1 the preparation of one-level shaking flask kind: the mother that just grown is planted and be inoculated into one-level shaking flask medium under aseptic condition, make bacterial classification piece swim in liquid level as far as possible, static cultivation is one day at 25 ℃, cultivates approximately one week and be placed on shaking table.Shaking table temperature is 25 ℃, rotating speed 160r/min.
1.2.2 the impact that different factors are cultivated yellow umbrella deep layer:
(1) impact that different temperatures is cultivated yellow umbrella deep layer: the shaking table that the secondary shaking flask of having inoculated is placed in respectively to 16 ℃, 19 ℃, 22 ℃, 25 ℃, 28 ℃, 31 ℃ is cultivated, and surveys Peloton density and mycelium dry weight (as follows) after four days.The mensuration of A, Peloton density: zymotic fluid is placed in to the graduated cylinder of 200mL, standing 5min, the volume ratio of calculating mycelium and zymotic fluid; The mensuration of B, mycelium dry weight: the baking oven that the mycelium after suction filtration is placed in to 60 ℃ dries to constant weight, and surveys its weight with analytical balance.
(2) impact that different pH cultivate yellow umbrella deep layer: it is 3.0,4.0,5.0,6.0,7.0,8.0,9.0 that the secondary shaking flask medium pH making is debugged respectively, inoculated and cultured after sterilizing.
(3) impact that different viscosities is cultivated yellow umbrella deep layer: the secondary shaking flask medium making is added respectively to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7% agar, and do control group not add the blank of agar.
(4) impact that different vaccination amount is cultivated yellow umbrella deep layer: the secondary shaking flask making is accessed respectively to 5%, 10%, 15%, 20% one-level shaking flask kind, cultivate and observe.
(5) impact that different bottled amounts are cultivated yellow umbrella deep layer: be respectively the one-level shaking flask kind of the secondary medium access 10% of 90mL, 120mL, 150mL, 180mL, 210mL, 240mL by bottled amount, survey result after cultivating.
(6) impact that different rotating speeds is cultivated yellow umbrella deep layer: be cultivation survey result on the shaking table of 90r/min, 120r/min, 150r/min, 180r/min, 210r/min, 240r/min at rotating speed respectively by the secondary shaking flask of inoculate.
Above experimental condition, except specified otherwise, all adopts the conical flask of 500mL, every bottled 150mL, and inoculum concentration is 10%, shaking table cultivation temperature is that 25 ℃, rotating speed are 160r/min.All in triplicate.
2. result and discussion:
2.1 temperature: Fig. 4 shows the result of different temperatures on yellow umbrella deep layer cultivation impact.
As can be seen from Figure 4, along with the rising of temperature, mycelium dry weight increases gradually, reaches maximum in the time of 28 ℃, and then the mycelial dry weight of rising along with temperature declines on the contrary; Therefore can tentatively judge that 28 ℃ is more suitable growth temperature.And the density of bacterium ball is at 19 ~ 25 ℃ higher, may be because bacterium ball vigor is stronger under lower temperature, bacterium ball furcella is around more to be caused.In order to further illustrate the otherness of mycelium dry weight between different temperatures, the mycelium dry weight under condition of different temperatures is carried out to variance analysis.
The pholiota adiposa mycelium dry weight variance analysis of table 1 different temperatures
Difference source SS df MS F F (0.05) F (0.01) Significance
Between group 87.92936 5 17.58587 58.74846 3.105875 5.064351 **
In group 3.592102 12 0.299342
Amount to 91.52146 17
As can be seen from Table 1: under different temperatures, between pholiota adiposa mycelium dry weight group is interior, difference is not remarkable, and each group difference is extremely remarkable.For further illustrating the difference between each group, then carry out multiple ratio between mean, the results are shown in Table 2.
The mycelium dry weight SSR check of table 2 different temperatures
As can be seen from Table 2, the mycelium dry weight difference between each temperature is all extremely remarkable, and mycelium dry weight 28 ℃ time is the highest, and mycelium dry weights obviously decline after 28 ℃, as can be seen here, and 28 ℃ of temperature that are best suited for mycelial growth.In conjunction with Peloton density growth curve, the temperature range that can be applicable to yellow umbrella growth is 19 ~ 28 ℃, and optimum temperature is 25 ~ 28 ℃.
2.2 pH: Fig. 5 shows that different pH cultivate the result of impact on yellow umbrella deep layer.
As can be seen from Figure 5, before pH5.0, mycelium dry weight increases gradually, and when pH5.0, mycelium dry weight is maximum, and then, along with the rising of pH, mycelium dry weight reduces gradually, and in pH4.0 ~ 6.0, mycelium dry weight maintains higher level; Peloton density also increases along with the increase of mycelium dry weight, reaches the highlyest when pH5.0, and Peloton density sharply reduces after pH6.0.As can be seen here, yellow umbrella is adapted at growing under slant acidity environment, tentatively can judge that its suitable pH is 4.0 ~ 6.0.In order to further illustrate the otherness of mycelium dry weight between different pH, it is carried out to variance analysis.
The pholiota adiposa mycelium dry weight variance analysis of the different pH of table 3
Difference source SS df MS F F (0.05) F (0.01) Significance
Between group 190.8548 6 31.80913 130.4999 2.847727 4.455842 **
In group 3.412476 14 0.243748
Amount to 194.2673 20
As can be seen from Table 3: under condition of different pH, between pholiota adiposa mycelium dry weight group is interior, difference is not remarkable, and each group difference is extremely remarkable.For further illustrating the difference between each group, then carry out multiple ratio between mean, the results are shown in Table 4.
The mycelium dry weight SSR check of the different pH of table 4
As can be seen from Table 4, during pH5.0, mycelium dry weight is maximum, but in pH5.0 ~ 6.0 o'clock, mycelium dry weight difference is not remarkable, 4.0 and 6.0 differences are extremely not remarkable, but 6.0 and 7.0 differences are extremely remarkable, in conjunction with Peloton density growth curve, can obtain, yellow umbrella is adapted at growing under slant acidity condition, and optimal pH is 5.0 ~ 6.0.
2.3 viscosity: Fig. 6 shows the impact that different viscosities is cultivated yellow umbrella deep layer
As seen from Figure 6, mycelium dry weight is maximum when viscosity 0.2%, and afterwards, mycelium dry weight constantly declines; And Peloton density also increases gradually at 0 ~ 0.2%, mycelium density is the highest to 0.2% time, and then along with the increase of viscosity, Peloton density reduces gradually, can tentatively judge thus, the agar that the range of viscosities that is applicable to yellow umbrella growth is 0 ~ 0.2%.As for Peloton density, after viscosity 0.2%, changing irregularly, is mainly due to the impact that is subject to viscosity, and the too high bacterium ball of viscosity can not precipitate fully, therefore the phenomenon rising has appearred 0.3,0.7% time in Peloton density.In order to further illustrate the otherness of mycelium dry weight between different viscosities, the mycelium dry weight under different viscosities condition is carried out to variance analysis.
The pholiota adiposa mycelium dry weight variance analysis of table 5 different viscosities
Difference source SS df MS F F (0.05) F (0.01) Significance
Between group 39.14639 7 5.592342 3.31325 2.657195 4.025935 *
In group 27.00595 16 1.687872
Amount to 66.15234 23
As can be seen from Table 5: under different viscosities, between pholiota adiposa mycelium dry weight group is interior, difference is not remarkable, and each group difference is remarkable.For further illustrating each group difference significance, then carry out multiple ratio between mean, the results are shown in Table 6.
The mycelium dry weight SSR check of table 6 different viscosities
As can be seen from Table 6, when mycelium dry weight is maximum, viscosity is 0.2%, but between them, difference is not remarkable, is therefore suitable for the viscosity of yellow umbrella deep layer cultivation for adding 0 ~ 0.5% agar; But from economic angle, consider, when making yellow umbrella liquid nutrient medium, needn't add agar.This cultivates from the deep layer of other edible mushrooms is different, is mainly because yellow umbrella self secretes a kind of mucosubstance in incubation, to have increased the viscosity of medium [4].
As can be seen from Figure 7, along with the increase of inoculum concentration, mycelium dry weight and Peloton density be corresponding increase all, but when 10% inoculum concentration, mycelium dry weight increasing degree is larger, and 15% time, Peloton density reaches maximum.Can tentatively judge, optimal inoculum concentration is 10% ~ 20%.In order to further illustrate the otherness of mycelium dry weight between different vaccination amount, the mycelium dry weight under different vaccination amount is carried out to variance analysis.
The pholiota adiposa mycelium dry weight variance analysis of table 7 different vaccination amount
Difference source SS df MS F F (0.05) F (0.01) Significance
Between group 71.99338 3 23.99779 33.33115 4.06618 7.590984 **
In group 5.759848 8 0.719981
Amount to 77.75323 11
As can be seen from Table 7: under different vaccination amount, between pholiota adiposa mycelium dry weight group is interior, difference is not remarkable, and each group difference is extremely remarkable.For further illustrating the difference between each group, then carry out multiple ratio between mean, the results are shown in Table 8.
The mycelium dry weight SSR check of table 8 different vaccination amount
As can be seen from Table 8, the mycelium dry weight significant difference of 20% and 15% inoculum concentration but do not reach utmost point significance level, 10% and 15% difference is not remarkable, considers economic factor, and 15% left and right is its suitable inoculum concentration.
2.5 bottled amount: Fig. 8 show the impact that different bottled amounts are cultivated yellow umbrella deep layer
As seen from Figure 8, when bottled amount is 90 ~ 180mL, mycelium dry weight changes little, and when bottled amount is 210mL, mycelium dry weight sharply increases and reaches maximum, during 240mL, declines to some extent; And Peloton density integral body is more steady, change little.Can preliminary judgement, the suitableeest bottled amount is 210 ~ 240mL/500mL.In order to further illustrate the otherness of mycelium dry weight between different bottled amounts, the mycelium dry weight of the bottled amount of difference is carried out to variance analysis.
The pholiota adiposa mycelium dry weight variance analysis of the different bottled amounts of table 9
Difference source SS df MS F F (0.05) F (0.01) Significance
Between group 386.6221 5 77.32441 508.2049 3.105875 5.064351 **
In group 1.825825 12 0.152152
Amount to 388.4479 17
As can be seen from Table 9: under different bottled amounts, between pholiota adiposa mycelium dry weight group is interior, difference is not remarkable, and each group difference is extremely remarkable.For further illustrating the difference between each group, then carry out multiple ratio between mean, the results are shown in Table 10.
The mycelium dry weight SSR check of the different bottled amounts of table 10
As can be seen from Table 10, when bottled amount is 210mL, mycelium dry weight is maximum, and significant difference between other bottled amount, therefore the bottled amount of 210mL/500mL is the bottled amount of the best of its Mycelium culture.This is mainly because yellow umbrella is to have a liking for oxygen bacterium, too high bottled amount impact ventilation, mycelia anoxic and poor growth; Too low bottled amount easily causes compared with large damage and affects its growth mycelia while shaking.
2.6 rotating speeds: Fig. 9 shows that different rotating speeds on the impact of yellow umbrella deep layer cultivation as seen from Figure 9, mycelium dry weight constantly increases along with the rising of rotating speed, during 150r/min, mycelium dry weight reaches the highlyest, between 150 ~ 180r/min, tends towards stability, on a declining curve after 180r/min; Peloton density is in rising trend when 90 ~ 150r/min, reaches the highlyest during 150r/min, and after 150r/min, Peloton density reduces gradually.Visible, the rotating speed of the most applicable Pholiota adiposa mycelia bulk-growth is 150 ~ 180r/min.In order to further illustrate the otherness of mycelium dry weight between different rotating speeds, the mycelium dry weight under different rotating speeds condition is carried out to variance analysis.
The pholiota adiposa mycelium dry weight variance analysis of table 11 different rotating speeds
Difference source SS df MS F F (0.05) F (0.01) Significance
Between group 17.47149 5 3.494298 219.6393 3.105875 5.064351 **
In group 0.190911 12 0.015909
Amount to 17.6624 17
As can be seen from Table 11: under different rotating speeds, between pholiota adiposa mycelium dry weight group is interior, difference is not remarkable, and each group difference is extremely remarkable.For further illustrating the otherness between each group, then carry out multiple ratio between mean, the results are shown in Table 12.
The mycelium dry weight SSR check of table 12 different rotating speeds
As can be seen from Table 12, between 150r/min and 180r/min, mycelium dry weight difference is not remarkable, but between they and other rotating speeds, difference is extremely remarkable, therefore can further determine that the rotating speed that the most applicable yellow umbrella is grown is 150 ~ 180r/min.This is mainly because rotating speed is too low, and dissolved oxygen affects mycelial growth not; Rotating speed too high to mycelial damage compared with large and be unfavorable for its growth.
3. conclusion:
By above result of the test, can be found out, the condition that the yellow umbrella deep layer of optimum is cultivated is: temperature is 25 ~ 28 ℃, and pH is 5.0 ~ 6.0, viscosity is for adding 0 ~ 0.5% agar (generally needn't add), inoculum concentration is 15% left and right, and bottled amount is 210mL/500mL, and rotating speed is 150 ~ 180r/min.
The research that the 5th joint, yellow umbrella liquid spawn fermentation tank are cultivated
1, material
1.1 bacterial strains: yellow umbrella, Shandong Province's edible mushroom work station provides
1.2 medium
1.2.1 slant medium: PDA
1.2.2 liquid nutrient medium: potato 20%, glucose 2%, yeast extract 0.2%, wheat bran 3%, MgSO 40.1%, KH 2pO 40.2%.
1.3 culture device: HZQ-Q type oscillator (Harbin Dong Lian Electronics Co., Ltd.), LS-B-50L type vertical pressure steam sterilization pan (Shanghai Medical Nuclear Instrument Factory) LRH-250-A type biochemical cultivation case (Guangdong medical apparatus and instruments factory) FA2004 type Shanghai electronic analytical balance (above electronic balance factory of Nereid section), accurate pH meter (Shanghai thunder magnetic), FMLT-52 type 5L desk-top fermentation cylinder (Guoqiang Biochemical Engineering Equipment Co., Ltd., Shanghai), 722 type spectrophotometers.
2, method
2.1 process routes: yellow umbrella preservation of bacteria strain → inclined-plane expands the research of cultivation → liquid spawn preparation (one-level shaking flask → secondary shaking flask) → 5L ferment tank
The preparation of 2.2 liquid spawns: it is 150mL that slant strains is inoculated in to 500mL(liquid amount) in conical flask, put into 25 ℃ of standing 24hr of insulating box, then at 25 ℃, the rotary shaking table shaken cultivation of 160r/min 5d, make liquid spawn standby (one-level shaking flask).In 500mL conical flask, pack 150mL medium into, the one-level shaking flask bacterial classification preparing is inoculated according to 10% inoculum concentration, at 25 ℃, the rotary shaking table shaken cultivation of 180r/min 4d, make liquid spawn standby (secondary shaking flask).
2.3 fermentation tank culture researchs:
2.3.1 5L fermentation tank culture: liquid amount 4L, rotating speed 160 r/min, throughput 6L/min, 25 ℃ of temperature, connect secondary shaking flask kind, inoculum concentration 10%, temperature is controlled at 25 ℃.Every 12hr sampling detects.
2.3.2 mycelium dry weight is measured: in fermentation process, by dry filter paper suction filtration for the whole culture fluids in each sample bottle, after repeatedly rinsing, be put in 60 ℃ of drying boxes and dry to constant weight with distilled water, measure dry weight.
2.3.3 reducing sugar test: film method is measured
2.3.4 pH value, DO pH-value determination pH: 5L fermentation tank is monitored automatically.
2.4 results and analysis
According to shake flask test result, carry out fermentation tank production test, with shake-flask seed, according to 10% inoculum concentration, being inoculated in fermentation tank cultivates, timing sampling observation is carried out in variation to the pH of fermentation process, DO value and mycelium production and reducing sugar, after getting, 5 batch datas obtain mean value, draw fermentation growth curve.As shown in Figure 10
2.4.1 fermentation tank culture mycelial growth curve
As seen from the figure, 0~24hr is the laundering period of yellow umbrella liquid spawn growth, and 36~60hr is in fast growing period, and growth quantity of mycelium significantly increases, and reducing sugar declines rapidly, enters the stable growth phase thereafter.After fermentation 72hr, mycelium production declines to some extent, and mycelium dry weight can reach 15.23mg/mL.
In incubation, pholiota adiposa mycelium is constantly grown, and forms bacterium ball, constantly consumes nutrient component in medium, and mycelium dry weight constantly raises, and at 60hr, reaches maximum, and now biomass is the highest, and it is thick that culture is.Continue fermented and cultured, mycelium senesces, autolyze, and biomass starts to decline.According to tracing analysis, fermentation termination should determine that in the time be 48~60hr.Although because now hypha biomass is not maximum, because the mycelia during this section is in the exponential phase that vitality is very vigorous, mycelia vigor is the strongest, after inoculation, mycelia is germination and growth very easily.
2.4.2 the change curve of pH value and oxygen dissolving value in fermentation process
5L fermentation tank culture, automatically record pH and dissolved oxygen change curve as Figure 11.
Yellow umbrella medium sterilization process makes pH decline approximately 0.5, and from shake flat experiment, the suitableeest initial pH is 5.5 left and right, thereby before sterilizing, the pH value of medium is adjusted to 6.0.From curve, the mycelial growth initial stage is because the peptone in the protease hydrolytic medium of thalline generation generates ammonium ion, make pH increase, during 40hr, pH appreciates the highest, along with the increase of biomass and the utilization of ammonium ion, and the accumulation of the organic acid in glucose utilization process declines pH, enter when exponential phase is 48hr and reduce as far as possible, along with exhausting of matrix, the increase of mycoprotein enzymic activity, in medium, amino nitrogen increases, and causes pH value to rise again, now mycelia be tending towards self-dissolving and metabolic activity a little less than.By dissolved oxygen curve, can be found out, before 48hr, dissolved oxygen is always in decline state, reaches minimumly during to 48hr, illustrates that now thalline is in exponential phase, and oxygen uptake rate is higher; After 48hr, dissolved oxygen rises to some extent, it's vigorous vegetative period has been past this explanation thalline, oxygen demand declines to some extent, starts to have secondary metabolite to form, and is also the period of biomass maximum, this and pH curve and the above dry weight curve that records thalli growth match, can tentatively judge thus, the incubation time of pholiota adiposa mycelium fermentation tank is about about 48hr, although that mycelium dry weight now can not reach is the highest, but bacterium ball is energetic, after inoculation, very easily sprout.Therefore controlled fermentation time well during fermentation tank culture, can pass through the pH of fermentation process and the observation of dissolved oxygen change curve simultaneously, judge that whether thalline is in suitable growing environment, this also shows under the primary condition of mycelial growth, need not to the pH of zymotic fluid and dissolved oxygen, control during the fermentation being suitable for.Thereby the cultivation of liquid spawn can be carried out in simple and easy fermentation tank.
3 discuss
The rule that in understanding thalli growth process, pH and dissolved oxygen change, on the one hand by the analysis to pH and dissolved oxygen variation, DO value low ebb period can increase dissolved oxygen by improving mixing speed or strengthening throughput approaching, make the required dissolved oxygen amount of thalli growth be not less than its critical value.On the other hand, can be used as the abnormal index of fermentation, while polluting aerobic property miscellaneous bacteria in fermentation, dissolved oxygen can drop near zero within a short period of time (2-5hr), after falling zero, do not go up for a long time, pH starts to decline very fast rather than rises, and can make like this our unusual circumstance early and takes measures.
The research of polysaccharide
The research of first segment, pholiota adiosapose polysaccharide extraction process
1. materials and methods:
1.1 materials:
1.1.1 bacterial classification: yellow umbrella, is provided by Shandong Province's edible mushroom work station.
1.1.2 mother culture media: add rich PDA: potato 20% glucose 2% wheat bran filtrate 5% peptone 0.5% V b10.001% KH 2pO 40.3% MgSO 40.25% agar 2%
1.1.3 one-level shaking flask medium: potato 20% glucose 2% peptone 0.25% KH 2pO 40.15% MgSO 40.25% V b10.001%
1.1.4 medicine: absolute ethyl alcohol, anthrone, sulfuric acid.
1.1.5 instrument and equipment: 722 type grating spectrophotometers (Shanghai analytical instrument factory), organize cracker, suction filtration device etc.
1.2 methods:
1.2.1 yellow destroying angel extraction method of polysaccharides:
Yellow destroying angel is pulverized à at 50 ℃ of drying in oven à and is weighed à hot water lixiviate à filtration à filtrate alcohol precipitation à fruitbody polysaccharide
1.2.2 the extraction of pholiota adiposa mycelium polysaccharide:
Zymotic fluid suction filtration à mycelium is pulverized à at 50 ℃ of baking oven inner drying à and is weighed à hot water lixiviate à filtration à filtrate alcohol precipitation à intracellular polyse
1.2.3 Methods in Determination of Polysaccaride Content:
The preparation of calibration curve solution: reference literature.
Sample determination: draw sample liquid 1.0mL, then add and prepare in advance to obtain anthrone-sulfuric acid solution (0.2g/100mL) 3.0mL, after mixing, put into rapidly frozen water, after it is cooling, put into again boiling water bath 10min, after taking out, put into immediately frozen water, after cooling, put room temperature and place 10min, at wavelength 620nm place, survey light absorption value, with distilled water, make blank.
1.1.4 the design of pholiota adiosapose polysaccharide Extraction technique:
Fruit body: solid-liquid ratio adopts 1:10,1:20,1:30, tetra-levels of 1:40, extraction temperature adopts 90 ℃, 100 ℃, 110 ℃, 120 ℃ four levels, and extraction time adopts 1h, 2h, 3h, tetra-levels of 4h to carry out respectively single factor experiment.
Mycelium: solid-liquid ratio adopts 1:20,1:25,1:30, tetra-levels of 1:35, extraction temperature adopts 80 ℃, 90 ℃, 100 ℃, 110 ℃ four levels, and extraction time adopts 1h, 2h, 3h, tetra-levels of 4h to carry out respectively single factor experiment.
2 results and discussion:
The mensuration of 2.1 polysaccharide:
Measuring the regression equation of polysaccharide calibration curve and recovery rate (mg/mL) equation of pholiota adiosapose polysaccharide is respectively:
Y=1.138X+0.0016
G=(Y-0.0016)2.5×10 5B/1.138
The light absorption value that Y----polysaccharide is measured at 620nm place;
Contained polysaccharide weight (ug) in every milliliter of filtrate of X-----;
The recovery rate of G-----polysaccharide (mg/mL);
Extension rate during B-----sample determination.
The impact of technological parameter on yellow destroying angel polysaccharide extract rate proposed in 2.2 hot dippings:
2.2.1 the impact of solid-liquid ratio: according to four levels of design, survey filtrate polysaccharide yield under 120 ℃, lixiviate 2h condition.Concrete outcome is shown in Figure 12.
As seen from Figure 12, the different yellow destroying angel polysaccharide extract rates of solid-liquid ratio have larger difference, along with the increase of solid-liquid ratio, pholiota adiosapose polysaccharide yield rises gradually, when solid-liquid ratio reaches 1:40, polysaccharide yield is the highest, and the larger solid-liquid ratio of this explanation contributes to the extraction of yellow destroying angel polysaccharide.
2.2.2 the impact of temperature: according to four levels of design, under the condition of solid-liquid ratio 1:40, lixiviate 2h, surveys filtrate polysaccharide yield.
Concrete outcome is shown in Figure 13:
Humid test result shows, along with temperature rises, yellow destroying angel polysaccharide extract rate rises gradually, in the time of 120 ℃, reach maximum, owing to being subject to condition restriction, cannot do the test higher than 120 ℃, add the words that improve again temperature on this temperature basis, desired appointed condition is very high, and the energy is caused to very large waste.Therefore under normal circumstances, 120 ℃ is the optimum extraction temperature of yellow destroying angel polysaccharide.
2.2.3 the impact of extraction time: under the condition of 120 ℃ of solid-liquid ratio 1:40, extraction temperature, extraction time the results are shown in Figure 14 to the impact of polysaccharide extract rate according to four levels of design.
As seen from Figure 14, extraction time is larger on the recovery rate impact of pholiota adiosapose polysaccharide.When 2h, reach maximum, its recovery rate is almost 2 times of 1h.Along with the prolongation of time, some polysaccharide can resolve into small-molecule substance and not easy-to-use ethanol precipitates, so along with the prolongation of extraction time, polysaccharide yield reduces on the contrary.
The impact of technological parameter on pholiota adiposa mycelium polysaccharide extract rate proposed in 2.3 hot dippings:
2.3.1 the impact of solid-liquid ratio: according to four levels of design, lixiviate 4h at 120 ℃, concrete outcome is shown in Figure 15:
Figure 15 shows, along with the rising of solid-liquid ratio, polysaccharide extract rate rises gradually, during to 1:30, reach the highest, but when solid-liquid ratio continues to raise, its polysaccharide yield increases hardly, considers the consumption of ethanol, the optimum condition that should select solid-liquid ratio 1:30 to extract for its polysaccharide.
2.3.2 the impact of temperature: according to four levels of design, lixiviate 4h under the condition of solid-liquid ratio 1:30, concrete outcome is shown in Figure 16:
Figure 16 shows, along with the rising of temperature, polysaccharide yield raises gradually, to 100 ℃, reaches the highest; But when temperature reaches 110 ℃, polysaccharide yield declines on the contrary, may be that too high some polysaccharide of temperature is degraded and polysaccharide yield is declined.Therefore 100 ℃ is the optimum temperature that its polysaccharide extracts.
2.3.3 the impact of time: under solid-liquid ratio 1:30,100 ℃ of conditions of extraction temperature, the impact that the time extracts yellow umbrella mycelial polysaccharides the results are shown in Figure 17 according to four levels of design.
Fig. 6 shows, along with the increase of extraction time, its polysaccharide extract rate increases, and reaches high extraction during 4h.Although along with the prolongation polysaccharide of time can be degraded, in the time of 100 ℃, the precipitation capacity of its polysaccharide is greater than its degradation amount, so polysaccharide yield can rise.This and fruitbody polysaccharide are different the extraction result of 120 ℃.
3 conclusions:
The test of some influence factors, has determined preferably extraction conditions when yellow destroying angel, mycelium polysaccharides are extracted.For fruit body, its optimum condition is solid-liquid ratio 1:40,120 ℃ of extraction temperatures, extraction time 2h; And mycelium, its optimum condition is solid-liquid ratio 1:30,100 ℃ of extraction temperatures, and extraction time 4h, its polysaccharide yield is the highest with this understanding.
Second section, pholiota adiposa mycelium polysaccharide delay the research of in vitro gastrocnemius fatigue
1 material
The yellow agaric kind of 1.1 medicines, is provided by Shandong Province's edible mushroom work station.Adopt liquid cultivating method, filter out preferably carbon source, nitrogenous source, design three factors, the orthogonal experiment of four levels, filters out the optimum medium of yellow umbrella, according to optimal case, cultivates, and obtains pholiota adiposa mycelium.Polysaccharide extracts in pholiota adiposa mycelium, mycelium after dried, crushed, by ratio of water to material 15:1 carry out filtering after water extraction, centrifugal, then extract is concentrated, with absolute ethyl alcohol, extract and obtain water-soluble polysaccharide.Reference production standard curve is pressed in thick measurement of the polysaccharide content, and the content that utilizes anthrone colorimetric method for determining to go out pholiota adiposa mycelium polysaccharide is 9.4525%, is mixed with the polysaccharide solution of 1mg/ml, 0.2mg/ml, 0.02mg/ml before experiment with ringer solution.Other reagent are commercially available chemical pure and analyze pure.
1.2 laboratory animal and grouping: bufo gargarizans Cantor (Bufo bufo gargarizans), 30, body weight 70-90g, male and female dual-purpose.Experiment is divided into control group and administration group at random by two specimen-gastrocnemius muscle of same toad.
1.3 instrument Lms-2BXing bis-road physiology record instrument (Sichuan Province Chengdu Instruement Factory product), SDQ-2 type two pass electronic stimulator (Bengbu, Anhui Province practical technique research institute product), muscle tone transducer (Hebei Gaobeidian city Xin Hang mechanical & electronic equipment corporation, Ltd product).The upper ware electronic balance of FA2004.Ma * 200g-(Shanghai City Jing Ke balance factory product).
2 methods
2.1 preparation of specimen
2.1.1 toad pair is ruined after marrow, separated gastrocnemius, removes sciatic nerve, and muscle connects sciatic nerve end and heel string end is pricked upper fine rule, is dipped in 10min in ringer solution.
2.1.2 a sample is inserted in the paraffin flesh groove that fills 20ml ringer solution, as a control group; Another sample is inserted and is filled 20ml containing in the paraffin flesh groove of polysaccharide solution, as administration group, soaks 20min simultaneously.Flesh groove temperature remains on 20-23 ℃.
2.2 contraction of muscle abilities are measured
The muscle bone nerve end of being punished for being related to is fixed on flesh trench bottom with fine rule, the fine rule of its heel string end is connected on tonotransducer, regulates muscle to trace as baseline in constant tension state, with stimulating electrode, touches muscle surface, giving continuous square wave stimulates, and on Er road instrument, records muscle curve.Stimulation parameter: DC 20v; The wide 0.3ms of ripple; Frequency of stimulation 1HZ
2.3 data processing
Record the time (t that shrinkage amplitude drops to maximum shrinkage amplitude 90% 0.9) drop to 50% time (t 0.5) and drop to 10% time (t 0.1).Contraction of muscle time when record stimulates 300S, diastolic time, and calculate the ratio (t of diastolic time and ST 300relax t 300contracting).Record be take S as unit, data with ± S represents, and carries out significance test of difference with paired t-test.
3 results
3.1 impacts of pholiota adiposa mycelium polysaccharide on the contraction of muscle duration:
Middle concentration group (0.2mg ml) can extend the contraction of muscle duration, at t 0.5time, with control group there was a significant difference meaning (p<0.05), at t 0.1time have a utmost point significant (p<0.01). therefore there is obvious delay fatigue effect (in Table 1).
The affect X ± S of table 1 pholiota adiposa mycelium polysaccharide on the contraction of muscle duration
Group n t 0.9 t 0.5 t 0.1
Control group 10 75.5±12.1 203.7±24.8 530.5±33
Low concentration group 10 92.0±7.2* 241.3±18.6* 608.7±47*
Control group 10 69.5±13.7 219.7±21.5 574.6±41.1
Middle concentration group 10 84.5±12.5* 285.7±13.6** 863.5±53.0**
Control group 10 76.7±16.03 221.0±21.86 552.2±44.8
High concentration group 10 79.0±21.2 240.9±24.8 591.4±67.9
* than P<0.05 * *, compare P<0.01 with control group with control group
3.2 pholiota adiposa mycelium polysaccharide are subject to the impact of electro photoluminescence contractility in the time of 300 seconds on muscle:
At in vitro gastrocnemius, be subject to electro photoluminescence in the time of 300 seconds, the polysaccharide of three concentration groups can improve t 300relax/t 300contracting ratio (table 2), the difference of high concentration group and control group has significant (P < 0.05).Show that pholiota adiposa mycelium polysaccharide has antifatigue effect.
Table 2 pholiota adiosapose polysaccharide is subject to the impact (X ± S) of electro photoluminescence contractility in the time of 300 seconds on muscle
Group n ST Diastolic time t 300Relax/t 300Contracting
Control group 10 0.091±0.035 0.909±0.035 11.51±4.86
Low concentration group 10 0.109±0.032 0.891±0.032 8.86±2.88
Control group 10 0.098±0.32 0.901±0.031 10.18±3.83
Middle concentration group 10 0.135±0.051 0.86±0.049 7.74±4.41
Control group 10 0.077±0.042 0.94±0.022 17.92±7.97
High concentration group 10 0.093±0.036 0.903±0.042 11.42±4.96*
* with control group than P < 0.05
4 discuss
The mechanism occurring about fatigue, Edward is grown (Edwards) nineteen eighty-three and has been proposed Quality Initiative theory, think that body is when moving, from central nervous system, spinal cord, neuromuscular tie-point until each subcellular structure of work flesh exists a kind of dynamic connection of integral body, as a chain, if while a certain local link of Quality Initiative being interrupted because of a certain or several factors. just may be because of the fatigue that causes out of hand.The effect of pholiota adiposa mycelium polysaccharide antifatigue may be also multidigit point.This experimental study shows: the fatigue of contraction that pholiota adiposa mycelium polysaccharide causes under the condition of electro photoluminescence isolated toad gastrocnemius sample in certain concentration range has retarding action.Show as the lasting ST of administration group muscle specimen and be longer than control group, tentatively illustrate that pholiota adiposa mycelium polysaccharide can act on this link of Skeletal Muscle Cell, by affecting myocyte's functional activity, bring into play antifatigue effect.
Contraction of muscle performance depends on institutional framework and the functional status of itself.A lot of experiments show, muscle continues violent contraction and brings out generation active oxygen, can cause in muscular tissue the functional disorders such as sarcoplasmic reticulum contractile protein.Modern medicine thinks, radical damage is one of major reason of muscular fatigue, and degree of injury is relevant with exercise intensity, and superoxide dismutase (SOD) can be removed the damage that radical pair body causes.There is bibliographical information [4], lycium barbarum polysaccharide can increase the SOD level in blood and muscle, mouse is exhausted to the recovery of motion is favourable.Aged mouse is at continuous gavage Ganoderma Polysaccharide after 10 days, SOD content in Mouse Blood red blood cell, liver cell, brain cell is compared all obviously raising this paper pholiota adiosapose polysaccharide the contractility of in vitro muscle is had to humidification with control group, delay fatigue, its mechanism also may be relevant therewith.
Another major reason of tired generation is the disorder of interior environment, as pH reduces, muscular tissue dehydration, causes the variation of osmotic pressure and electrolyte concentration and causes fatigue.Originally studies show that, the thick polysaccharide of higher concentration is compared with control group, can shorten muscle and reach the tired time, analyzing its reason may be the osmotic pressure of polysaccharide Change Example culture fluid in isolated experiment, make skeletal muscle tissue dehydration, in extracellular fluid, various ion concentrations change, and have broken the stable state of environment in myocyte, also myocyte's the function that may be other various composition influences of containing in thick polysaccharide, has accelerated the fatigue of muscle.In experiment afterwards, should, further by polysaccharide purification, observe the tired impact that sterling polysaccharide causes under electro photoluminescence isolated toad gastrocnemius.
The research of yellow umbrella liquid strain cultivation system
1, the research of different culture media on Pholiota adiposa mycelia growth and fruiting impact
1 materials and methods
1.1 material
1.1.1 for examination bacterial classification: yellow umbrella, Yin Zi Shandong Province edible mushroom work station.
1.1.2 mother culture media: take respectively potato, glucose, sucrose, brown sugar, peptone, agar etc. does mother culture media by different proportion as main component, sterilizing 20min at 121 ℃, under aseptic condition, to entering in the culture dish of sterilizing, concrete formula is as table 1:
Table 1 Mother culture based formulas
Formula Potato Wheat bran Glucose Sucrose Brown sugar Peptone KH 2PO 4 MgSO 4 V B1 Agar
1 20 2 0.5 0.25 0.15 0.001 2
2 20 1 1 0.5 0.25 0.15 0.001 2
3 20 2 0.5 0.25 0.15 0.001 2
4 20 2 0.5 2
5 30 2 0.5 0.25 0.15 2
6 20 2 0.5 0.001 2
Note: above content is mass percent.Lower same.
1.1.3 liquid nutrient medium: take potato, glucose, brown sugar, peptone, yeast extract, wheat bran etc. does liquid nutrient medium as main component by different proportion, packs 500mL conical flask into, and every bottle approximately fills 150mL, sterilizing 30min at 121 ℃, concrete formula is as table 2:
Table 2 liquid culture based formulas
Formula Potato Glucose Brown sugar Peptone Yeast extract Wheat bran KH 2PO 4 MgSO 4 V B1
1 20 2 0.5 0.25 0.15 0.001
2 20 2 0.5 0.25 0.15 0.001
3 20 2 0.5 0.25 0.15 0.001
4 20 2 5 0.25 0.15 0.001
1.1.4 planting material medium: choose conventional raw material cotton seed hulls, wood chip is basis, prepares in varing proportions planting material, material: water=1:1.3, packs in Cans thereafter, sterilizing 2hr at 121 ℃, every bottle approximately fills siccative 0.11Kg, specifically fills a prescription in Table 3:
Table 3 planting material culture medium prescription
Formula Cotton seed hulls Wood chip Wheat bran Quicklime White sugar
1 78 20 1 1
2 50 28 20 1 1
3 28 50 20 1 1
4 78 20 1 1
5 39 39 20 1 1
1.2 method
1.2.1 the screening of mother culture media: meet a 0.5cm under aseptic condition 2the bacterial classification of size, in flat board, is placed in 25 ℃ of incubators and cultivates, and establishes 10 repetitions, observes and record the upgrowth situation of mycelia for every group.
1.2.2 the screening of liquid nutrient medium:
Under aseptic condition, by inoculation shovel inoculation shaking flask, every bottle approximately meets 4 ~ 5 0.5cm 2the mycelium of size, makes mycelium float on liquid level, is put in standing cultivation 24hr in 25 ℃ of incubators, is then put in 25 ℃ of shaking tables and cultivates, and rotating speed is 160r/min, establishes 5 repetitions for every group, observes the upgrowth situation of bacterium ball.
(1), after there is bacterium ball, under every day aseptic condition, random bacterium ball of picking is inoculated on the flat board of PDA and is placed under 25 ℃ of constant temperature and cultivates.Observe the growth rate of bacterium ball on flat board, to determine bacterium ball vigor size, measure mycelial percent by volume and mycelial dry weight is determined mycelial growth amount, thus definite fermentation termination.
(2) after fermentation ends, zymotic fluid is poured into standing 30min in the graduated cylinder of 200mL, observed the shared volume ratio of bacterium ball.
(3) dry to constant weight among bacterium ball being inserted after filtering fermentation liquor to the baking oven of 60 ℃, weigh mycelium dry weight.
1.2.3 the screening of planting material medium:
To under the slant strains aseptic condition of having grown, be connected in Cans one of every bottle graft, every about 1cm 2size, is put in the culturing room of 25 ℃ and cultivates, and establishes 40 repetitions for every group, observes and record upgrowth situation, pollution rate and the full bottle number of days etc. of mycelia.
1.2.4 fruiting experiment:
The mushroom canopy that the Cans of sending out full bacterium is placed in to 15 ℃ of left and right is managed fruiting, observes and record the indexs such as aobvious flower bud time, fruiting time, fruit body proterties and biological transformation ratio.
2 results and analysis
The screening of 2.1 mother culture medias: the growing state of mycelia on different mother culture medias is as shown in the table:
The growing state of the yellow umbrella of table 4 on different mother culture medias
Experimental group Mycelia form Colony diameter (cm) Average daily long speed (cm/d)
1 Yellow, dense 5.34 0.54
2 Shallow white, sparse 5.16 0.53
3 Shallow white, sparse 4.65 0.48
4 Yellow, dense 4.57 0.47
5 White, denseer 4.05 0.45
6 Pale yellow, dense 5.87 0.58
6 the effect of filling a prescription is as can be seen from the above results best, next is 1,2, has proved that the effect of use brown sugar is best, and this explanation Pholiota adiposa mycelia is suitable for lower C/N ratio, higher C/N ratio is unfavorable for its growth on the contrary, and this conclusion and the C/N ratio result of the test that we do match.In addition because brown sugar composition is not single, the different kinds of ions itself containing can meet the demand of mycelial growth, therefore do not add mineral salt mycelia, still grow finely, and the price of brown sugar is relatively cheap, therefore should not selects the medium that conventional phosphorus content is high to do mother culture media.So group 6 is the best mother culture medias that filter out.
The screening of 2.2 liquid nutrient mediums:
(1) mensuration of yellow umbrella fermentation termination: the mensuration of fermentation termination divides mycelial growth amount and two indexs of growth vigor to measure, concrete data are in Table 5:
The mensuration of table 5 amount of growth and growth vigor
Project The 5th day The 6th day The 7th day The 8th day The 9th day
The shared volume ratio of bacterium ball (%) 39.3 55.2 86.2 100 91.6
Mycelium dry weight (g) 0.6881 0.6921 1.0625 1.3268 1.1981
Sprout time (hr) 10 9 7 8 9
Growth rate (cm/d) 2.23 2.73 4.07 3.82 3.06
As can be seen from the above table, before 8d, mycelium production is always in rising trend, to 8d mycelium production, reach the highest, thereafter the prolongation mycelium production along with the time starts to decline, this explanation mycelia starts self-dissolving, and bacterium ball vigor is the highest at 7d, so its fermentation termination should be 7 ~ 8d, if be used for, do bacterial classification, select 7d for best; If be used for, produce mycelium, select 8d for best.
(2) the bacterium ball growing state of each formula: measure the growing state of bacterium ball when fermentation termination, filter out liquid nutrient medium optimization formula, in Table 6
The bacterium ball growing state of table 6 different liquids medium
Formula The shared volume ratio of bacterium ball (%) Mycelium dry weight (g) Bacterium nodule number (/mL) The bacterium nodule number of φ >=2mm (individual/mL) Pellet form
1 100 1.3268 29 29 Little and even
2 96.4 1.1803 14 13 Not of uniform size
3 100 1.3304 27 27 More even
4 100 1.3487 Cannot count Cannot count Very little and muddy
As seen from the above table, the effect of formula 1 and formula 3 is all good, no matter from mycelium dry weight or bacterium nodule number amount, the two difference is little, and the glucose in formula 1 and the price of peptone will be far away higher than brown sugar and yeast extracts in formula 3, though the dry weight of formula 4 is heavier, but its fermentation time is longer, and the individuality of bacterium ball is especially little, is unfit to do bacterial classification, therefore fill a prescription 3 for optimization formula.
The screening of 2.3 planting material medium:
(1) bacterial classification at the growing state of the planting material of various formulas in Table 7
The growing state of table 7 mycelia on different planting material medium
Formula Sprout time (hr) Mycelia form Average daily long speed (cm/d) The full bottle time (d) Pollution rate (%)
1 22 ++ 0.22 39 5.0
2 19 +++ 0.26 36 2.5
3 16 ++++ 0.31 32 2.5
4 14 + 0.20 41 10.0
5 17 ++++ 0.27 35 7.5
Note: ++++representing that mycelial growth is sturdy, dense, color and luster is yellow,
As can be seen from the above table: although slow its average daily long speed of the sprout time of formula 3 is the fastest, full bottle shortest time and pollution rate are minimum, and although formula 4 sprouts the fastest but growing way is the poorest, the long speed of day bacterium is the slowest and pollution rate is the highest, this illustrates that yellow umbrella is a kind of aerobic wood saprophytic bacteria, wood chip is its suitable growth raw material, but because pure wood chip gas permeability is very poor, be unfavorable for its growth, therefore want the better and more rich cotton seed hulls of nutrition of more additional gas permeabilities to make raw material, the putting in order of indices of this point mycelial growth from five kinds of formulas also can be found out.
(2) the fruiting situation of each formula is in Table 8:
The fruiting situation of the different planting material medium of table 8
Formula Budding the time (d) Average yield (Kg/ bottle) Gross yield (Kg) Biological efficiency (%)
1 49 0.10 3.80 91
2 45 0.12 4.68 109
3 41 0.15 5.85 136
4 51 0.14 5.04 127
5 44 0.13 4.81 118
As seen from the above table, the biological efficiency of formula 3,4 is higher, illustrate that wood chip is the suitableeest culturing raw material of yellow umbrella, but as can be seen from Table 7, the long speed of formula 4 is the slowest, the full bottle time is also the longest, therefore wood chip and cotton seed hulls also should keep certain proportioning just can make the growth of its mycelia and biological efficiency reach best, in set formula, can find out that the indices of formula 3 is for best, therefore formula 3 is best plant formulation.
3 conclusions
As can be seen from the test results, yellow umbrella the most practical most economical Mother culture based formulas is: potato 20%, brown sugar 2%, peptone 0.5%, Cobastab 10.001% ,agar 2%; Liquid culture based formulas is: potato 20%, brown sugar 2%, yeast extract 0.5%, KH 2pO 40.25%, MgSO 4,0.15%, Cobastab 10.001%; The optimization formula of planting material is: cotton seed hulls 28%, wood chip 50%, wheat bran 20%, white sugar 1%, quicklime 1%.
Certainly, about the formula of yellow umbrella medium, we just conduct a preliminary study, as for further research after whether existing medium preferably and formula to need.
2, yellow umbrella liquid spawn sends out the research of bacterium feature and planting type
1. materials and methods
1.1 for the yellow umbrella of examination bacterial classification, and Yin Zi Shandong Province edible mushroom work station, makes respectively test tube kind and liquid strain.
1.2 cotton seed hulls medium: cotton seed hulls 86%, wheat bran 10%, white sugar 1%, quicklime 3%.
The production of 1.3 seeding tank kinds
The formula of liquid nutrient medium: potato 20%, brown sugar 1.5%, glucose 1%, wheat bran 3.5%, peptone 0.2%, KH 2pO 40.2%, MgSO 40.1%, V b10.0001%, bubble enemy 0.03%, by formula, medium prepared, pack in fermentation tank, and bacterium 0.5hr at 121 ℃, the shaking flask kind that the access of sterile working has after cooling been grown, inoculum concentration is to cultivate 4 ~ 5 days at 10 ~ 15%, 25 ℃.
1.4 method
Composts or fertilisers of cultivating is added to water by formula and mix, material: water=1:1.2 ~ 1.4, stewing heap 1hr, makes moisture soak into composts or fertilisers of cultivating, pack thereafter, sack specification is: 17cm * 33cm * 0.04cm.Every bag of middle part, make a call to the aperture of a diameter 1.5 ~ 2cm, sterilizing 2hr at 121 ℃.Cooling inoculation after sterilizing thoroughly, sterile working during inoculation.Every bag of liquid spawn meets 10 ~ 15ml, and every of test tube kind connects 5 ~ 6 bags, respectively connects 100 bags, puts into 25℃ culturing room and cultivates, and observes mycelium germination situation, and line measures mycelial growth rate, repeats 3 times, records purseful number of days and statistics pollution sack number.
The screening of 1.5 high-yield culturing modes
The bacterium bag of sending out full mycelia is moved in booth and carried out fruiting experiment, adopt following three kinds of modes to cultivate: the sleeping covering method that buries of (1) de-bag; (2) the vertical covering method that buries of de-bag section; (3) the vertical covering method that buries of de-bag.In triplicate, 30 bags of each random samplings are arranged in furrow in every processing.Furrow cuckoo lattice are long 6.5m, wide 0.9m, dark 0.3m.Bag is spaced apart 1 ~ 2cm with bag, with fine earth, fills, and then fills with flood once.After water infiltration, cover Nutrition Soil 1 ~ 2cm.The formula of Nutrition Soil is soil: sand=7:3, more additional 1/10000 Nitrogen, Phosphorus and Potassium (as vegetable garden soil or humus soil, can not add Nitrogen, Phosphorus and Potassium).During fruiting, temperature of shed is controlled at 5 ~ 20 ℃ as far as possible, and relative air humidity rests in 85% ~ 95%.The fruiting situation of every kind of planting type of observed and recorded, fruiting output and fruit body quality etc.
2. result
The difference of 2.1 bacterium features
2.1.1 the difference of sprout time
Pholiota adiposa mycelia is sprouted slower, and the time of sprouting after liquid spawn picking bag is 24hr, and the material feeding time is 60hr, and the time that test tube kind is sprouted is 48hr, and the material feeding time is 96hr.
2.1.2 the difference of mycelial growth rate
It is slow that the more general edible mushroom of Pholiota adiposa mycelia growth rate is wanted, growth rate average every day of the 4.5mm of liquid spawn mycelia, and solid spawn mycelial growth rate is 2.5mm
2.1.3 the difference of bacterial classification purseful number of days
Yellow umbrella liquid spawn purseful number of days is 33 ~ 35 days, and the fastest 28 days is purseful, average 32 days.Solid spawn needs 45 ~ 50 days, average 46 days.Differ over 10 days.
2.1.4 the difference of pollution rate
Yellow umbrella add up in 100 bags, only chosen 4 bags of pollutions, pollution rate 5% left and right.And in 100 bags of solid spawn, having 11 bags of pollutions, pollution rate is 11% left and right.Specifically see the following form:
Liquid spawn and solid spawn send out bacterium comparison
The difference of 2.2 planting types
The following table that the results are shown in by three kinds of training methods of cotton seed hulls composts or fertilisers of cultivating planting pholiota adipose.As can be seen from the table, vertical to bury Casing ways the highest to take off bag for biological efficiency.Reach 136.0%, but from fruit body proterties, difference is not remarkable.Different planting types is difference slightly, and wherein to stand, to bury the fruit body proterties of earthing formula best.
The contrast of the yellow umbrella output of different planting
Planting type Average every bag of output (Kg/ bag) Biological efficiency (%) Cap amount/stem amount
The sleeping covering method that buries 0.686 91.5 10.93
The disconnected covering method that buries 0.910 121.3 11.55
The vertical covering method that buries 1.020 136.0 15.21
3. discuss
Because liquid spawn has mobility, its mycelium pellet and mycelia fragment can be scattered and be sprouted at the different parts of bacterium bag, and growth point is many, sprouts fast.Therefore after inoculation, mycelia can grow by the different parts rapid spread in bag, has so just greatly reduced the chance that miscellaneous bacteria infects, and has reduced pollution rate.Thereby just shortened a bacterium time, thus just there is listed situation in table, thus the whole production cycle was shortened about 10 days.
Low form kind during yellow umbrella belongs to, therefore be applicable to autumn, winter, cultivation in the early spring.Under fruiting condition, temperature is lower, and fruit body color and luster is more golden yellow, meat is thick, and this should be noted that in the arrangement of yellow umbrella planting time.
The yellow umbrella of cottonseed shell cultivation will obtain good quality and high output, not only needs rational culture material formula and processing method, but also will have with it supporting high-yield culturing mode.Fruiting body yield is not only relevant with breediness, and obviously relevant to planting type.Stand and bury the Quality and yield that covering method can significantly improve yellow destroying angel, this may be relevant with mycelial growth direction, but also need further discussion.
accompanying drawing explanation
Fig. 1 is different nitrogen sources and the affect figure of different carbon source on mycelia; Fig. 2 is the affect figure of carbon source concentration on the cultivation of mycelium deep layer; Fig. 3 is the affect figure of nitrogen concentration on the cultivation of mycelium deep layer; Fig. 4 is that different temperatures is cultivated the result figure of impact on yellow umbrella deep layer; Fig. 5 is the affect figures of different PH on the cultivation of pholiota adiposa mycelium deep layer; Fig. 6 is the affect figure of different viscosities on the cultivation of mycelium deep layer; Fig. 7 is the affect figure of inoculum concentration on yellow umbrella deep layer cultivation; Fig. 8 is the affect figure of bottled amount on yellow umbrella deep layer cultivation; Fig. 9 is the affect figure of rotating speed on yellow umbrella deep layer cultivation; Figure 10 is the variation diagram of yellow umbrella 5L fermentation tank biomass and reducing sugar; Figure 11 is the variation diagram of PH and dissolved oxygen in yellow umbrella fermentation process; Figure 12 is the affect figure of solid-liquid ratio on yellow destroying angel polysaccharide extracting process; The affect figure of Figure 13 extraction temperature on yellow destroying angel polysaccharide extracting process; The affect figure of Figure 14 extraction time on yellow destroying angel polysaccharide extracting process; Figure 15 is the affect figure of solid-liquid ratio on yellow destroying angel polysaccharide extracting process; Figure 16 is the affect figure of extraction temperature on yellow destroying angel polysaccharide extracting process; Figure 17 is the affect figure of extraction time on yellow destroying angel polysaccharide extracting process.

Claims (10)

1. the research of yellow umbrella liquid spawn and polysaccharide, it is characterized in that yellow umbrella liquid nutrient medium the screening of suitable carbon nitrogen source, the research of bacterium feature and planting type is sent out in the research that the impact that the orthogonal test research of yellow umbrella liquid nutrient medium, different concentration of carbon and nitrogen sources are cultivated yellow umbrella deep layer, the research of yellow umbrella deep layer condition of culture, yellow umbrella liquid spawn fermentation tank are cultivated, the research of pholiota adiosapose polysaccharide extraction process, the research of pholiota adiosapose polysaccharide antifatigue, different culture media on Pholiota adiposa mycelia growth and the impact of fruiting, yellow umbrella liquid spawn.
2. the yellow umbrella liquid nutrient medium as claimed in claim 1 screening of suitable carbon nitrogen source, it is characterized in that by the test to the relevant formula of design, filter out carbon source, nitrogenous source and the C/N ratio of the most applicable yellow umbrella growth, result is that optimum carbon source is glucose, optimum nitrogenous source is peptone, wheat bran, beef extract, and optimum C/N ratio is 40:1.
3. the orthogonal test research of yellow umbrella liquid nutrient medium as claimed in claim 1, it is characterized in that filtering out preferably carbon source, nitrogenous source, design the orthogonal experiment of three factor four levels, screen the optimum medium of yellow umbrella, optimum medium is potato 20%, brown sugar 1%, yeast extract 0.2%, MgSO40.1%, KH2PO40.2%, VB10.001%, by this testing program, cultivate, the output of pholiota adiposa mycelium is 17.52g/L.
4. the impact that different concentration of carbon and nitrogen sources as claimed in claim 1 is cultivated yellow umbrella deep layer, it is characterized in that the carbon source concentration that suitable yellow umbrella deep layer is cultivated is 2.0%, 3.0% and 2.5%, suitable nitrogen source concentration is 0.3%, 0.1% and 0.2%, and optimum medium is potato 20%, glucose 2.0%, peptone 0.1%, MgSO40.10%, KH2PO40.20%, vitamin B1 0.001%.
5. the research of yellow umbrella deep layer condition of culture as claimed in claim 1, it is characterized in that by yellow umbrella strain cultivation being carried out to the setting of different temperatures, different pH value, different rotating speeds, different bottled amount, different viscosities and different vaccination amount, the optimum condition that records yellow umbrella deep layer cultivation is: temperature is 25 ~ 28 ℃, pH is 5.0 ~ 6.0, rotating speed is 150 ~ 180r/min, bottled amount is 210mL/500mL, and viscosity is for adding 0 ~ 0.5% agar (generally needn't add), and inoculum concentration is 15% left and right.
6. the research that yellow umbrella liquid spawn fermentation tank as claimed in claim 1 is cultivated, is characterized in that by finding out the test of 5L fermentation tank, the pH value that yellow umbrella deep layer is cultivated first rises and declines afterwards, rises again thereafter; Dissolved oxygen is along with mycelial growth declines gradually, after minimum point, start again slow rise, mycelial dry weight is the trend of rising always, reaches after maximum and just no longer increases, reducing sugar declines always, and the terminal that can tentatively judge thus yellow umbrella fermentation is 60hr left and right.
7. the research of pholiota adiosapose polysaccharide extraction process as claimed in claim 1, is characterized in that result of study shows, the optimum condition that fruitbody polysaccharide extracts: solid-liquid ratio 1:40,120 ℃ of extraction temperatures, extraction time 2h; And the optimum condition that mycelium polysaccharides extracts: solid-liquid ratio 1:30,100 ℃ of extraction temperatures; Extraction time 4h.
8. the research of pholiota adiosapose polysaccharide antifatigue as claimed in claim 1, it is characterized in that yellow umbrella high concentration group (1mg/ml) polysaccharide can extend maximum shrinkage amplitude and drop to for 10% time, middle concentration group (0.2mg/ml) polysaccharide and low concentration group (0.02mg/ml) polysaccharide can extend amplitude peak and drop to for 90%, 50%, 10% time, therefore, pholiota adiposa mycelium polysaccharide has delay fatigue effect.
9. the impact of different culture media as claimed in claim 1 on Pholiota adiposa mycelia growth and fruiting, it is characterized in that the upgrowth situation on different culture media according to yellow umbrella, screened the mother culture media that is suitable for its growth, liquid nutrient medium and planting material medium, result shows, the optimization formula of mother culture media is: potato 20%, brown sugar 2%, peptone 0.5%, vitaminB10 .001%, agar 2%, the optimization formula of liquid nutrient medium is: potato 20%, brown sugar 2%, yeast extract 0.5%, KH2PO4 0.25%, MgSO4 0.15%, vitaminB10 .001%, the optimization formula of planting material medium is: cotton seed hulls 28%, wood chip 50%, wheat bran 20%, white sugar 1%, quicklime 1%.
10. yellow umbrella liquid spawn as claimed in claim 1 sends out the research of bacterium feature and planting type, the sprout time, growth rate, purseful time and the pollution rate that it is characterized in that yellow umbrella liquid spawn are clearly better than solid spawn, and the planting type of yellow umbrella liquid spawn is best to take off the vertical mode of burying of bag.
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Cited By (5)

* Cited by examiner, † Cited by third party
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104478547A (en) * 2014-11-03 2015-04-01 河北大学 Method for utilizing vitex negundo var ineica scrap to culture pholiota adiposa
CN104940242A (en) * 2015-06-01 2015-09-30 吉林农业大学 Pleurotus citrinopileatus Singer polysaccharide protein compound, preparation method and use thereof
CN105368895A (en) * 2015-10-10 2016-03-02 南京农业大学 Method for preparing dinghu scale toadstool intracellular and extracellular polysaccharide with antioxidant activity
CN111187723A (en) * 2019-09-30 2020-05-22 贵州科学院 Method for producing mycelium and extracellular polysaccharide by liquid culture of large-cap small-skin umbrella
CN113637594A (en) * 2021-10-18 2021-11-12 山东蓬勃生物科技有限公司 Pholiota adiposa strain YX1, culture method and application thereof

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