CN104710206A - Preparation method for volvariella volvacea liquid strain - Google Patents
Preparation method for volvariella volvacea liquid strain Download PDFInfo
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Abstract
Disclosed is a preparation method for volvariella volvacea liquid strain. Volvariella volvacea is a kind of mushroom which grows in tropical and subtropical areas in hot summer, and is an important edible fungus in our country. The yield of the volvariella volvacea in our country is the top in the world, and the volvariella volvacea mainly grows in provinces such as Guangdong and Fujian in our country. In recent years, production of volvariella volvacea dramatically develops in East China and North China zones. The names of strains are relatively confused, and the conventional seed production and cultivation methods at the present can't satisfy the need of an edible fungus product in the market. A large-scale industrial production mode for producing some edible fungus mycelia has been a new way of edible fungus production, and thus fermentation technology applied to an edible fungus is born at the right moment. For screening excellent varieties suitable for cultivation and preferable production guide, comprehensive research is carried out to six normal strains in a certain zone, wherein the research is about characteristics, such as a growth speed of a hypha, antagonism between strains, relative activity of a MC enzyme, isozyme and fungus resistance, of the six strains.
Description
Technical field
A method prepared by straw mushroom liquid strain, belongs to edible mushroom cultivation field.
Background technology
Straw mushroom [Volvariella volvacea (Bull. ex Fr.) Sing.], has another name called luxuriant pin mushroom, Volvariella volvacen, numb mushroom, bar mushroom, Nanhua mushroom, tribute mushroom, the raw mushroom of family etc., is abroad " Chinese mushroom " also known as straw mushroom.Be under the jurisdiction of Mycophyta (Eumycota), Basidiomycetes (Basidiomycetes), Agaricales (Agaricales), Guang Bing mushroom section (Pluteaceae), luxuriant pin mushroom belongs to (Volvariella).Straw mushroom is the edible mushrooms that subtropical and tropical zones is extensively cultivated.It is delicious flavour not only, and nutritive value is very high.According to surveying and determination, dried agaric mushroom protein content is 37.13%, apparently higher than general vegetables, the fresh straw mushroom of every hectogram is containing Catergen 06.27 milligram, the shaddock more famous than rich vitamin C, orange, tomato, capsicum are all high, occupy first of fruit and vegetable, in addition, also containing the material such as 17 seed amino acids of 8 seed amino acids comprising needed by human in straw mushroom.
Straw mushroom is cold in nature, taste is sweet, have invigorate the spleen and benefit qi, clear heat reduces phlegm and internal heat, the fertile child of hair-cream, protect effect of liver and strengthening stomach and removing toxic substances, often eat straw mushroom, be conducive to hypotensive, antitumor, enhancing body is disease-resistant, the healing of accelerated in wounds.Modern medicine study shows, straw mushroom contains isomerism protein, can strengthen human immunologic function, reduces cholesterol level, prevention of arterial gruel type.Plant the material of albumen containing a kind of people of crying in straw mushroom, have antitumous effect, it also contains nitrogen extract and purine bases, again can the growth of anticancer.
China is the productive consumption big country of straw mushroom, and in addition, the cultivation of the state straw mushrooms such as Japan, Thailand, Vietnam, Malaysia is also very general.The past straw mushroom production area of China mainly concentrates on the southern coastal provinces such as Guangdong, Zhejiang, Fujian.Over nearly 20 years, the straw mushroom of China produces and has a great development, and northern each province utilizes wheat straw, cotton seed skin, planted the bacterium chaff of other mushroom carries out Volvaria volvacea cultivation, creates many sections cultivation mode of applicable boreal climate feature.The sale of straw mushroom product mainly contains 2 aspects: one is be processed into the factors such as salt marsh peeling straw mushroom outlet Japan, and this product sells well for many years in the international market.Another selling market of straw mushroom is domestic, and the home sale market potential of straw mushroom is huge.Guangzhou fruit and vegetable company, just near the wholesale vegetable market of the south of the River, Guangzhou, has built maximum straw mushroom wholesale market, Guangzhou (causeway straw mushroom trade market).Along with improving constantly of people's living standard, the northerly sales volume of straw mushroom can also increases year by year.
The opportunities and challenges brought to edible mushrooms industry in the face of entry to WTO and present situation backward in technique, we must have breakthrough technically and innovate.Therefore this is in the urgent need to replacing the cultivation of solid spawn with liquid spawn.We are conceived to the research carrying out straw mushroom submerged fermentation technology, change traditional method of producing with solid spawn.We test finally studied successfully straw mushroom liquid strain large-scale production new technology through researchs in a few years, repeatedly; this technology not only can shorten the production of hybrid seeds time of bacterial classification greatly; improve strain quality; reduce bacterial classification production cost; improve Fruitbody, quality product and commodity value etc., and less investment, technology is easily grasped; be applicable to China's national situation, there is very high promotional value.
Summary of the invention
1. straw mushroom liquid culture carbon nitrogen source is preferred
1 materials and methods
1.1 material
1.1.1 bacterial classification straw mushroom V
110, draw from edible mushrooms institute of Jiangsu Gaoyou City.
1.1.2 mother culture media
Potato 20%, glucose 2%, wheat bran 3%, peptone 0.15%, KH
2pO
40.2%, MgSO
47H
2o 0.15%., agar 2%.
1.1.3 one-level Shake flask medium
Potato 5%, sucrose 2%, peptone 0.15%, wheat bran 3%, KH
2pO
40.1%, MgSO
40.05%, VB
10.001%
1.1.4 carbon source is for examination substratum
Peptone 0.5%, KH
2pO
40.05%, MgSO
40.1%, V
b10.001%, carbon source.Carbon source is wherein replaced respectively with glucose, lactose, fructose, maltose, sucrose, the red sugar and starch of 2%.
1.1.5 nitrogenous source is for examination substratum
Glucose 2%, KH
2pO
40.05%, MgSO
40.1%, V
b10.001%, nitrogenous source.Use the peptone of 0.5%, extractum carnis, yeast extract paste, KNO respectively
3, glycine and L-glutamic acid replaces nitrogenous source wherein.
.
1.1.6 inorganic salt are for examination substratum
By glucose 2%, peptone 0.5%, VB
1mgSO in 0.001% substratum
47H
2o, uses the ZnSO of 0.2% respectively
4, KH
2pO
4, CaCI
2, MgSO
47H
2o and Fe
2(SO
4)
3replace.
1.1.7 agricultural byproducts hydrolyzed solution is for examination substratum:
By wheat bran 4%, glucose 2%, potato 5%, KH
2pO
40.1%, MgSO
40.05%, VB
1wheat bran in 0.001% substratum, uses the crushed maize of equivalent, soya bean slag and starch to replace respectively.
1.2 method
1.2.1 medium preparing
Potato is slitting shape after peeling, and puts into water together with wheat bran, boils rear maintenance about 25min.Get filtrate after eight layers of filtered through gauze, then add each medicine respectively, constant volume.While hot substratum is distributed into test tube, loading amount is 1/4 ~ 1/3 of test tube, and at 0.15mPa, 121 DEG C of sterilizing 0.5h take out pendulum inclined-plane.Liquid nutrient medium is sub-packed in 500ml Erlenmeyer flask, every bottled 150ml, 121 DEG C of sterilizing 0.5h..
1.2.2 actication of culture
Be connected on test tube slant by the straw mushroom bacterial classification aseptic technique of preservation, cultivate about one week at 30 DEG C, now mycelia covers with inclined-plane, can use.
1.2.3 the preparation of liquid spawn
1.2.3.1 one-level shaking flask is inoculated: 0.5 close ㎝ × 0.5 ㎝ of size is got in aseptic technique mother from inclined-plane plants block, is inoculated in (bacterium block is thin, is with substratum less, makes bacterium block swim on liquid level as far as possible) in liquid nutrient medium.Every bottle graft kind 3 pieces.25 DEG C of standing 24h, are then placed in 30 DEG C, and 220 r/min Leftward/rightward rotating shaking tables are cultivated about 5 days.
1.2.3.2 inoculate second-level shake flask aseptic technique, inoculum size is 10%, and to be placed on rotary shaker 30 DEG C, 220r/min cultivates 3 days.Often group establishes 3 repetitions.
1.2.4 result treatment
1. fermented liquid is poured in graduated cylinder by the mensuration of bacterium sphere volume ratio, after fully static, measure bacterium sphere volume, calculates its volume ratio.
2. the size measurement transfer pipet of bacterium ball is drawn 1ml fermented liquid respectively and is put into 3 culture dish from graduated cylinder, serves as a contrast with squared paper below culture dish, measures bacterium spherical diameter.
3. after fermented liquid filters with absorbent cotton by the mensuration of mycelium morphology factor, then with distilled water flushing several, drain, then dry to constant weight in 60 DEG C of baking ovens, weigh with electronic analytical balance.
2 results and analysis
2.1 different carbon sources are on the impact of straw mushroom liquid culture
2.1.1 the selection result (see table 1) of the suitableeest carbon source:
table 1 different carbon source is on the impact of straw mushroom liquid culture
From table 1, can find out that straw mushroom is relatively more extensive to the utilization of carbon source, monose, Shuan Tang, polysaccharide all can utilize.On the substratum taking sucrose as carbon source, mycelium growth vigor is best, and bacterium ball growth fraction is more even.On the substratum being carbon source with glucose, maltose, straw mushroom growth is also better, but good not as good as growing way on the substratum that glucose is carbon source.On the substratum taking starch as carbon source, straw mushroom growth is poor.
2.1.2 variance analysis
As shown in Table 1, straw mushroom, on the substratum being carbon source with glucose, fructose, lactose, brown sugar, maltose, sucrose, Zulkovsky starch, all grows better.By carrying out variance analysis to its dry weight, filter out preferably carbon source.Variance analysis is carried out to dry mycelial weight in 7 kinds of substratum, the results are shown in Table 2.
table 2 carbon source substratum mycelium dry weight analysis of variance table
| Difference source | SS | df | MS | F value | F(0.05) | F(0.01) |
| Between group | 0.233 | 6 | 0.0388 | 19.5107 | 2.8477 | 4.4558 |
| In group | 0.0279 | 14 | 0.0020 | |||
| Amount to | 0.2609 | 20 |
SS-sum of squares of deviations, df-degree of freedom, MS-is all square, F
(0.05)-F threshold value (as follows)
As shown in Table 2, carbon source F value 19.5107 > F
(0.05)=2.8477, and F value 4.1001 > F
(0.01)=4.4558, can find out straw mushroom each for trying to utilize in substratum the ability of carbon source different, difference is extremely remarkable.In order to further illustrate the significance of difference between substratum, carrying out again the multiple comparisons between mean, having the results are shown in Table 3.
table 3 carbon source substratum mycelium dry weight SSR master meter
Checked as can be seen from mycelium dry weight SSR: sucrose, maltose, between glucose and brown sugar, difference is not remarkable, and sucrose, maltose, glucose and, significant difference between fructose, Zulkovsky starch, sucrose and fructose, lactose, Zulkovsky starch difference are extremely remarkable.From the known sucrose of mycelium dry weight be most suitable for straw mushroom growth carbon source, be secondly maltose and glucose, and starch is unsuitable for the growth of straw mushroom.
2.2 different nitrogen sources are on the impact of straw mushroom submerged fermentation
2.2.1 the selection result of the suitableeest nitrogenous source:
table 4 different nitrogen sources is on the impact of straw mushroom liquid culture
As shown in Table 4, straw mushroom grows better in yeast extract paste, peptone, extractum carnis substratum, and pellet form is better; In inorganic nitrogen-sourced substratum, growth is very fast, but bacterium ball is uneven, has furcella; On L-glutamic acid and glycine medium, mycelial growth is slow, and bacterium ball is little.Totally it seems, straw mushroom grows and is obviously better than other nitrogen source medium in yeast extract paste, peptone, extractum carnis substratum.
2.2.2 variance analysis
As shown in Table 4, straw mushroom all can grow preferably in compound nitrogen source and inorganic nitrogen-sourced substratum, but there is difference in pellet form, size, carries out variance analysis to its dry weight, filters out preferably nitrogenous source.Variance analysis is carried out to dry mycelial weight in 7 kinds of substratum, the results are shown in Table 5:
table 5 nitrogen source medium mycelium dry weight analysis of variance table
| Difference source | SS | df | MS | F | F(0.05) | F(0.01) |
| Between group | 0.88 | 6 | 0.1467 | 72.092 | 2.8477 | 4.4558 |
| In group | 0.0285 | 14 | 0.0020 | |||
| Amount to | 0.9085 | 20 |
As shown in Table 5, nitrogenous source F value 72.092 > F(0.05)=2.8477, and F value > F(0.01)=4.4558.Can find out, straw mushroom utilizes the ability of nitrogenous source different, and difference is extremely remarkable.
table 6 nitrogen source medium mycelium dry weight SSR master meter
Checked as can be seen from table 6 mycelium dry weight SSR: between peptone, extractum carnis, yeast extract paste and saltpetre, ammonium nitrate, difference is not remarkable, and two seed amino acid differences are not remarkable; And peptone, extractum carnis, yeast extract paste and ammonium nitrate, glycine and L-glutamic acid difference are extremely remarkable.It can thus be appreciated that, straw mushroom grows better on the substratum being nitrogenous source with peptone, yeast extract paste, extractum carnis, and grow poor on the substratum taking glycine, L-glutamic acid as single nitrogenous source, so straw mushroom utilizes the ability of compound nitrogen source more much better than than amino acid, this is consistent with its result on solid medium.
2.3 different inorganic salt are on the impact of straw mushroom liquid culture
2.3.1 the selection result of the suitableeest inorganic salt:
the different inorganic salt of table 7 are on the impact of straw mushroom liquid culture
As can be seen from Table 7, straw mushroom is containing MgSO
4, Fe
2(SO
4)
3, KH
2pO
4and ZnSO
4substratum on growth better, wherein, containing KH
2pO
4substratum on grow best, containing CaCI
2substratum on grow the poorest.Visible straw mushroom mycelia necessary for growth K
+and Mg
2+plasma, to Ca
2+demand little.
2.3.2 variance analysis
As can be seen from table 7, the mycelium dry weight difference to some extent of different inorganic salt, carries out variance analysis to its dry weight, filters out the inorganic salt being relatively applicable to straw mushroom growth.Variance analysis is carried out to dry mycelial weight in 5 kinds of substratum, the results are shown in Table 8.
table 8 different inorganic salt mycelium dry weight analysis of variance table
| Difference source | SS | df | MS | F value | F(0.05) | F(0.01) |
| Between group | 0.3825 | 4 | 0.0956 | 10.4209 | 3.4780 | 5.9943 |
| In group | 0.0918 | 10 | 0.0092 | |||
| Amount to | 0.4742 | 14 |
As shown in Table 8, the F value 10.4209>F (0.05)=3.478 of inorganic salt, and F value > F (0.01)=5.9943, this shows, straw mushroom biomass on different minimal medium is different, there is pole significant difference.In order to further illustrate the significance of difference between substratum, carrying out again the multiple comparisons between mean, having the results are shown in Table 9.
table 9 different inorganic salt mycelium dry weight SSR master meter
As shown in Table 9, KH
2pO
4with ZnSO
4, CaCI
2, MgSO
4, Fe
2(SO
4)
3significant difference, and ZnSO
4with Fe
2(SO
4)
3, CaCI
2difference is not remarkable, with MgSO
4, KH
2pO
4significant difference.By SSR
0.01can find out: KH
2pO
4with ZnSO
4, CaCI
2, Fe
2(SO
4)
3difference is extremely remarkable, with MgSO
4difference is not remarkable.Therefore, straw mushroom growth needs KH
2pO
4and MgSO
4participation.
2.4 agricultural byproducts hydrolyzed solutions are on the impact of straw mushroom liquid culture
2.4.1 the selection result of agricultural byproducts hydrolyzed solution:
the different agricultural byproducts hydrolyzed solution of table 10 is on the impact of straw mushroom liquid culture
2.4.2 variance analysis
As can be seen from Table 10, the mycelium dry weight difference to some extent of different agricultural byproducts hydrolyzed solution, variance analysis is carried out to its dry weight, filters out the suitableeest carbon-nitrogen ratio (see table 11):
table 11 different agricultural byproducts hydrolyzed solution substratum mycelium dry weight analysis of variance table
| Difference source | SS | df | MS | F | F(0.05) | F(0.01) |
| Between group | 0.0553 | 3 | 0.0184 | 9.0993 | 4.0662 | 7.5910 |
| In group | 0.0162 | 8 | 0.0020 | |||
| Amount to | 0.0716 | 11 |
As shown in Table 11, the F value 9.0993>F (0.05)=4.0662 of different agricultural byproducts hydrolyzed solution, and F value > F (0.01)=7.5910, this shows, straw mushroom biomass on different agricultural byproducts hydrolyzed solution substratum is different, there is pole significant difference.In order to further illustrate the significance of difference between substratum, carrying out again the multiple comparisons between mean, having the results are shown in Table 9.
table 12 different agricultural byproducts hydrolyzed solution mycelium dry weight SSR master meter
As shown in Table 12, wheat bran and soya bean slag substratum, soya bean slag and maize powder medium, between Semen Maydis powder and starch culture-medium, difference is remarkable, and wheat bran and Semen Maydis powder, difference is very remarkable between starch.It can thus be appreciated that straw mushroom grows better on the substratum being raw material with wheat bran and soya bean slag, and grows poor on the substratum taking starch as raw material.
3 discuss
Through above-mentioned experiment and analysis, can draw the following conclusions: straw mushroom all can well grow on the substratum taking sucrose as carbon source, polysaccharide substratum grows poor, and optimum carbon source is sucrose.Compound nitrogen source substratum grows and is all better than inorganic nitrogen-sourced and two seed amino acid substratum, and grow best on the substratum being nitrogenous source with peptone, yeast extract paste, extractum carnis.Straw mushroom is containing KH
2pO
4, MgSO
4, Fe
2(SO
4)
3substratum on well-grown, and containing KH
2pO
4substratum on grow best.The substratum that straw mushroom is prepared at agricultural-food hydrolyzed solution all well can grow, can larger biomass be obtained.And substratum prepared by wheat bran is most suitable for straw mushroom growth, maximum biomass can be obtained.
2. the orthogonal test research of straw mushroom liquid nutrient medium
1 materials and methods
1.1 material
1.1.1 strains tested: straw mushroom V
110, draw from edible mushrooms institute of Jiangsu Gaoyou City.
1.1.2 substratum
A. mother culture media: potato 20%, glucose 1%, wheat bran 3%, peptone 0.15%, agar 2%, KH
2pO
40.2%, MgSO
47H
2o 0.15%.
B. one-level Shake flask medium: potato 5%, sucrose 2%, peptone 0.15%, wheat bran 3%, KH
2pO
40.1%, MgSO
40.05%, VB
10.001%
C. carbon source is for examination substratum: peptone 0.5%, KH
2pO
40.05%, MgSO
40.1%, V
b10.001%, carbon source.Carbon source is wherein replaced respectively with glucose, lactose, fructose, maltose, sucrose, the red sugar and starch of 2%.
D. nitrogenous source is for examination substratum: glucose 2%, KH
2pO
40.2%, MgSO
40.1%, V
b10.001%, nitrogenous source.Use the peptone of 0.2%, extractum carnis, yeast extract paste, KNO respectively
3, glycine and L-glutamic acid replaces nitrogenous source wherein.
.
E. inorganic salt are for examination substratum: glucose 2%, peptone 0.5%, VB
10.001%.Inorganic salt are wherein replaced respectively with zinc sulfate, calcium chloride, magnesium sulfate, ferric sulfate and the potassium primary phosphate of 0.2%.
F. agricultural byproducts hydrolyzed solution is for examination substratum: by wheat bran 4%, glucose 1%, potato 5%, KH
2pO
40.1%, MgSO
40.05%, VB
1wheat bran in 0.001% substratum, uses the crushed maize of equivalent, soya bean slag and starch to replace respectively.
G. orthogonal test culture medium prescription: potato 5%, KH
2pO
40.2%, V
b10.001%, carbon source 1%, nitrogenous source 0.2%, agricultural-food 3%, inorganic salt 0.1%.Wherein carbon source, nitrogenous source, inorganic salt, agricultural byproducts are in table 13.
table 13 level of factor table
1.2 method
1.2.1 actication of culture: the bacterial classification aseptic technique of preservation is seeded to inclined-plane mother culture media, cultivates about 7d at 30 DEG C.
1.2.2 one-level shaking flask is inoculated: aseptic technique is planted from inclined-plane mother and got 0.5 ㎝
2the bacterial classification block of size, is inoculated in and is equipped with in the one-level shaking flask of substratum, and every bottle graft kind 3 pieces makes bacterial classification block swim on liquid level as far as possible.Leave standstill 24hr under 30 DEG C of conditions, be then placed in 30 DEG C, the rotary shaker of about 220 r/min is cultivated about 5 days.
1.2.3 inoculating carbon source, nitrogenous source, inorganic salt and agricultural byproducts hydrolyzed solution shaking flask loads in the shaking flask of 500mL by formula C, D, E, F by ready-made substratum, every bottle about fills 150mL, sterilizing 30min at 121 DEG C, after cooling, the cultivation that cultured one-level shaking flask kind accesses various composition is concentrated by aseptic technique, inoculum size is 10%, then puts into 30 DEG C, 3d cultivated by the shaking table of 220r/min.Observe and record the upgrowth situation of bacterium ball, weighing mycelial dry weight.
1.2.4 orthogonal test
(1) test design is from carbon source, nitrogenous source, inorganic salt and agricultural byproducts hydrolyzed solution for examination cultivation results, chooses good three kinds of carbon and nitrogen sources, inorganic salt, adds three kinds of good agricultural byproducts, design four factor three hydraulic test schemes, be shown in Table 3.
(2) result treatment
The mensuration of mycelium morphology factor by after fermented liquid suction filtration, then with distilled water flushing several, drains, then dries to constant weight in 60 DEG C of baking ovens, weigh with electronic analytical balance.
2 results and analysis
2.1 carbon sources, nitrogenous source, inorganic salt, agricultural-food test results and analysis
From Fig. 1-4, straw mushroom mycelia utilizes the order of nitrogenous source to be peptone > extractum carnis > yeast extract paste > ammonium nitrate > ammonium sulfate > ammonium chloride, the order of carbon source is utilized to be sucrose > maltose > glucose > lactose > fructose > brown sugar > starch, the order of inorganic salt is utilized to be potassium primary phosphate > magnesium sulfate > ferric sulfate > sodium-chlor > calcium chloride, the order of agricultural-food hydrolyzed solution is utilized to be wheat bran > soya bean slag > Semen Maydis grit > starch, so the suitableeest nitrogenous source of straw mushroom mycelial growth is peptone, extractum carnis, yeast extract paste, the suitableeest carbon source is glucose, maltose, sucrose, the suitableeest inorganic salt are potassium primary phosphates, magnesium sulfate, ferric sulfate, the suitableeest agricultural-food are wheat bran, soya bean slag, Semen Maydis grit.Select 3 kinds of the suitableeest nitrogenous sources, carbon source, inorganic salt and agricultural-food wherein to carry out orthogonal test, filter out the optimum combination of liquid culture based formulas.
2.2 orthogonal experiments and variance analysis
As can be seen from Fig. 5 analysis, carbon source has the greatest impact to straw mushroom mycelial growth, is secondly nitrogenous source and agricultural-food, and inorganic salt impact is minimum, and this is consistent with the condition that most of hypha of edible fungus grows.Take dry mycelial weight as reference index, the best of breed of culture medium prescription is A
1b
3c
3d
1, that is: sucrose 1%+ peptone 0.2%+ wheat bran 3%+MgSO
40.1%.
3 discuss
Known by testing above and analyzing, straw mushroom can utilize various carbon and nitrogen sources more widely, and the optimization formula of its liquid nutrient medium is potato 5%, sucrose 1%, peptone 0.2%, wheat bran 3%, MgSO
40.1%, KH
2pO
40.2%, V
b10.001%.As for the how many impact on mycelium production of content of each composition in formula, by further experimental study.
3. different concentration of carbon and nitrogen sources is on the impact of straw mushroom deep drainpipe
1. materials and methods
1.1 material
1.1.1 bacterial classification straw mushroom V
110, draw from edible mushrooms institute of Jiangsu Gaoyou City.。
1.1.2 mother culture media potato 20%, glucose 1%, wheat bran 3%, peptone 0.15%, KH
2pO
40.1%, MgSO
47H
2o 0.05%., agar 2%.
1.1.3 one-level Shake flask medium potato 20%, wheat bran 3%, glucose 2%, peptone 0.2%, KH
2pO
40.25%, MgSO
40.15%, vitamins B
10.001%.
1.1.4 carbon source concentration is for examination substratum potato 5%, peptone 0.2%, KH
2pO
40.25%, MgSO
40.15%, vitamins B
10.001%, glucose.Wherein the concentration of glucose is respectively 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%.
1.1.5 nitrogen concentration is for examination substratum potato 5%, glucose 2%, KH
2pO
40.25%, MgSO
40.15%, vitamins B
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 test substratum chooses comparatively suitable glucose (2.0%, 2.5%, 3.0%) and peptone (0.1%, 0.2%, 0.3%), and conventional inorganic salt MgSO
4+ KH
2pO
4(0.10%+0.20%, 0.15%+0.25%, 0.20%+0.30%) carries out orthogonal test.
1.2 method
1.2.1 medium preparing potato is cut into small pieces after peeling, and puts into water together with wheat bran, boils rear maintenance about 30min, after 8 layers of filtered through gauze, gets filtrate and adds other compositions successively, constant volume after other component dissolves.Solid medium is distributed into test tube, and loading amount is 1/4 ~ 1/5 of test tube, 121 DEG C of sterilizing 30min, takes out pendulum inclined-plane.Liquid nutrient medium is sub-packed in 500mL Erlenmeyer flask, every bottled 150mL, 121 DEG C of sterilizing 30min.
1.2.2 the bacterial classification aseptic technique of preservation is connected to inclined-plane by actication of culture, and cultivate about 5d at 30 DEG C, now mycelia covers with inclined-plane, can use.
1.2.3 the preparation aseptic technique of one-level shaking flask kind is got size from inclined-plane and is about 0.5cm
2mother plant block, be inoculated in liquid nutrient medium, make bacterium block swim on liquid level as far as possible.Every bottle graft kind 3 pieces.25 DEG C of quiescent culture 24h, are then placed in 30 DEG C, about 220r/min rotary shaker are cultivated about 5 d.
1.2.4, under inoculating second-level shake flask aseptic technique, accessed in second-level shake flask by the one-level shaking flask kind Sterile pipette grown, inoculum size is 10%, is placed on rotary shaker, 30 DEG C, and 220r/min cultivates 3 ~ 4 d.Often group establishes 3 repetitions.
1.2.5 the mensuration of result treatment (1) Peloton density.Fermented liquid is poured in graduated cylinder, after fully static, measure bacterium sphere volume, calculate its volume ratio.(2) the mensuration of growth quantity of mycelium.By fermented liquid with after filter paper suction filtration, then with distilled water flushing several, drain, then dry to constant weight in 60 DEG C of baking ovens, weigh with electronic analytical balance.
2 results and analysis
2. 1 carbon source concentration is on the impact of straw mushroom deep drainpipe
the carbon source of table 15 different concns is on the impact of straw mushroom deep drainpipe
| Sucrose concentration (%) | 0.5 1 1.5 2 2.5 3 3.5 |
| Bacterium spherical diameter/mm | 2-3 2-3 2-4 2-4 2-4 2-4 3-4 |
| Dry mycelial weight/g (100ml) -1 | 0.326 0.484 0.578 0.625 0.583 0.473 0.412 |
As shown in Table 15, straw mushroom grows in the liquid nutrient medium being carbon source with the glucose of different concns, 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, to concentration be 2.0% reach the highest, then along with the rising mycelium dry weight of concentration reduces.This illustrates the growth of the neither suitable Mycelia of Straw Mushroom, Volvariel volvacea of lower or higher carbon source concentration.And the Peloton density difference of each concentration is little, just there is obvious minimizing under a high concentration condition.
Variance analysis is carried out to mycelium dry weight in 7 kinds of different concns carbon source substratum, the results are shown in Table 16.
the carbon source substratum mycelium dry weight variance analysis of table 16 different concns
As can be seen from Table 16: the carbon source F=e > F of different concns
0.05=2.8477, but F=3.4678 < F
0.01=4.4558, straw mushroom is described each for trying to utilize in substratum the ability of the carbon source of different concns different, significant difference.For further illustrating the significance of difference between substratum, then carrying out the multiple comparisons between mean, the results are shown in Table 17.
table 17 different concns carbon source substratum mycelium dry weight SSR checks
As can be seen from Table 17: 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 0.5%, 3.5% time is not remarkable yet, but the mycelium dry weight difference of carbon source concentration when 2.0% and 3.5% is significant, the better carbon source concentration of therefore Mycelia of Straw Mushroom, Volvariel volvacea growth is 1.5%, 2.0% and 2.5%..
2.2 nitrogen concentrations are on the impact of straw mushroom deep drainpipe
the nitrogenous source of table 18 different concns is on the impact of straw mushroom deep drainpipe
| Peptone concentration (%) | 0.1 0.2 0.3 0.4 0.5 0.6 0.7 |
| Bacterium spherical diameter/mm | 1-2 2-3 2-3 3-4 3-4 4-5 5-6* |
| Dry mycelial weight/g (100ml) -1 | 0.564 0.597 0.615 0.587 0.553 0.497 0.481 |
As can be seen from Table 18, when straw mushroom grows in the liquid nutrient medium of the peptone containing different concns, when the lower concentration of 0.1%, 0.2%, 0.3%, mycelium dry weight is comparatively large, and Peloton density is comparatively large, and the diameter of bacterium ball is relatively little; And high density 0.4%, 0.5%, 0.6%, 0.7% time, mycelium growing way obviously weakens, Peloton density is downward trend, and mycelium dry weight also diminishes, and bacterium spherical diameter increases relatively.
Variance analysis is carried out to Mycelia of Straw Mushroom, Volvariel volvacea dry weight in 7 kinds of different concns nitrogen source medium, the results are shown in Table 19.
the nitrogen source medium mycelium dry weight variance analysis of table 19 different concns
As can be seen from Table 19: the nitrogenous source F=12.2156 > F of different concns
0.05=2.8477, but F=12.2156 > F
0.01=4.4558, straw mushroom is described each for trying to utilize in substratum the ability of different concns nitrogenous source different, difference is extremely remarkable.For further illustrating the significance of difference between each concentration, then carrying out the multiple comparisons between mean, the results are shown in Table 20.
table 20 different concns nitrogen source medium mycelium dry weight SSR checks
As can be seen from Table 20: the concentration of nitrogen is 0.3%, 0.4%, 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 0.1%, 0.2%, 0.4%, 0.5%, but the mycelium dry weight between concentration 0.1% and 0.3% exists significant otherness yet.Therefore the better nitrogen concentration of suitable Mycelia of Straw Mushroom, Volvariel volvacea growth is 0.2%, 0.3%, 0.4%, and now Mycelia of Straw Mushroom, Volvariel volvacea growth is vigorous, and bacterium ball is of moderate size, evenly, can obtain more mycelium.
2.3 orthogonal test
2.3.1 test design
Foundation the results of univariate logistic analysis above, selects 3 suitable carbon sources, nitrogenous source, wheat bran concentration and conventional inorganic salt MgSO
4with KH
2pO
4different concns matched combined carries out the test design (see table 21) of four factor three levels.Therefrom choose the arranging scheme of optimum carbon source concentration, nitrogenous source, wheat bran concentration and inorganic salt concentration, determine the optimum medium of this bacterial classification.
table 21 level of factor
2.3.2 orthogonal experiments:
Analyzed from Fig. 6: the R value of A factor (carbon source) is maximum, the R value of B factor (nitrogenous source) is less, and the R value of C factor (inorganic salt) and C factor is minimum, can judge thus, carbon source concentration has the greatest impact to straw mushroom deep drainpipe, and the impact of inorganic salt concentration is minimum.As can be seen from analyzing further, take mycelium dry weight as reference index, various the concentrating of nutrients best of breed is A
2b
1c
3d
1, i.e. sucrose 2%, peptone 2%, wheat bran 4%, inorganic salt MgSO
40.05%, KH
2pO
40.1%.
3. conclusion
Known by testing above and analyzing, straw mushroom can adapt to the carbon and nitrogen sources of different concns, wheat bran and inorganic salt more widely, but the optimization formula of its liquid nutrient medium is sucrose 2%, peptone 2%, wheat bran 4%, inorganic salt MgSO
40.05%, KH
2pO
40.1%, then add VB
10.001%.Optimum concn as other carbon and nitrogen sources, agricultural-food hydrolyzed solution and inorganic salt needs later further experiment.
4. the research of straw mushroom deep drainpipe condition
1. materials and methods
1.1 material
1.1.1 bacterial classification: straw mushroom V
110, draw from edible mushrooms institute of Jiangsu Gaoyou City.
1.1.2 mother culture media potato 20%, glucose 2%, wheat bran 3%, peptone 0.15%, KH
2pO
40.2%, MgSO
47H
2o 0.1%., agar 2%.
1.1.3 one-level Shake flask medium potato 5%, sucrose 2%, peptone 0.15%, wheat bran 3%, KH
2pO
40.1%, MgSO
40.05%, VB
10.001%
1.1.4 second-level shake flask substratum potato 20%, glucose 2%, peptone 0.3%, KH
2pO
40.1%, MgSO
40.05%, V
b10.001%
1.2 method
1.2.1 the preparation of one-level shaking flask kind: cultured test tube stock is got 0.5cm
2bacterium block, aseptically accesses 500ml and fills in the one-level Shake flask medium of 150ml nutrient solution, make bacterial classification block swim in liquid level, static gas wave refrigerator one day at 25 DEG C, and to be placed on temperature be 30 DEG C, rotating speed 220r/min as far as possible.About 5d cultivated by shaking table, as liquid spawn.
1.2.2 test the preparation of shaking flask: the Erlenmeyer flask all adopting 500mL, every bottled second-level shake flask substratum 150mL, inoculum size is 10%, and shaking table culture temperature is 30 DEG C, rotating speed is 220r/min.All in triplicate.
1.3 Different factor are on the impact of straw mushroom deep drainpipe:
1.3.1 differing temps is on the impact of straw mushroom deep drainpipe: the second-level shake flask inoculated is placed in the shaking table cultivation of 24 DEG C, 27 DEG C, 30 DEG C, 33 DEG C, 36 DEG C and 39 DEG C respectively.
1.3.2 different pH is on the impact of straw mushroom deep drainpipe: being debugged respectively by the second-level shake flask medium pH made is 4.0,5.0,6.0,7.0,8.0 and 9.0, inoculation culture after sterilizing.
1.3.3 different viscosity is on the impact of straw mushroom deep drainpipe: the second-level shake flask substratum made is added respectively the agar of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and do control group with the blank of not adding agar.
1.3.4 different vaccination amount is on the impact of straw mushroom deep drainpipe: the second-level shake flask made is accessed respectively the one-level shaking flask kind of 1%, 5%, 10%, 15%, 20%, cultivates and observes.
1.3.5 different bottled amount is on the impact of straw mushroom deep drainpipe: the one-level shaking flask kind by bottled amount being the secondary medium access 10% of 90mL, 120mL, 150mL, 180mL, 210mL, 240mL respectively, surveys result after cultivation.
1.3.6 different rotating speeds is on the impact of straw mushroom deep drainpipe: by the second-level shake flask inoculated at rotating speed be respectively 100r/min, 130r/min, 160r/min, 190r/min, 220r/min, 250r/min shaking table on cultivate survey result.
1.4 measuring methods: under the condition arranged, cultivate and survey Peloton density and mycelium dry weight afterwards in three days.
1.4.1 the mensuration of Peloton density: graduated cylinder fermented liquid being placed in 200mL, leaves standstill 5min, calculates the volume ratio of mycelium and fermented liquid.
1.4.2 the mensuration of mycelium dry weight: the baking oven that the mycelium after suction filtration is placed in 60 DEG C is dried to constant weight, surveys its weight with analytical balance.
2. result and discussion:
2.1 temperature are on the impact of straw mushroom deep drainpipe:
the impact that table 23 differing temps grows Mycelia of Straw Mushroom, Volvariel volvacea
| Temperature (DEG C) | 24℃ 27℃ 30℃ 33℃ 36℃ 39℃ |
| Bacterium spherical diameter/mm | 2-3 2-3 3-4 3-4 4 5-6 |
| Dry mycelial weight/g (100ml) -1 | 0.209 0.326 0.647 0.638 0.528 0.316 |
As can be seen from Table 23, along with the rising of temperature, mycelium dry weight increases gradually, reaches maximum when 30 DEG C, then along with the mycelial dry weight of rising of temperature declines on the contrary; Therefore tentatively can judge 30 DEG C is the growth temperature be comparatively suitable for.And the size of bacterium ball also increases to some extent along with the increase of temperature, mainly the ratio of little bacterium ball reduces.In order to further illustrate the otherness of mycelium dry weight between differing temps, variance analysis being carried out to the mycelium dry weight under condition of different temperatures, the results are shown in Table 24.
the Mycelia of Straw Mushroom, Volvariel volvacea dry weight variance analysis of table 24 differing temps
| Difference source | SS | df | MS | F | F （0.05） | F （0.01） | Significance |
| Between group | 0.4982 | 5 | 0.0996 | 69.3753 | 3.1059 | 5.0643 | ** |
| In group | 0.0172 | 12 | 0.0014 | ||||
| Amount to | 0.5155 | 17 |
As can be seen from Table 24: under differing temps, significant difference between in Mycelia of Straw Mushroom, Volvariel volvacea dry weight group, and each group difference is extremely remarkable.For further illustrating the difference between each group, then carrying out the multiple comparisons between mean, the results are shown in Table 25.
the mycelium dry weight SSR of table 25 differing temps checks
As can be seen from Table 25,30 DEG C all extremely remarkable with the mycelium dry weight difference of 24 DEG C, 27 DEG C, 36 DEG C, 39 DEG C, and 30 DEG C are not remarkable with the mycelium dry weight difference of 33 DEG C, and mycelium dry weight when 30 DEG C is the highest, after 30 DEG C, mycelium dry weight declines, as can be seen here, 30-33 DEG C is best suited for the temperature of Mycelia of Straw Mushroom, Volvariel volvacea growth.Consider economic factor, the temperature range of straw mushroom growth is generally located at 30-31 DEG C.
2.2 pH affect straw mushroom deep drainpipe:
the impact that table 26 initial pH value grows Mycelia of Straw Mushroom, Volvariel volvacea
| PH value | 4.0 5.0 6.0 7.0 8.0 9.0 |
| Bacterium spherical diameter/mm | 1-1.5 1.8 1.5-2 2-3 1-1.2 does not have ball |
| Dry mycelial weight/g (100ml) -1 | 0.162 0.495 0.593 0.567 0.256 0.036 |
As can be seen from Table 26, before pH6.0, mycelium dry weight increases gradually, and when pH6.0, mycelium dry weight is maximum, and then along with the rising of pH, mycelium dry weight reduces gradually, and between pH6.0 ~ 7.0, mycelium dry weight maintains higher level; The diameter of bacterium ball also increases along with the increase of mycelium dry weight, reaches the highest when pH7.0, and after pH7.0, bacterium ball size sharply reduces.As can be seen here, straw mushroom grows under being adapted at neutral environment, tentatively can judge that its comparatively suitable pH is 6.0 ~ 7.0.In order to further illustrate the otherness of mycelium dry weight between different pH, variance analysis is carried out to it.
the Mycelia of Straw Mushroom, Volvariel volvacea dry weight variance analysis of the different pH of table 27
| Difference source | SS | df | MS | F | F （0.05） | F （0.01） | Significance |
| Between group | 0.8257 | 5 | 0.1651 | 139.0986 | 3.1059 | 5.0634 | ** |
| In group | 0.0142 | 12 | 0.0012 | ||||
| Amount to | 0.8400 | 17 |
As can be seen from Table 27: under condition of different pH, significant difference between in Mycelia of Straw Mushroom, Volvariel volvacea dry weight group, and each group difference is extremely remarkable.For further illustrating the difference between each group, then carrying out the multiple comparisons between mean, the results are shown in Table 28.
table 28
the mycelium dry weight SSR of different pH checks
As can be seen from Table 28, during pH6.0, mycelium dry weight is maximum, but when pH6.0 ~ 7.0, mycelium dry weight difference is remarkable, pH6.0 and 4.0,5.0,8.0,9.0 differences are extremely remarkable, we also did pH6.3 ~ 6.5 and tested, and mycelium dry weight is higher than pH6.0, and bacterium ball size does not have difference.Therefore, the optimal pH of straw mushroom liquid culture is about 6.5, close to neutral, and unlike the meta-alkalescence reported.
2.3 viscosity affect straw mushroom deep drainpipe:
the impact that table 29 agar concentration grows Mycelia of Straw Mushroom, Volvariel volvacea:
| Agar concentration (%) | 0 0.1 0.2 0.3 0.4 0.5 |
| Bacterium spherical diameter/mm | 5-6 3-4 2-2.5 2 1.5-2 2-4* |
| Dry mycelial weight/g (100ml) -1 | 0.582 0.603 0.631 0.563 0.554 0.495 |
As can be seen from Table 29, mycelium dry weight is maximum when viscosity 0.2%, and afterwards, mycelium dry weight constantly declines; And bacterium spherical diameter is all significantly less than contrast in the substratum adding agar, reduce gradually between 0.1 ~ 0.4%, to 0.4% time bacterium spherical diameter minimum, the ratio of bead is maximum, then along with the increase of viscosity, in nutrient solution, the quantity of bacterium ball reduces, and occurs irregular bulk, can tentatively judge thus, the range of viscosities being applicable to straw mushroom growth is the agar of 0.1 ~ 0.2%.In order to further illustrate the otherness of mycelium dry weight between different viscosity, variance analysis is carried out to the mycelium dry weight under different viscosity condition.
the Mycelia of Straw Mushroom, Volvariel volvacea dry weight variance analysis of table 30 different viscosity
| Difference source | SS | df | MS | F | F （0.05） | F （0.01） | Significance |
| Between group | 0.0326 | 5 | 0.0065 | 9.3997 | 3.1059 | 5.0643 | ** |
| In group | 0.0083 | 12 | 0.0007 | ||||
| Amount to | 0.0409 | 17 |
As can be seen from Table 30: under different viscosity, between in Mycelia of Straw Mushroom, Volvariel volvacea dry weight group, difference is not remarkable, and each group difference is remarkable.For further illustrating each group difference significance, then carrying out the multiple comparisons between mean, the results are shown in Table 31.
the mycelium dry weight SSR of table 31 different viscosity checks
As can be seen from Table 31, when mycelium dry weight is maximum, viscosity is 0.2%, 0.2 and 0,0.3 significant difference, but 0.1 and 0.2, difference between 0.3 and 0.4 is remarkable, therefore, the viscosity being suitable for straw mushroom deep drainpipe is the agar of interpolation 0.1 ~ 0.4%.Consider that viscosity is too low, bacterium ball is comparatively large, and the bacterium ball ratio of minor diameter is also low, is unfavorable for inoculation, and waste is compared in interpolation more, therefore; When making straw mushroom liquid nutrient medium, generally should add the agar of 0.2%, if add a certain amount of agricultural-food hydrolyzed solution, also can add agar.
2.4 inoculum sizes affect kind amount to straw mushroom deep drainpipe:
the impact that table 32 inoculum size grows Mycelia of Straw Mushroom, Volvariel volvacea
| Inoculum size (%) | 1％ 5％ 10％ 15％ 20％ |
| Bacterium spherical diameter/mm | 4 3-4 2-3 2-3 2* |
| Dry mycelial weight/g (100ml) -1 | 0.315 0.475 0.631 0.614 0.570 |
As can be seen from table 32, along with the increase of inoculum size, all corresponding increase of mycelium dry weight and Peloton density, but when 10% inoculum size, mycelium dry weight increasing degree is comparatively large, when reaching 15%, by declined, but not obvious.Can tentatively judge, optimal inoculum size is 10% ~ 15%.In order to further illustrate the otherness of mycelium dry weight between different vaccination amount, variance analysis is carried out to the mycelium dry weight under different vaccination amount.
the Mycelia of Straw Mushroom, Volvariel volvacea dry weight variance analysis of table 33 different vaccination amount
| Difference source | SS | df | MS | F | F （0.05） | F （0.01） | Significance |
| Between group | 0.1949 | 4 | 0.0487 | 46.6938 | 3.4780 | 5.9943 | ** |
| In group | 0.0104 | 10 | 0.0010 | ||||
| Amount to | 0.2054 | 14 |
As can be seen from Table 33: under different vaccination amount, between in Mycelia of Straw Mushroom, Volvariel volvacea dry weight group, difference is not remarkable, and each group difference is extremely remarkable.For further illustrating the difference between each group, then carrying out the multiple comparisons between mean, the results are shown in Table 34.
the mycelium dry weight SSR of table 34 different vaccination amount checks
As can be seen from Table 34, the inoculum size of straw mushroom is when 20% and 15%, and mycelium dry weight difference is extremely remarkable, but between 10% and 15%, difference is not remarkable, considers economic factors, and about 10% is its suitable inoculum size.
2.5 bottled amounts are on the impact of straw mushroom deep drainpipe
the impact that table 36 liquid amount grows Mycelia of Straw Mushroom, Volvariel volvacea
| Liquid amount (ml) | 90 120 150 180 210 240 |
| Bacterium spherical diameter/mm | 4-5 3-4 2-3 2-3 2-4 2-4 |
| Dry mycelial weight/g (100ml) -1 | 0.326 0.483 0.598 0.625 0.531 0.343 |
As can be seen from Table 36, when bottled amount is 120 ~ 210mL, mycelium dry weight change is little, declines obviously during 240mL; And the change of bacterium spherical diameter is little.Can preliminary judgement, the suitableeest bottled amount is 150 ~ 210mL/500mL.In order to further illustrate the otherness of mycelium dry weight between different bottled amount, variance analysis is carried out to the mycelium dry weight of the bottled amount of difference.
the Mycelia of Straw Mushroom, Volvariel volvacea dry weight variance analysis of the different bottled amount of table 37
| Difference source | SS | df | MS | F | F （0.05） | F （0.01） | Significance |
| Between group | 0.2546 | 5 | 0.0509 | 17.4495 | 3.1059 | 5.0643 | ** |
| In group | 0.0350 | 12 | 0.0029 | ||||
| Amount to | 0.2896 | 17 |
As can be seen from Table 37: under different bottled amount, significant difference between in Mycelia of Straw Mushroom, Volvariel volvacea dry weight group, and each group difference is extremely remarkable.For further illustrating the difference between each group, then carrying out the multiple comparisons between mean, the results are shown in Table 38.
the mycelium dry weight SSR of the different bottled amount of table 38 checks
As can be seen from Table 38, when bottled amount is 180mL, mycelium dry weight is maximum, and between 150ml and 210ml bottled amount, difference is not remarkable, therefore the bottled amount of 189mL/500mL is the bottled amount of the best of its Mycelium culture.This is mainly because straw mushroom is addicted to oxygen bacterium, too high bottled amount impact ventilation, mycelia anoxic and poor growth; Too low bottled amount easily causes larger damage to mycelia and affects its growth when shaking.
2.6 rotating speeds are on the impact of straw mushroom deep drainpipe:
the impact that table 39 different rotating speeds grows Mycelia of Straw Mushroom, Volvariel volvacea
As can be seen from Table 39, mycelium dry weight constantly increases along with the rising of rotating speed, and during 220r/min, mycelium dry weight reaches the highest, tends towards stability between 190 ~ 250r/min, on a declining curve after 190r/min; Bacterium spherical diameter is few at below 160r/min bacterium ball, and general bacterium ball increases, adhesion, occurs irregular bulk, and more than 160r/min bacterium ball increases, and during to 250r/min, bacterium ball starts to reduce, and size is uneven, out-of-shape.Visible, the rotating speed of the most applicable Mycelia of Straw Mushroom, Volvariel volvacea growth is 190 ~ 220r/min.In order to further illustrate the otherness of mycelium dry weight between different rotating speeds, variance analysis is carried out to the mycelium dry weight under different rotating speeds condition.
the Mycelia of Straw Mushroom, Volvariel volvacea dry weight variance analysis of table 40 different rotating speeds
| Difference source | SS | df | MS | F | F （0.05） | F （0.01） | Significance |
| Between group | 0.6299 | 4 | 0.1575 | 217.2724 | 3.4780 | 5.9943 | ** |
| In group | 0.0072 | 10 | 0.0007 | ||||
| Amount to | 0.6372 | 14 |
As can be seen from Table 41: under different rotating speeds, significant difference between in Mycelia of Straw Mushroom, Volvariel volvacea dry weight group, and each group difference is extremely remarkable.For further illustrating the otherness between each group, then carrying out the multiple comparisons between mean, the results are shown in Table 42.
the mycelium dry weight SSR of table 42 different rotating speeds checks
As can be seen from Table 42, between different rotating speeds, Mycelia of Straw Mushroom, Volvariel volvacea dry weight difference is extremely remarkable, and when rotating speed is 220 r/min, straw mushroom degree mycelium dry weight is the highest, therefore, can determine that the rotating speed that the most applicable straw mushroom grows is 150 ~ 180r/min.This is mainly because rotating speed is too low, and mycelia is easy fracture not, can not form bead, and dissolved oxygen is inadequate in addition, affects mycelial growth; Rotating speed is too high comparatively large to mycelial damage, and is unfavorable for that it grows.
3. conclusion:
As can be seen from above test-results, the condition of optimum straw mushroom deep drainpipe is: temperature is 30 ~ 33 DEG C, and pH is 6.0 ~ 7.0, and viscosity is the agar of interpolation 0.2%, and inoculum size is about 10 ~ 15%, and bottled amount is 180mL/500mL, and rotating speed is about 220r/min.
5. the research of straw mushroom liquid strain fermentor cultivation
1, material
1.1 bacterial strains:straw mushroom V
110, draw from edible mushrooms institute of Jiangsu Gaoyou City.
1.2 substratum
1.2.1 slant medium:pDA
1.2.2 Shake flask medium:potato 5%, sucrose 2%, peptone 0.2%, wheat bran 4%, MgSO
40.1%, KH
2pO
40.2%, V
b10.001%, pH value nature.
1.2.3 fermentation tank culture medium:starch slurry 5%, Semen Maydis powder 5%, glucose 0.3%, peptone 0.3%, VB
10.001%, MgSO
40.05%, bubble puts 0.03-0.05%, KH
2pO
40.1%, pH7.5
1.3 culture device:hZQ-Q type vibrator (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 (upper Nereid section electronic balance factory), accurate pH meter (Shanghai thunder magnetic), FMLT-52 type 5L desk-top fermentation cylinder (Guoqiang Biochemical Engineering Equipment Co., Ltd., Shanghai).
2, method
2.1 operational paths:straw mushroom preservation of bacteria strain → actication of culture, inclined-plane enlarged culturing → one-level shaking flask → second-level shake flask → 5L fermentor tank or the research of 100L ferment tank
2.2 actication of culture:the straw mushroom bacterial classification be stored in refrigerator is inoculated on slant medium, with 32 DEG C of incubators, cultivates 4-5 days.
the preparation of 2.3 liquid spawns:get three pieces of 0.5m
2slant strains to be inoculated in 500mL(liquid amount be 150mL) in Erlenmeyer flask, be placed in 25 DEG C of thermostat containers and leave standstill 24hr, then at 32 DEG C, 180r/min rotary shaker shaking culture 5d, make liquid spawn (one-level shaking flask) for subsequent use.In 500mL Erlenmeyer flask, load 150mL substratum, by the one-level shaking flask bacterial classification prepared according to 10% inoculum size inoculate, at 32 DEG C, 220r/min rotary shaker shaking culture 3d, make liquid spawn (second-level shake flask) for subsequent use.
2.4 Submerged fermentation researchs:
2.4.1 5L fermentor cultivation:5L fermentor tank loads 4L fermentation tank culture medium, connects second-level shake flask kind by 10% inoculum size, and in 30 DEG C, 220 r/min aerated culture, every 12hr sampling detects mycelial growing state.
2.4.2 100L fermentor cultivation:100L fermentor tank loads 60L substratum, and connect second-level shake flask kind by 10% inoculum size, the pressure in fermentor tank maintains 0.025Mpa, and temperature is 32-34 DEG C of cultivation, and every 12hr sampling detects mycelial growing state.
2.4.3 mycelium dry weight is measured:in fermenting process, 5L fermentor tank is by the whole nutrient solutions in bottle, and with the filter paper suction filtration of drying, 100L fermentor tank, gets 100ml nutrient solution at every turn, with the filter paper suction filtration of drying; Then after repeatedly rinsing with distilled water, be put in 60 DEG C of loft drier and dry to constant weight, measure dry weight.
2.4.4 pH value measures:5L fermentor tank is monitored automatically.
2.5 results and analysis
2.5.1 fermentor cultivation mycelial growth curve:
As can be seen from Fig. 7 and Fig. 8, the increment of the fermentor tank mycelia of 5L and 100L is all double logarithmic curve relation with incubation time, 0 ~ 12hr is the adaptive phase of straw mushroom liquid strain growth, 12hr grows later and accelerates gradually, 24 ~ 48hr speed of growth is the fastest, slow down gradually subsequently, after fermentation 60hr, mycelium production declines to some extent, and mycelium dry weight can reach 1.1021g/100mL.
In culturing process, Mycelia of Straw Mushroom, Volvariel volvacea constantly grows, and forms a large amount of bacterium ball, and constantly consume substratum Middle nutrition composition, mycelium dry weight constantly increases, and reach maximum at 48 ~ 60hr, now biomass is the highest, and culture is thick.Continue fermentation culture, nutritive substance is poor, and mycelium senesces, autolyze, and biomass starts to decline.According to tracing analysis, fermentation termination should determine that in the time be 48 ~ 54hr.Because now although hypha biomass is not maximum, because the mycelia during this section is in the very vigorous logarithmic phase of vitality, mycelia vigor is the strongest, and after inoculation, mycelia is germination and growth very easily.
2.5.2 the change curve of fermentation time and pH value
5L fermentor cultivation, automatically record pH change curve as Fig. 9.
From shake flask test, the suitableeest initial pH value of straw mushroom liquid culture is 6 ~ 7, and substratum makes pH decline about 0.5 at sterilization process, thus before sterilization, the pH value of substratum is adjusted to 7 ~ 7.5.From curve, the mycelial growth initial stage, because envrionment conditions is suitable for, mycelia itself has the ability of certain adjust ph, thus makes pH value be in more stable state. along with mycelia Metabolic activity is constantly strengthened, and the reasons such as Accumulation of Organic Acids, pH value is declined, after mycelia stops growing, pH value rises again, and now mycelia is tending towards self-dissolving and Metabolic activity is more weak.This matches with the dry weight curve recording thalli growth, and can tentatively judge thus, the incubation time of Mycelia of Straw Mushroom, Volvariel volvacea fermentor tank is about about 48 ~ 54hr, although mycelium dry weight now can not reach the highest, very easily sprouts after inoculation.Therefore fermentation time should be controlled well during fermentor cultivation, simultaneously by the observation of the pH of fermenting process, judge whether thalline is in suitable growing environment, this also shows need not control the pH of fermented liquid during the fermentation under the starting condition being suitable for mycelial growth.
3 discuss
By understand straw mushroom silk in fermentor tank and the rule of pH change in mycelial growth process, the Best Times of fermentor cultivation straw mushroom can be grasped on the one hand, carry out the preparation work of inoculation in time, access when mycelia activity is the highest and cultivated.On the other hand, the progress of fermenting can be judged, can be used as the index that fermentation is abnormal.
Accompanying drawing explanation
Fig. 1 is the effect diagram of different carbon source to Mycelia of Straw Mushroom, Volvariel volvacea dry weight; Fig. 2 is the effect diagram of different nitrogen sources to Mycelia of Straw Mushroom, Volvariel volvacea dry weight; Fig. 3 is the effect diagram of different inorganic salt to Mycelia of Straw Mushroom, Volvariel volvacea dry weight; Fig. 4 is the effect diagram of different agricultural-food to straw mushroom; Fig. 5 is L9 (3
4) orthogonal experiments figure; Fig. 6 is L9 (3
4) orthogonal experiments figure; Fig. 7 is the biological spirogram of straw mushroom 5L fermentor tank; Fig. 8 is the biological spirogram of straw mushroom 100L fermentor tank; Fig. 9 is straw mushroom 5L fermentor tank Ph change curve.
Claims (2)
1. the method prepared of straw mushroom liquid strain, is characterized in that the optimization formula of straw mushroom liquid nutrient medium is sucrose 2%, peptone 2%, wheat bran 4%, inorganic salt MgSO
40.05%, KH
2pO
40.1%, then add VB
10.001%.
2. the method prepared of straw mushroom liquid strain, is characterized in that the condition of optimum straw mushroom deep drainpipe is: temperature is 30 ~ 33 DEG C, and pH is 6.0 ~ 7.0, viscosity is the agar of interpolation 0.2%, inoculum size is about 10 ~ 15%, and bottled amount is 180mL/500mL, and rotating speed is about 220r/min.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106497798A (en) * | 2016-10-27 | 2017-03-15 | 稼圃麟（廊坊）生物科技发展有限公司 | Edible fungus species are carried out with fluid medium and the propagation method of rapid expansion breeding |
| CN109628318A (en) * | 2018-12-14 | 2019-04-16 | 上海市农业科学院 | A kind of confirmation method of 9715 strain liquid Spawn incubation terminal of straw mushroom |
| CN110214626A (en) * | 2019-07-16 | 2019-09-10 | 灌南县人民政府蔬菜办公室 | A kind of straw mushroom cultural method |
| CN112341268A (en) * | 2020-10-29 | 2021-02-09 | 吴迪 | Culture solution for cultivating mushrooms and preparation method thereof |
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| CN102523917A (en) * | 2011-12-14 | 2012-07-04 | 广东省微生物研究所 | Method for cultivating straw mushroom |
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| CN101195806A (en) * | 2007-12-04 | 2008-06-11 | 西宁市城西区食用菌研究所 | Industrial production method of cordyceps mushroom |
| CN101699969A (en) * | 2009-11-05 | 2010-05-05 | 张纪明 | Submerged fermentation culture method of straw mushroom liquid strain and culture medium thereof |
| CN102523917A (en) * | 2011-12-14 | 2012-07-04 | 广东省微生物研究所 | Method for cultivating straw mushroom |
| CN102550293A (en) * | 2012-02-03 | 2012-07-11 | 连云港市农业科学院 | Method for liquid fermentation cultivation of Agaricus bisporus strain |
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| CN106497798A (en) * | 2016-10-27 | 2017-03-15 | 稼圃麟（廊坊）生物科技发展有限公司 | Edible fungus species are carried out with fluid medium and the propagation method of rapid expansion breeding |
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| CN109628318A (en) * | 2018-12-14 | 2019-04-16 | 上海市农业科学院 | A kind of confirmation method of 9715 strain liquid Spawn incubation terminal of straw mushroom |
| CN110214626A (en) * | 2019-07-16 | 2019-09-10 | 灌南县人民政府蔬菜办公室 | A kind of straw mushroom cultural method |
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| CN112341268A (en) * | 2020-10-29 | 2021-02-09 | 吴迪 | Culture solution for cultivating mushrooms and preparation method thereof |
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