CN108812057A - A kind of fluid nutrient medium and cultural method for being cultivated in collybia albuminosa room - Google Patents
A kind of fluid nutrient medium and cultural method for being cultivated in collybia albuminosa room Download PDFInfo
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- CN108812057A CN108812057A CN201810663585.3A CN201810663585A CN108812057A CN 108812057 A CN108812057 A CN 108812057A CN 201810663585 A CN201810663585 A CN 201810663585A CN 108812057 A CN108812057 A CN 108812057A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of fluid nutrient medium and cultural method for cultivating in collybia albuminosa room, the fluid nutrient medium include the component of following mass concentration：100~200g/L of potato, 5~15g/L of carbohydrate, 3~5g/L of nitrogen source, 20~30g/L of wheat bran, 10~20g/L of corn flour, 0.1~0.15g/L of vitamin B compound, 2.5~4g/L of microelement, 5~10g/L of ant nest.Present invention combination SPSS orthonormal design of experiments and experiment of single factor are optimized the solid state rheology condition of collybia albuminosa using the average value of collybia albuminosa bacterium ball growth diameter and growth rate as index.Collybia albuminosa dry mycelial weight is suitable for large-scale production up to 15.91g/L under condition of culture after optimization.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of fluid nutrient medium and training for cultivating in collybia albuminosa room
The method of supporting.
Background technique
Collybia albuminosa (Termitomyces sp.) also known as chicken silk mushroom, chicken bacterium, handle of umbrella mushroom, three altar bacterium belong to Basidiomycotina
(Basidiomycotina), Hymenomycetes (Hymenomycetes), Agaricales (Agaricales), from Zhe San section
(Lyophyllaceae), Termitomyces (Termitomyces R.Heim).The species are a kind of and Macrotermitinaes
(Macrotermitinae) the famous delicious edible fungus of insect symbiosis, due to being similar to umbrella outside its fructification, therefore also known as umbrella
Belong to.Collybia albuminosa has special growing environment, that is, is only grow on termitarium, it is necessary to be in termitarium with symbiosis of termite
Dominant bacteria.Once dead, collybia albuminosa also will be unable to survive termite in termitarium in useless nest, therefore, under natural conditions, chicken
The fructification of fir bacterium can not be grown in the case where no termitarium.Fungus garden in termitarium is the secretion by termite,
Such as saliva forms with canebreak stick to each other, active skull cap components (such as amino acid) rich in.Meanwhile with its
He compares edible fungus, and ant nest has the characteristics that high humility, high carbon dioxide concentration.These are all undoubtedly collybia albuminosa and are forming son
The main original that the higher reason of environmental requirement and collybia albuminosa fructification can not temporarily be obtained by artificial culture in terms of entity
Cause.
Meanwhile collybia albuminosa is a kind of rare medical edible fungal, delicious in taste, nutritive value and medical value are higher,
With enhancing immune function of human body, it is anti-inflammatory, prevention intestinal cancer, blood-nourishing, moisturize, strengthening the spleen and stomach and other effects.It has been investigated that collybia albuminosa is also
Amino acid rich in, saponin, the various actives substances such as polysaccharide, protein, brain glycosides, cumarin, ergosterol, but due to chicken
Fir bacterium and symbiosis of termite, fructification must be grown on live nest and the disadvantages of by season and big climatic effects, at present collybia albuminosa
And artificial cultivation is not implemented.Therefore, most of scholar selects other approach to cultivate to play chicken fir as far as possible collybia albuminosa
The nutritive value of bacterium.
The indoor culture of fungi mainly has solid culture and liquid fermentation and culture two ways.Liquid fermentation is used on the market
The mycelium of production has the characteristics that with short production cycle, yield is big, easily-controllable, therefore liquid culture fungal mycelium has become food
The common means with field of medicaments.Although currently, obtaining the existing related report of collybia albuminosa mycelium using liquid state fermentation culture technique
Road, but in view of collybia albuminosa is to the particular/special requirement of natural habitat, the yield of liquid culture is simultaneously not very high.The former base of collybia albuminosa
Last stage is with jack bacterium formal distribution in termitarium.Jack bacterium stablizes growth and later period collybia albuminosa in nest
It is high humidity, a high concentration CO that the successful sprouting of entity, which requires termite ant nest,2, constant temperature ecological environment.Therefore, we are to wet
Degree, temperature, CO2Concentration explores the growth effect of collybia albuminosa mycelia；After determining optimal solid culture condition, with
The solid spawn that the method obtains is used for liquid fermentation and culture, and further carries out to liquid fermentation medium and condition of culture
Screening and optimization.
Summary of the invention
Present invention aim to address above-mentioned the deficiencies in the prior art, provide a kind of liquid for cultivating in collybia albuminosa room
Culture medium and cultural method.
The present invention is achieved by the following technical solutions：
A kind of fluid nutrient medium for being cultivated in collybia albuminosa room, the component including following mass concentration：
100~200g/L of potato, 5~15g/L of carbohydrate, 3~5g/L of nitrogen source, 20~30g/L of wheat bran, corn flour 10
~20g/L, 0.1~0.15g/L of vitamin B compound, 2.5~4g/L of microelement, 5~10g/L of ant nest.
Preferably, the carbohydrate is at least one of maltose, microcrystalline cellulose, soluble starch.
Preferably, the nitrogen source is yeast powder and/or peptone.
It preferably, include calcium, magnesium, sodium, potassium in the microelement.
Preferably, the culture medium includes the component of following mass concentration：
Potato 200g/L, maltose 15g/L, yeast powder 5g/L, wheat bran 30g/L, corn flour 20g/L, vitamin B compound
0.15g/L, CaCl20.6g/L, MgSO40.4g/L, KH2PO41.5g/L, NaH2PO40.5g/L, ant nest 5g/L.
A kind of cultural method that the fluid nutrient medium is used to cultivate in collybia albuminosa room, includes the following steps：
(1) jack bacterium in picking ant nest, is seeded on PDA solid medium, cultivates in 28 DEG C of constant incubators, instead
Multiple scribing line culture, obtains the collybia albuminosa strain of purifying；
(2) the collybia albuminosa mycelia of purifying is connected in PDA liquid medium, cultivates, obtains in 28 DEG C of isothermal vibration shaking tables
Obtain collybia albuminosa bacterium ball；
(3) by collybia albuminosa bacterium ball in humidity 70~90%, CO2Concentration 0.1~0.2%, the training of 24~28 DEG C of temperature of solid
It supports and is cultivated in base, obtain collybia albuminosa original seed；
(4) collybia albuminosa original seed is inoculated into fluid nutrient medium, in initial ph value 4.00, liquid amount 20~40% (v/v),
Inoculum concentration 0.5~1.5% (v/v), 25~28 DEG C of temperature, shaking speed 180r/min are cultivated 7~9 days.
Preferably, solid culture condition described in step (3) is humidity 80%, CO226 DEG C of concentration 0.15%, temperature.
Preferably, the liquid amount is 30% (v/v), and inoculum concentration is 1% (v/v), and temperature is 28 DEG C, cultivation cycle 9
It.
The beneficial effects of the present invention are：
Present invention combination SPSS orthonormal design of experiments and experiment of single factor, with the average value of collybia albuminosa bacterium ball growth diameter
It is index with growth rate, the solid state rheology condition of collybia albuminosa is optimized.The result shows that carrying out collybia albuminosa solid state rheology
When, humidity 80%, CO2Concentration 0.15%, 26 DEG C of temperature of condition of culture can effectively facilitate the growth of collybia albuminosa bacterium ball.It is most suitable
Fluid nutrient medium component be potato 200g/L, maltose 15g/L, yeast extract powder 5g/L, wheat bran 30g/L, corn flour 20g/L,
Vitamin B compound 0.15g/L, CaCl20.6g/L, MgSO40.4g/L, KH2PO41.5g/L, NaH2PO40.5g/L, ant nest
5g/L；Optimal culture conditions are initial ph value 4.00, liquid amount 30% (v/v), inoculum concentration 1% (v/v), 28 DEG C of temperature, shaking table
Revolving speed 180r/min, cultivation cycle 9 days, collybia albuminosa dry mycelial weight was suitable for up to 15.91g/L under the condition of culture after optimization
Large-scale production.
Detailed description of the invention
Fig. 1 is each factor of collybia albuminosa solid state rheology in the embodiment of the present invention 2 to the influence diagram of bacterium bulb diameter.
Fig. 2 is difference CO in the embodiment of the present invention 22The influence diagram that concentration grows collybia albuminosa bacterium ball.
Fig. 3 is the relational graph of the first suboptimization each factor and mycelium dry weight in the embodiment of the present invention 3.
Fig. 4 is the relational graph of the second suboptimization each factor and mycelium dry weight in the embodiment of the present invention 4.
Fig. 5 is the relational graph of third suboptimization each factor and mycelium dry weight in the embodiment of the present invention 5.
Fig. 6 is the relational graph of the 4th suboptimization each factor and mycelium dry weight in the embodiment of the present invention 6.
Fig. 7 is the relational graph of difference pH and mycelium dry weight in the embodiment of the present invention 7.
Fig. 8 is the relational graph of different vaccination amount and mycelium dry weight in the embodiment of the present invention 7.
Fig. 9 is the relational graph of different liquid amounts and mycelium dry weight in the embodiment of the present invention 7.
Figure 10 is the relational graph of different temperatures and mycelium dry weight in the embodiment of the present invention 7.
Figure 11 is the variation diagram of optimization front and back bacterium ball growth diameter in the embodiment of the present invention 8.
Figure 12 is the growth curve chart of culture medium and PDA basal medium after optimizing in the embodiment of the present invention 8.
Specific embodiment
To be best understood from the present invention, below with reference to examples and drawings, the invention will be further described, following embodiment
It is only that the present invention will be described rather than is limited to it.Raw material used in embodiment are conventional commercial.
1 collybia albuminosa strain separating of embodiment and purifying
Termite ant nest (Odontotermes formosanus) picks up from Jiangyin, is taken out the fungus garden for being covered with jack bacterium, Yu Cai
The collection same day is aseptically put into rapidly -20 DEG C of refrigerators and protects as early as possible in the jack bacterium to sterile centrifugation tube on picking fungus garden
It deposits.Meanwhile being seeded in PDA culture medium with transfer needle picking jack bacterium in superclean bench, it is placed in 28 DEG C of constant temperature incubations
It is cultivated in case, when jack bacterium surface grows hairy mycelia, mycelia picking is subjected to scribing line culture to new plate.It is repeatedly several
It is secondary, obtain the collybia albuminosa strain of purifying.
The optimization of 2 collybia albuminosa solid culture condition of embodiment
(1) preparation of original seed：The collybia albuminosa mycelia of purifying is connected in PDA liquid medium, is shaken in 28 DEG C of isothermal vibrations
Culture 5d is carried out in bed, the bacterium ball of acquisition is tested for solid culture condition optimizing.The strain obtained after optimization is that collybia albuminosa is former
Kind.
(2) preparation of PDA culture medium：In potato (peeling) 200g, glucose 20g, agar 20g, water 1000ml ratio
Carry out preparation, the packing of culture medium.It dispenses and is placed on 115 DEG C of sterilizing 30min in autoclave, after sterilizing is completed, culture medium
Inverted plate when being cooled to certain temperature, it is spare.
(3) optimization of condition of culture：Choose CO in this part23 concentration, humidity, temperature factors, utilize SPSS software design
Orthogonal experiment, for CO2Concentration optimization has chosen 0.05%, 0.1%, 0.15%3 level；Humidity optimum option 70%,
80%, 90%3 levels；24 DEG C of temperature selection, 26 DEG C, 28 DEG C of 3 levels；5 repetitions of each level；Factor level table is as follows
Table 1, cultivation cycle 7d.Then, according to Orthogonal experiment results, for CO2Concentration choose 0.10%, 0.15%, 0.20%,
0.25%, 0.30%5 horizontal progress experiment of single factor.
1 collybia albuminosa solid state rheology orthogonal optimization factor level of table
(4) it is inoculated with：With oese from the collybia albuminosa original seed in (1) 1 collybia albuminosa bacterium ball of picking (diameter about 0.2~
It 0.5cm) is inoculated into the center of plate, is placed in carbon dioxide incubator and cultivates, according to orthogonal design table (table 1), if 9 kinds
Processing, every group of each 5 repetition.
(5) measurement of growth amount：After culture 7 days, bacterium bulb diameter is measured with ruler, subtracts bacterium ball original diameter, as
The net growth diameter of bacterium ball.
This experiment is with CO2Concentration (A), humidity (B) and temperature (C) are three variables, just using 23.0 software design of SPSS
Experiment is handed over, the optimization of condition of culture is carried out using the net growth diameter of bacterium ball as index, concrete outcome is shown in Table 2 and table 3.It can therefrom see
Out, it is A that Group III quadratic sum, which is worth descending sequence,>B>C, it is A that showing, which influences maximum factor to experimental result, followed by
B is finally C.This illustrates CO2Concentration has certain importance to the growth of collybia albuminosa mycelia, this can be subsequent collybia albuminosa
Culture provide a new approaches.
2 collybia albuminosa solid state rheology orthogonal experiment data analytical table of table
Effect is examined between 3 collybia albuminosa solid state rheology main body of table
a.R2=0.818 (R after adjustment2=0.273)
Meanwhile being in conjunction with the optimal level that the optimal level that table 4 and Fig. 1 can be seen that A factor is 0.15%, B factor
The optimal level of 80%, C factor is 26 DEG C, i.e., optimal condition of culture combination is A3B2C2, i.e. carbon dioxide 0.15%, humidity
80%RH, 26 DEG C of temperature.To collybia albuminosa culture 7 days under the condition of culture, the net growth diameter of mycelia has reached about 1.1cm.
The marginal average value of 4 each factor of collybia albuminosa solid state rheology of table
It is found in conjunction with above-mentioned analysis of experimental data, CO2Concentration is affected to collybia albuminosa bacterium ball growth.Therefore, into one
5 CO of step setting2Concentration gradient is to screen the most suitable CO of collybia albuminosa2Concentration.Figure it is seen that working as CO2When concentration is 0.20%,
The average value (having a net increase of long diameter) of collybia albuminosa bacterium bulb diameter reaches maximum, about 0.75cm；And from the point of view of bacterium bulb diameter growth rate,
Work as CO2When concentration is 0.15%, the growth rate of bacterium bulb diameter reaches maximum, and about 39.29%, this shows in the CO2Under concentration, chicken
The speed of growth of fir bacterium is very fast.So using bacterium ball growth rate as main indicator, the most suitable CO of collybia albuminosa bacterium ball solid culture2It is dense
Degree is 0.15%.
First suboptimization of 3 collybia albuminosa liquid fermentation condition of embodiment
(1) preparation of level-one kind：The collybia albuminosa original seed optimized in embodiment 2 is transferred in fresh PDA fluid nutrient medium,
It is crushed with high-speed homogenization machine moderate strength, about 10~20 seconds (the broken time can be appropriately extended when strain is more), in 28 DEG C
5d, as level-one kind are cultivated in constant temperature oscillation shaking table, for the screening of indoor liquid fermentation medium and the optimization of condition of culture.
(2) preparation of culture medium：When carrying out collybia albuminosa first time Medium of shaking flask fermentation optimizing components, added according to table 5
The carbon source of variety classes and content, while adding peptone 2g, MgSO41g, KH2PO42g, K3PO41g, ant nest 10g, water
1000mL.Every group is all provided with 3 repetitions, shares 18 groups every time, dispenses after the completion of preparing, dispenses and be placed in autoclave 115 DEG C
Sterilize 30min, sterilizing, and cooling is spare.
5 collybia albuminosa first time Medium of shaking flask fermentation optimizing factors water-glass (g/L) of table
(3) it is inoculated with：The collybia albuminosa level-one kind obtained in (1) is continued in superclean bench equal with high-speed homogenization crusher machine
Even, with the strain of sterile pipette tips absorption 1% in prepared culture medium, inoculation finishes the perseverance for being placed on 28 DEG C, 180r/min
Warm shaking table carries out shaken cultivation.
(4) measurement of biomass：After culture 7 days, collybia albuminosa shake flask fermentation product is filtered with Medium speed filter paper, is obtained
Mycelium together with filter paper is placed in 60 DEG C of electric drying oven with forced convections, and drying to constant weight, and weighing subtracts filter paper own wt, as ferments
The mycelial dry weight of collybia albuminosa afterwards.
It is arranged according to orthogonal experiment factor level table, respectively with glucose (A), sucrose (B), sodium carboxymethylcellulose (C),
Microcrystalline cellulose (D), maltose (E), 6 factors of soluble starch (F) refer to as variable using mycelial dry weight as measurement
Mark the first suboptimization carried out liquid fermentation, data analysis result such as table 6.It is handled through 23.0 statistical software of SPSS, it can by table 7
To find out, descending Group III quadratic sum value is D>E>F>B>C>Influence degree of the influence factors such as A, D, E, F and B to experiment
It is big compared with other factors, and C and A is minimum to the influence degree of experiment；D, the P value of E is much smaller than 0.05, illustrates two factors pair of D, E
The influence of experimental result has a significant difference, and influence of the C and A factor to experimental result and indifference.
6 first suboptimization orthogonal experiment data analytical table of table
The inspection of effect between 7 first suboptimization main body of table
a.R2=0.967 (R after adjustment2=0.888)
Wherein the optimal level of A factor is 5g/L it can be seen from table 8 and Fig. 3, and the optimal level of B factor is 15g/L, C
The optimal level of factor is 10g/L, and the optimal level of D factor is 5g/L, and E factor optimal level is 15g/L, F factor it is optimal
Level is 15g/L, i.e., best medium is A at subassembly1B3C2D3E3F3.(the microcrystalline cellulose and can due to consideration that D, F factor
Soluble starch) occur incomplete dissolution phenomena during the experiment, the high phenomenon of vacation of experimental result is caused, so this time screening
E and B is selected to carry out subsequent screening and optimizing afterwards.
The marginal average value of 8 first each factor of suboptimization of table
Second suboptimization of 4 collybia albuminosa liquid fermentation condition of embodiment
(1) preparation of culture medium：Various composition is added according to table 9, while adding what embodiment 3 first time optimal screening went out
Preferably medium component and water 1000mL carry out second of Medium of shaking flask fermentation optimizing components of collybia albuminosa.Every group is all provided with 3
It repeats, shares 18 groups every time, dispensed after the completion of preparing, dispense and be placed on 115 DEG C of sterilizing 30min in autoclave, sterilized, it is cooling
It is spare.
9 collybia albuminosa of table, second of Medium of shaking flask fermentation optimizing factors water-glass (g/L)
(2) it is inoculated with：Collybia albuminosa level-one kind is continued in superclean bench to use high-speed homogenization crusher machine uniform, with sterile rifle
Head draws 1% strain in prepared culture medium, and inoculation finishes and is placed on 28 DEG C, the constant-temperature table of 180r/min shakes
Swing culture.
(3) measurement of biomass：After culture 7 days, collybia albuminosa shake flask fermentation product is filtered with Medium speed filter paper, is obtained
Mycelium together with filter paper is placed in 60 DEG C of electric drying oven with forced convections, and drying to constant weight, and weighing subtracts filter paper own wt, as ferments
The mycelial dry weight of collybia albuminosa afterwards.
According to the setting of orthogonal experiment factor level table, respectively with potato (A), maltose (B), sucrose (C), peptone
(D), ant nest extracting solution (E), microelement (F) (NaH2PO4、MgSO4、CaCl2、KH2PO4) it is that culture medium constituent is matched
Than, the second suboptimization carried out liquid fermentation using mycelial dry weight as measurement index, data result such as table 10.Pass through SPSS
23.0 statistical softwares carry out data processing, and as can be seen from Table 11, descending Group III quadratic sum value is B>A>D>F>C=E,
From the point of view of numerical values recited, the influence factors such as B, A and D are big compared with other factors to the influence degree of experiment, and C and E is to the shadow of experiment
The degree of sound is minimum；From the point of view of conspicuousness size in table, it can be seen that the P value of A, B, D, F are respectively less than 0.05, illustrate in the factor water
The flat lower experimental result of setting has significant difference, while R after adjustment2=0.951 illustrates that model-fitting degree is preferable.
10 second suboptimization orthogonal experiment data analytical table of table
The inspection of effect between 11 second suboptimization main body of table
a.R2=0.985 (R after adjustment2=0.951)
The optimal level of A factor is 200g/L it can be seen from table 12 and Fig. 4, and the optimal level of B factor is 10g/L, C
The optimal level of factor is 0g/L, and the optimal level of D factor is 6g/L, and the optimal level of E factor is 0g/L, F factor it is optimal
Level is (0.5:0:0.5:0.5) g/L, i.e., optimal culture horizontal combination is A3B3C1D3E1F3, i.e. potato 200g/L, maltose
10g/L, peptone 6g/L, calcium and magnesium potassium sodium ratio are 0.5:0:0.5:0.5.Wherein, the optimal level of factor C and E screening is 0g/
L, consideration optimize again.
The marginal average value of 12 second each factor of suboptimization of table
The third suboptimization of 5 collybia albuminosa liquid fermentation condition of embodiment
(1) preparation of culture medium：Various composition is added according to table 13, while adding potato 200g/L, MgSO40.5g/L,
KH2PO41g/L, NaH2PO41g/L, ant nest 10g/L carry out collybia albuminosa third time Medium of shaking flask fermentation optimizing components.Every group
3 repetitions are all provided with, share 18 groups every time, is dispensed after the completion of preparing, is dispensed and be placed on 115 DEG C of sterilizing 30min in autoclave, go out
Bacterium, cooling are spare.
13 collybia albuminosa third time Medium of shaking flask fermentation optimizing factors water-glass (g/L) of table
(2) it is inoculated with：Collybia albuminosa level-one kind is continued in superclean bench to use high-speed homogenization crusher machine uniform, with sterile rifle
Head draws 1% strain in prepared culture medium, and inoculation finishes and is placed on 28 DEG C, the constant-temperature table of 180r/min shakes
Swing culture.
(3) measurement of biomass：After culture 7 days, collybia albuminosa shake flask fermentation product is filtered with Medium speed filter paper, is obtained
Mycelium together with filter paper is placed in 60 DEG C of electric drying oven with forced convections, and drying to constant weight, and weighing subtracts filter paper own wt, as ferments
The mycelial dry weight of collybia albuminosa afterwards.
In conjunction with last optimization, maltose (A) is chosen again, sucrose (B), peptone (C), while adding yeast leaching
Powder (D), wheat bran (E), 6 factors of corn flour (F) equally carry out third as variable using mycelial dry weight as measurement index
Suboptimization screening, data result such as the following table 14.Data processing is carried out by 23.0 statistical software of SPSS, is shown in Table 15.95%
Under confidence level, F value and conspicuousness can be seen that influence of 4 factors such as A, D, E, F to experimental result all has significantly from table
Property, wherein the conspicuousness of A, F factor is more obvious, and the P value of B and C factor is all larger than 0.05, shows the factor to experimental result
Influence it is not significant；Therefore, it is known that maltose, corn flour, wheat bran and yeast powder are big compared with other factors to the influence degree of experiment,
Sucrose, peptone are smaller to the influence degree of experiment, which will provide important foundation to subsequent optimization.
14 third suboptimization orthogonal experiment data analytical table of table
After 15 third suboptimization of table between main body effect inspection
a.R2=0.976 (R after adjustment2=0.919)
The optimal level that can be seen that wherein A factor from table 16 and Fig. 5 is 15g/L, and the optimal level of B factor is 10g/
The optimal level of L, C factor is 3g/L, and the optimal level of D factor is 5g/L, and E factor optimal level is 30g/L, and F factor is most
It is 20g/L that excellent water is flat, i.e., best medium is A at subassembly3B3C2D3E3F3。
The marginal average value of each factor after 16 third suboptimization of table
4th suboptimization of 6 collybia albuminosa liquid fermentation condition of embodiment
(1) preparation of culture medium：Various composition is added according to table 17, while adding potato 200g/L, maltose 15g/L,
Yeast powder 5g/L, wheat bran 30g/L, corn flour 20g/L carry out the 4th Medium of shaking flask fermentation optimizing components of collybia albuminosa.Every group
3 repetitions are all provided with, share 18 groups every time, is dispensed after the completion of preparing, is dispensed and be placed on 115 DEG C of sterilizing 30min in autoclave, go out
Bacterium, cooling are spare.
The 4th Medium of shaking flask fermentation optimizing factors water-glass (g/L) of 17 collybia albuminosa of table
(2) it is inoculated with：Collybia albuminosa level-one kind is continued in superclean bench to use high-speed homogenization crusher machine uniform, with sterile rifle
Head draws 1% strain in prepared culture medium, and inoculation finishes and is placed on 28 DEG C, the constant-temperature table of 180r/min shakes
Swing culture.
(3) measurement of biomass：After culture 7 days, collybia albuminosa shake flask fermentation product is filtered with Medium speed filter paper, is obtained
Mycelium together with filter paper is placed in 60 DEG C of electric drying oven with forced convections, and drying to constant weight, and weighing subtracts filter paper own wt, as ferments
The mycelial dry weight of collybia albuminosa afterwards.
In conjunction with preceding optimization several times, the main component of culture medium determines substantially, this time mainly for microelement, vitamin
Class and ant nest leaching liquor are optimized once again, and the data that orthogonal experiment obtains are shown in Table 18.It is carried out by SPSS23.0 statistical software
Data processing is analyzed as shown in table 19, it can be seen that the P value of 6 factors such as A, B, C, D, E, F is all larger than 0.05, illustrates this
A little factors are not significant to the influence of this experimental result under respective horizontal, at the same from the average value of table 20 can be seen that each value it
Between differ smaller, since the variable of this suboptimization inherently belongs to microelement, so we take each factor in an experiment
Optimal level is as final content in the medium.
The 4th suboptimization orthogonal experiment analytical table of table 18
Effect is examined between the 4th suboptimization main body of table 19
a.R2=0.790 (R after adjustment2=0.286)
Data can be seen that under the setting of this factor level from table 20 and Fig. 6, and wherein the optimal level of A factor is
The optimal level of 0.15g/L, B factor is 0.6g/L, and the optimal level of C factor is 0.4g/L, and the optimal level of D factor is
The optimal level of 0.5g/L, E factor is 1.5g/L, and the optimal level of F factor is 5g/L, i.e. best medium is at subassembly
A3B3C2D1E3F1, i.e. vitamin B compound 0.15g/L, CaCl20.6g/L, MgSO40.4g/L, NaH2PO40.5g/L,
KH2PO41.5g/L, ant nest leaching liquor 5g/L.
The marginal average value of the 4th each factor of suboptimization of table 20
In summary four suboptimization as a result, final determining collybia albuminosa Liquid Culture based component is：Potato 200g/L, malt
Sugared 15g/L, yeast extract powder 5g/L, wheat bran 30g/L, corn flour 20g/L, vitamin B compound 0.15g/L, CaCl2 0.6g/
L, MgSO40.4g/L, KH2PO41.5g/L, NaH2PO40.5g/L, ant nest 5g/L.The optimization of subsequent liquid fermentation culture conditions
It will be carried out on the basis of this culture medium prescription.
The optimization of 7 condition of culture of embodiment
It chooses four pH value, temperature, inoculum concentration, liquid amount factors and carries out experiment of single factor.Wherein, optimize for pH value and select
3.0,4.0,5.0,6.0,7.0 5 levels are taken；Inoculum concentration optimum option 0.5%, 1%, 1.5% (v/v) three level；Dress
Liquid measure optimum option 20%, 30%, 40% (v/v) three level；28 DEG C of temperature selection, 25 DEG C, 22 DEG C of three levels；Each water
3 repetitions of flat setting, while optimal setting period 3d, 5d, 7d, 9d, 11d of inoculum concentration and liquid amount are distinguished in different cycles
Obtain a data.
1.pH value
Different microorganisms need different pH value during growth and breeding, only find most suitable pH value, growth
The speed of breeding can just be speeded, and yield can just increase.
During high-temperature sterilization, relevant chemical reaction may be occurred between medium component, pH value also can be with
Change, this research configuration culture medium before sterilization after pH value variation as shown in table 21, data can from table
Out, pH value generally can all slightly decrease after sterilizing.
The variation of the sterilizing of table 21 front and back pH value
In addition, as can see from Figure 7, when initial ph value is equal to 4.0, mycelium dry weight highest, as pH value constantly increases
Greatly, mycelium dry weight is gradually reduced, but it is more slow to reduce trend；And when pH value is reduced to 3.0, corresponding to mycelium
Sudden drop phenomenon occurs for dry weight, drops to 2.64 ± 0.12g/L by 13.25 original ± 0.17g/L, illustrates strong acid environment very not
It is suitble to the mycelial growth of collybia albuminosa, and weakly acidic condition is relatively suitble to the growth of thallus.Therefore, from this suboptimization it can be concluded that, most
The initial ph value for being suitble to collybia albuminosa mycelial growth is 4.0.
2. inoculum concentration
It is concentrated on the 9th day from figure 8, it is seen that dry mycelial weight caused by three different vaccination amounts reaches peak value.When
When inoculum concentration is 0.5%, dry mycelial weight reached 12.14 ± 0.24g/L of maximum value at the 9th day, when inoculum concentration is 1%, mycelia
Yield gap of the body the 7th day and the 9th day is unobvious, respectively 11.64 ± 0.20g/L and 11.84 ± 0.92g/L, and works as and connect
When kind amount is 1.5%, dry mycelial weight reached 11.48 ± 1.03g/L of maximum value, but the mycelial yield gap with the 7th day at the 9th day
Nor very big.So preferably selection is cultivated 9 days by 0.5% inoculation.But when due to 0.5% inoculation, production in the 9th day
Amount compared with the 7th day compared to amplification be not it is fairly obvious, therefore, different inoculations can be selected according to actual needs in actual production
Amount.For example, when strain supply abundance, optional 1.0% inoculum concentration, the period 7 days；It, can if strain supply is inadequate
Select 0.5% inoculum concentration, the period 7 days, or the period is appropriately extended.
3. liquid amount
Liquid amount mainly influences volume dissolved oxygen coefficient (KLA), and liquid amount is bigger, and dissolved oxygen coefficient is smaller, and thallus can not be fine
Growth；Liquid amount is fewer, and dissolved oxygen coefficient is also bigger, but liquid amount is fewer, the evaporation rate of solution in fermentation production process
It can be faster.Therefore, in actual production, the liquid amount of shaking flask or fermentor is also important to one for obtaining target product yield
Factor.
From fig. 9, it can be seen that mycelium dry weight caused by 30% liquid amount reached peak value at the 5th day, it is
14.3 ± 0.4g/L, and it is apparently higher than the mycelium dry weight of other test groups.The produced mycelia of 20% and 40% liquid amount point
Peak value is not reached the 7th day and the 9th day, produced mycelium dry weight changes width wherein 40% liquid amount was at the 7th day and the 9th day
Degree less, respectively 13.1 ± 0.2 and 13.2 ± 0.3g/L, but 30% liquid amount list when the peak value of the two was still less than the 5th day
Generated dry mycelial weight in the volume of position.Therefore, through above-mentioned analysis it is found that 30% liquid amount can be chosen in actual production
(v/v) it ferments for thallus.
4. temperature
Temperature is a critically important factor in microbial cultivation process, and the variation of temperature can cause in microbial body
The numerous biochemical reactions carried out, preference temperature can stimulate microorganism to grow, and uncomfortable temperature such as high temperature can make to have
The intracorporal enzyme inactivation of machine, low temperature can then reduce or stop the metabolism of microorganism, when temperature is too low, then it is dead will lead to cell
It dies.As can be seen from Figure 8, mycelium dry weight highest when cultivation temperature is 28 DEG C, reaches about 13.25 ± 0.17g/L, and 22 DEG C
Mycelium dry weight it is minimum, only about 3.88 ± 0.40g/L.Therefore, the optimum temperature when culture of collybia albuminosa shaking flask is 28 DEG C.
The production of 8 growth curve of embodiment
(1) the growth curve comparison of collybia albuminosa solid state rheology optimization front and back
Solid state rheology optimization is carried out to collybia albuminosa bacterium ball according to the design scheme of embodiment 2, is most suitable with resulting result
Condition of culture, the PDA culture medium before selection optimization carry out culture observation to collybia albuminosa bacterium ball in natural conditions (normal), meanwhile,
It is formula with the resulting optimum medium ingredient of embodiment 3~7, in CO2Solid state rheology is carried out to collybia albuminosa bacterium ball in incubator,
The setting period 30 days, every the bacterium ball growth value of measurement in 2 days, each 5 repetitions；It is bent that growth is drawn using bacterium bulb diameter as index
Line compares and analyzes the growth curve of the culture medium after optimization and the culture medium before optimization.
In summary liquid state fermentation and solid state rheology condition optimizing as a result, culture medium is in humidity after selection collybia albuminosa optimization
80%, 26 DEG C of temperature, CO2It is cultivated under the conditions of concentration 0.15%, draws the net growth diameter of collybia albuminosa bacterium ball and growth rate becomes
Change figure, and is compared with the growing state under its natural conditions in PDA culture medium.It can be seen from figure 11 that after optimization
Culture medium is that collybia albuminosa bacterium ball is promoted to grow on the whole compared to culture medium before optimizing in carbon dioxide incubator.Optimizing
On culture medium, the speed of preceding 6d growth is slower, this stage is lag phase, but rises in 6d, the diameter of bacterium ball growth and
Growth rate has apparent rising, is logarithmic phase；And before optimization on culture medium, the lag phase of collybia albuminosa bacterium ball experienced 9d,
Logarithmic growth phase is initially entered from 10d.In general, into the chicken after logarithmic phase, grown on the culture medium after optimization
Fir bacterium bacterium mean diameter of a ball is apparently higher than culture medium before optimization.
(2) the growth curve comparison before and after collybia albuminosa shake flask fermentation culture optimization
It is optimized according to shake flask fermentation of the design scheme of embodiment 3~7 to collybia albuminosa, with obtained most suitable culture
Based component is that formula combines optimal culture conditions, carries out shaking flask culture to collybia albuminosa, sets the period 17 days, is starting with the 3rd day
It is period, subsequent every the biomass of measurement in 2 days, every group of 3 repetitions；It is not optimised simultaneously with same setting method measurement
Biomass in PDA basal medium draws growth curve by index of biomass, by after optimization culture medium be not optimised
The growth curve of PDA culture medium compares and analyzes.
According to the optimization of the above fermentation medium and condition of culture, we compare culture medium and PDA after collybia albuminosa optimization
The comparison of basal medium growth curve, concrete outcome are shown in Figure 12.As seen from Figure 12, the collybia albuminosa of two kinds of culture medium cultures
Mycelium dry weight when reaching peak value at the 9th day, there is no shortenings with basal medium compared with for the culture medium after illustrating optimization
Collybia albuminosa dry mycelial weight reaches the cultivation cycle of peak value, but the peak difference of two kinds of culture mediums is away from larger, at the 9th day, PDA culture
The mycelium of base culture is only 7.75 ± 1.00g/L, and after optimizing the mycelium dry weight of culture medium culture up to 15.91 ±
0.28g/L.Therefore, in industrialized production process, the culture medium after can choose optimization carries out 9 days harvest bacterium of fermenting and producing
Filament.
Conclusion：In conjunction with SPSS orthonormal design of experiments and experiment of single factor, with the average value of collybia albuminosa bacterium ball growth diameter
It is index with growth rate, the solid state rheology condition of collybia albuminosa is optimized.The result shows that carrying out collybia albuminosa solid state rheology
When, humidity 80%, CO2Concentration 0.15%, 26 DEG C of temperature of condition of culture can effectively facilitate the growth of collybia albuminosa bacterium ball.It is most suitable
Fluid nutrient medium component be potato 200g/L, maltose 15g/L, yeast extract powder 5g/L, wheat bran 30g/L, corn flour 20g/L,
Vitamin B compound 0.15g/L, CaCl20.6g/L, MgSO40.4g/L, KH2PO41.5g/L, NaH2PO40.5g/L, ant nest
5g/L；Optimal culture conditions are initial ph value 4.00, liquid amount 30% (v/v), inoculum concentration 1% (v/v), 28 DEG C of temperature, shaking table
Revolving speed 180r/min, cultivation cycle 9 days, collybia albuminosa dry mycelial weight was up to 15.91g/L under the condition of culture after optimization.
Embodiment described above is only that preferred embodiments of the present invention will be described, not to model of the invention
It encloses and is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical side of the invention
The various changes and improvements that case is made, should fall within the scope of protection determined by the claims of the present invention.
Claims (8)
1. a kind of fluid nutrient medium for being cultivated in collybia albuminosa room, which is characterized in that the component including following mass concentration：
100~200g/L of potato,
5~15g/L of carbohydrate,
3~5g/L of nitrogen source,
20~30g/L of wheat bran,
10~20g/L of corn flour,
0.1~0.15g/L of vitamin B compound,
2.5~4g/L of microelement,
5~10g/L of ant nest.
2. a kind of fluid nutrient medium for being cultivated in collybia albuminosa room according to claim 1, it is characterised in that：The carbon
Hydrate is at least one of maltose, microcrystalline cellulose, soluble starch.
3. a kind of fluid nutrient medium for being cultivated in collybia albuminosa room according to claim 1, it is characterised in that：The nitrogen
Source is yeast powder and/or peptone.
4. a kind of fluid nutrient medium for being cultivated in collybia albuminosa room according to claim 1, it is characterised in that：It is described micro-
It include calcium, magnesium, sodium, potassium in secondary element.
5. a kind of fluid nutrient medium for being cultivated in collybia albuminosa room according to claim 1, which is characterized in that the training
Feeding base includes the component of following mass concentration：
Potato 200g/L,
Maltose 15g/L,
Yeast powder 5g/L,
Wheat bran 30g/L,
Corn flour 20g/L,
Vitamin B compound 0.15g/L,
CaCl20.6g/L,
MgSO40.4g/L,
KH2PO41.5g/L
NaH2PO40.5g/L,
Ant nest 5g/L.
6. a kind of cultural method of described in any item fluid nutrient mediums of Claims 1 to 5 for cultivating in collybia albuminosa room, special
Sign is, includes the following steps：
(1) jack bacterium in picking ant nest, is seeded on PDA solid medium, cultivates in 28 DEG C of constant incubators, draws repeatedly
Line culture obtains the collybia albuminosa strain of purifying；
(2) the collybia albuminosa mycelia of purifying is connected in PDA liquid medium, is cultivated in 28 DEG C of isothermal vibration shaking tables, obtain chicken
Fir bacterium bacterium ball；
(3) by collybia albuminosa bacterium ball in humidity 70~90%, CO2In concentration 0.1~0.2%, 24~28 DEG C of temperature of solid medium
Culture obtains collybia albuminosa original seed；
(4) collybia albuminosa original seed is inoculated into fluid nutrient medium, in initial ph value 4.00, liquid amount 20~40% (v/v), inoculation
0.5~1.5% (v/v) is measured, 25~28 DEG C of temperature, shaking speed 180r/min are cultivated 7~9 days.
7. the cultural method cultivated in collybia albuminosa room according to claim 6, it is characterised in that：It is solid described in step (3)
Body condition of culture is humidity 80%, CO226 DEG C of concentration 0.15%, temperature.
8. the cultural method cultivated in collybia albuminosa room according to claim 6, it is characterised in that：The liquid amount is 30%
(v/v), inoculum concentration is 1% (v/v), and temperature is 28 DEG C, cultivation cycle is 9 days.
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| CN111345199A (en) * | 2019-06-20 | 2020-06-30 | 四川乌蒙山四季菌业有限责任公司 | Black skin termitomyces liquid strain culture solution and preparation method thereof |
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| CN107517733A (en) * | 2017-09-29 | 2017-12-29 | 贵州棒棒食用菌产业有限公司 | A kind of artificial collybia albuminosa cultural method |
| CN107896824A (en) * | 2017-11-29 | 2018-04-13 | 四川山水美地农业投资有限公司 | A kind of cultural method of Termitomyces albuminosus with black skin |
| CN108029458A (en) * | 2017-11-29 | 2018-05-15 | 四川山水美地农业投资有限公司 | A kind of preparation method of Termitomyces albuminosus with black skin liquid spawn |
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| CN105695338A (en) * | 2016-01-25 | 2016-06-22 | 四川保兴现代农业科技股份有限公司 | Liquid spawn propagation method and field bionic cultivation method of termitomyces albuminosus |
| CN107517733A (en) * | 2017-09-29 | 2017-12-29 | 贵州棒棒食用菌产业有限公司 | A kind of artificial collybia albuminosa cultural method |
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