CN108739056A - Application of the wheatfeed in inhibiting Phellinus fermentation mycelium aging - Google Patents
Application of the wheatfeed in inhibiting Phellinus fermentation mycelium aging Download PDFInfo
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- CN108739056A CN108739056A CN201810349591.1A CN201810349591A CN108739056A CN 108739056 A CN108739056 A CN 108739056A CN 201810349591 A CN201810349591 A CN 201810349591A CN 108739056 A CN108739056 A CN 108739056A
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- wheatfeed
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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
- A01G18/20—Culture media, e.g. compost
Abstract
The present inventor passes through the study found that application of the wheatfeed in inhibiting Phellinus fermentation mycelium aging.The application process is：Wheatfeed is added in Phellinus fermentation medium, a concentration of 5-10g/L of the wheatfeed in Phellinus fermentation medium.The inventors found that addition wheatfeed can inhibit the aging of mycelia, continue to grow.Mycelial biomass, bioactivity can be improved using wheatfeed.
Description
Technical field
The invention belongs to Phellinus fermentation arts, and in particular to application of the wheatfeed in delay Phellinus fermentation mycelium aging.
Background technology
Phellinus bacterium is a kind of very precious macro fungi with important medical value, is Basidiomycotina
(Basidiomyctina).It is grown on Japan, Philippine, Australia, the few places such as China and North America.Phellinus is often parasitic
On the dead wood of mulberry tree, fructification be it is perennial, it is wooden.Have so far from Han dynasty in the use of China, Phellinus bacterium
More than 2000 years history.《Compendium of Materia Medica》Phellinus energy " relieving the five internal organs, suitable flatus, toxin expelling gas " is recorded, it is civil using it as one
Kind controls the miraculous cure of hepatopathy, cancer, is known as " strain superfine product ".
Due to the particularity of Phellinus own growth, cause natural Phellinus quantity very rare, it can hyoscine in nature
Resource it is limited, it is difficult to become stable industrial products source, it is very unfavorable to the development of Phellinus industry.
Phellinus artificial cultivation is extremely difficult, and condition of culture is harsh, and growth cycle is up to 3-4, is also unfavorable for industrializing
Large-scale production.Therefore obtaining mycelium using liquid fermentation and culture is particularly important.Studies have shown that liquid fermentation and culture obtains
It obtains mycelial various active constituents and is similar to fructification.Moreover, during Submerged liquid culturation, hyphal cell can be anti-
It answers in device (fermentation tank or big triangular flask) to be in optimum temperature, Optimum pH, is grown under the conditions of most suitable carbon, nitrogen ratio etc., breathed
Metabolism exhaust gas caused by effect can discharge in time again, therefore metabolism is vigorous, and mycelia growth is rapid, can in asking in short-term
To obtain a large amount of mycelium, have the production time it is short, it is efficient, it is at low cost the advantages that.Meanwhile it can also be generated in zymotic fluid more
Sugar, alkaloid, terpenoid, sterol, glucoside, phenols, enzyme, nucleic acid, amino acid, vitamin, plant hormone and have antibiosis
The several physiological active substances such as the various compounds of element effect.These substances respectively to angiocarpy, liver, nervous system, kidney,
The human bodies organ disease such as sexual organ has prophylactic treatment effect, and with work(such as anticancer, anti-inflammatory, antibacterial, anti-aging, antiulcers
Effect.
In existing production technology and document report, the biomass of Phellinus fermentation mycelium is not high, and not by mycelium
Bioactivity as evaluation phellinus igniarius mycelium index.
Invention content
The present inventor passes through the study found that wheatfeed has the application inhibited in Phellinus fermentation mycelium aging.
The application process is：Wheatfeed is added in Phellinus fermentation medium.
In a preferred embodiment of the invention, a concentration of 5-10g/L of the wheatfeed in Phellinus fermentation medium.
The inventors found that addition wheatfeed can inhibit the aging of mycelia, continue to grow.Utilize wheatfeed
It is high to obtain mycelial biomass height, bioactivity.
In a specific embodiment of the invention, which includes following component by weight：Glucose 10-
15 parts, 15-20 parts of sucrose, 15-25 parts of corn flour, 10-20 parts of dregs of beans, 0-10 parts of yeast powder, 5-10 parts of wheatfeed, MgSO4·
7H20.25-1 parts of O, 2 parts of CaCl2,0.1-0.5 parts of VB1, a concentration of 4-8 parts of ramulus mori water extract.
Fermentation medium component (g/L) provided by the invention：Glucose 10-15, sucrose 15-20, corn flour 15-25, beans
Dregs of rice 10-20, yeast powder 0-10, wheatfeed 5-10, MgSO4·7H2O 0.25-1、CaCl22, VB1 0.1-0.5, ramulus mori water carry
A concentration of 4-8g/L of object.Utilize this culture medium shaker fermentation technological parameter：Inoculum concentration 5-15%；24-30 DEG C of cultivation temperature, shakes
Bed rotating speed 140-180rpm.Using the culture medium prescription and technological parameter of the present invention, phellinus igniarius mycelium biology can be greatly improved
Amount and bioactivity, mycelial biomass reach as high as 3.0%, and other biological activity index is respectively：Adenosine 0.26%, polysaccharide
2.24%, ergosterol 0.37%, mannitol 12.45%.
Specific implementation mode
1 carbon source of embodiment is screened
1.1 culture medium
Shake-flask seed culture medium：Glucose 15-25g/L, peptone 5-15g/L, MgSO4·7H2O 0.5g/L, KH2PO4
2g/L, VB10.1g/L, pH 6.0.
Carbon source screens basal medium：Peptone 5-15g/L, MgSO4·7H2O 0.5g/L, KH2PO42g/L,
VB10.1g/L, pH 6.0.
1.2 test method
Level liquid Spawn incubation：150mL shake-flask seed culture mediums are filled in 500mL triangular flasks, take cultured inclined-plane
Parent species access triangular flask, are placed under 26-32 DEG C of constant-temperature table, and rotating speed 140-180r/min cultivates 7-10d, to get level liquid
Strain.
Secondary liquid Spawn incubation：150mL shake-flask seed culture mediums are filled in 500mL triangular flasks, draw level liquid bacterium
Kind, inoculum concentration 15-30%.It is placed under 26-32 DEG C of constant-temperature table, rotating speed 140-180r/min, cultivates 2-3d.
Shake flask fermentation culture：Cultured secondary liquid strain is taken, is 10% according to inoculum concentration, is inoculated in 5 kinds respectively not
With carbon source culture medium (i.e. carbon source screen basal medium in add sucrose, glucose, lactose, corn flour, maltose respectively,
Additive amount is 20-30g/L), it is placed under 26-32 DEG C of constant-temperature table, rotating speed 140-180r/min, cultivates 7-10d, 3 parallel examinations
It tests, the influence that analysis different carbon source grows phellinus igniarius mycelium obtains two kinds of optimum carbon sources.
1.3 carbon source screening experiment results
Experiment is chosen glucose, sucrose, lactose, corn flour and maltose and is added as different carbon source, is surveyed after culture is ripe
The percentage composition for determining nucleosides in mycelium, ergosterol, mannitol, polysaccharide and total nitrogen, referring to table 1.3.
Under 1.3 different carbon source culture of table in bacterium powder every Biological indicators percentage composition
Nucleotide：Using corn flour as the content of adenosine, guanosine and uridine in the bacterium powder of carbon source culture and other carbon source phases
Increase than conspicuousness, wherein adenosine increases 50.0%, 39.5%, 67.9% than glucose, sucrose, lactose and maltose respectively
With 52.4%；Guanosine has increased separately 55.9%, 89.4%, 55.4% and 76.4%；Uridine increased separately 33.0%,
24.0%, 52.8% and 42.6%.
Ergosterol：It is apparently higher than lactose and maltose using corn as content in the mycelium of carbon source culture, is carried respectively
It is high by 14.5% and 15.6%, and the then difference unobvious compared with glucose, sucrose, wherein increase 1.28% than sucrose, but
8.67% is but had dropped than glucose.
Mannitol：Using the content of corn flour as mannitol in the bacterium powder of carbon source culture be apparently higher than with glucose, sucrose,
Lactose and maltose have increased separately 15.2%, 11.4%, 16.9% and 20.7% as carbon source culture medium.
Polysaccharide：Using maltose as the polyoses content highest of carbon source culture, respectively than glucose, sucrose, lactose and corn
Powder increases 65.1%, 57.7%, 46.5% and 46.4%；And using corn flour as polyoses content and grape in carbon source culture
Sugar, sucrose are apparent compared to increasing, and have increased separately 34.9% and 21.2%, no significant difference compared with lactose.
Total nitrogen：It is trained using corn flour as total nitrogen content in the bacterium powder of carbon source culture with glucose, sucrose, lactose and maltose
Base is supported compared to trend is significantly increased, has increased separately 18.0%, 15.2%, 24.5% and 23.3%.
It is analyzed according to data above, using corn flour as nucleosides, the content of mannitol and grape in the bacterium powder of carbon source culture
Sugar, sucrose, lactose are compared with maltose and are had a clear superiority, and not notable for the content maize powder medium advantage of ergosterol
But still there is increased trend；Bacterium powder content advantage using maltose as the middle polysaccharide of carbon source culture is notable, hence it is evident that is higher than other
Carbon source culture medium.
2 nitrogen source of embodiment is screened
2.1 culture medium
Shake-flask seed culture medium：Glucose 15-25g/L, peptone 5-15g/L, MgSO4·7H2O 0.5g/L, KH2PO4
2g/L, VB10.1g/L, PH 6.0.
Nitrogen source screens basal medium：Glucose 15-25g/L, MgSO4·7H2O 0.5g/L, KH2PO42g/L,
VB10.1g/L, PH 6.0.
2.2 test method
Level liquid strain is with secondary liquid Spawn incubation method as 2.1.Shake flask fermentation culture：Take cultured two
Grade liquid spawn, is 10% according to inoculum concentration, is inoculated in 6 kinds of different nitrogen sources culture mediums respectively and (screens basis culture in nitrogen source
Dregs of beans, peptone, yeast powder, urea, beef extract and wheat bran, additive amount 10-25g/L are added in base respectively), it is placed in 26-32
Under DEG C constant-temperature table, rotating speed 140-180r/min cultivates 7-10d, and 3 parallel tests, analysis different carbon source is to phellinus igniarius mycelium
The influence of growth obtains two kinds of optimum nitrogen sources.
2.3 nitrogen source screening experiment results
2.3.1 the nitrogen source screening experiment result under identical incubation time
Experiment is chosen six kinds of dregs of beans, peptone, yeast powder, urea, beef extract and wheat bran and is added as different nitrogen sources, culture
The percentage composition (table 2.3.1) of nucleosides, ergosterol, mannitol, polysaccharide and total nitrogen in bacterial strain is measured after same time.
Under table 2.3.1 nitrogen source primary screening cultures in bacterium powder every Biological indicators percentage composition
Note："/" indicates that biomass can not measure very little
According to table as can be seen that in using peptone, urea and beef extract as the culture medium of nitrogen source with dregs of beans, yeast powder and
Wheat bran is compared, and bacterial strain growing way is obviously suppressed or even stops growing, and yield conspicuousness reduces.
Ergosterol：It to be apparently higher than yeast using dregs of beans as the content of adenosine and ergosterol in the bacterium powder of nitrogen source culture
Powder and wheat bran, wherein adenosine have increased separately 12.9% and 40.6%；Ergosterol has increased separately 27.3% and 44.5%.
Mannitol：The content of mannitol is apparently higher than other two kinds of nitrogen sources in using wheat bran as the bacterium powder of nitrogen source culture, respectively
It is higher by 12.1% and 17.5% than dregs of beans and yeast powder.
Total nitrogen：Total nitrogen content highest in using dregs of beans as the bacterium powder of nitrogen source, increases than yeast powder and wheat bran respectively
16.4% and 61.7%；And be polyoses content in the bacterium powder of nitrogen source compared with other two nitrogen sources using yeast powder, conspicuousness increases,
Respectively 45.5% and 15.0% are increased than dregs of beans and wheat bran.
2.3.2 the nitrogen source screening experiment result under identical maturity
It is tested according to 2.3.1, it is found that strain growth maturity differs greatly under different nitrogen sources culture medium, therefore set with bacterium
Strain culture maturity carries out second of nitrogen source screening experiment for fermentation termination.Experiment choose dregs of beans, three kinds of yeast powder and wheat bran
It is added as different nitrogen sources, the percentage of nucleosides, ergosterol, mannitol, polysaccharide and total nitrogen in bacterial strain is measured after culture is ripe
Content (table 2.3.2)
Under table 2.3.2 nitrogen source postsearch screening cultures in bacterium powder every Biological indicators percentage composition
Using dregs of beans as adenosine in the bacterium powder of nitrogen source and ergosterol, the conspicuousness compared with other two kinds increases, wherein adenosine point
27.1%, 64.8% is not increased than yeast powder, wheat bran；Ergosterol has increased separately 19.0%, 56.0%.
Total nitrogen content is apparently higher than wheat bran in using dregs of beans and yeast powder as the bacterium powder of nitrogen source culture, has increased separately 69.5%
With 70.6%, and dregs of beans and yeast powder are then not significantly；Polyoses content highest in using yeast powder as the bacterium powder of nitrogen source culture, compares respectively
Dregs of beans and wheat bran increase 41.9% and 59.3%.
From the analysis above, we can see that using dregs of beans as adenosine, ergosterol and total nitrogen content highest in the bacterium powder of nitrogen source culture, with
Other nitrogen sources, which are compared, has significant advantage, and notable using yeast powder as polyoses content advantage in the bacterium powder of nitrogen source culture, hence it is evident that
Higher than other nitrogen sources.The content of mannitol does not show any trend, and content is not much different in each nitrogen source.So choosing
Dregs of beans and yeast powder are compound nitrogen source.
The screening of 3 carbon source optimal combination of embodiment and carbon-nitrogen ratio
3.1 orthogonal experiment
3.1.1 experimental program
According to carbon nitrogen source the selection result, three kinds of experimental selection corn flour, sucrose and glucose carbon sources are as compounded carbons, choosing
Two kinds of dregs of beans, yeast powder nitrogen sources are selected as compound nitrogen source, using SPSS Software for Design orthogonal tests, quadrature factor and level are shown in Table
3.1.1,5 factor+4 is horizontal, determines carbon nitrogen source optimal combination and optimum proportioning.
Table 3.1.1 orthogonal tests factor and content (g/L)
According to above-mentioned configuration, every group is both needed to that MgSO is added the carbon-nitrogen ratio of assay medium4·7H2O 0.5g/L, KH2PO4
1g/L, PH 6.0 is placed under 26-32 DEG C of constant-temperature table, rotating speed 14-180r/min cultures.
3.1.2 experimental result
Table 3.1.2 lists under carbon-nitrogen ratio screening and culturing every Biological indicators content (%) in bacterium powder
Table 3.1.2
It is analyzed using SPSS softwares, determines the best two groups of schemes of carbon-nitrogen ratio：
A groups：Glucose 10-15g/L, sucrose 10-15g/L, corn flour 10-15g/L, dregs of beans 10-20g/L, yeast powder 10-
15g/L、MgSO4·7H2O 0.5g/L, KH2PO41g/L, PH 6.0,
B groups：Glucose 10-15g/L, sucrose 15-20g/L, corn flour 15-25g/L, dregs of beans 10-20g/L, yeast powder 10-
15g/L、MgSO4·7H2O 0.5g/L, KH2PO41g/L, PH 6.0,
The above culture medium sterilizes 30min at 121 DEG C, and inoculation is placed under 26-32 DEG C of constant-temperature table, rotating speed 140-180r/
Min is cultivated to fermenting-ripening, in triplicate.
3.2 orthogonal experiments are verified
3.2.1 experimental program
It is matched according to carbon nitrogen as a result, two groups of experimental selection A, B is verified, three parallel, repeats three times, selects best
Scheme.
3.2.2 experimental result
Every Biological indicators content (%) in bacterium powder in the orthogonal verification test of table 3.2.1 carbon-nitrogen ratios
It is analyzed using SPSS softwares, there were significant differences on polysaccharide for A groups and B groups, and B groups are significantly higher than A groups, other indexs do not have
Significant difference, so selection B groups：Glucose 10-15g/L, sucrose 15-20g/L, corn flour 15-25g/L, dregs of beans 10-
20g/L, yeast powder 10-15g/L, MgSO40.5g/L, KH2PO41g/L, PH 6.0.
The screening of 4 inorganic salts of embodiment and vitamin and microelement
4.1 orthogonal test
4.1.1 experimental program
Carbon nitrogen optimum proportioning formulated in combination inorganic salts and vitamin and microelement the screening liquid training drawn with embodiment 3
Support base.MgSO is added in vitamin4·7H2O 0.5g/L, KH2PO41g/L.Using L9(34) orthogonal design, determine inorganic salts and
Vitamin type and its optimal addition, improve the component of fluid nutrient medium.Inorganic salts, vitamin type and its matter of addition
Amount is shown in Table 4.1 and table 4.2 respectively.
4.1 L of table9(34) orthogonal test factor and content (g/L)
4.2 L of table9(34) orthogonal test factor and level (g/L)
Culture medium sterilizes 30min at 121 DEG C, and inoculation is placed under 26-32 DEG C of constant-temperature table, rotating speed 140-180r/min trainings
It supports to fermenting-ripening, in triplicate.
4.1.2 experimental result
Every Biological indicators content (%) in bacterium powder under 4.5 small-scale inorganic salt screening and culturing of table
| Biological indicators | Adenosine | Polysaccharide | Ergosterol | Mannitol | Mycelia yield |
| 1 | 0.188 | 5.218 | 0.216 | 12.917 | 2.957 |
| 2 | 0.247 | 1.991 | 0.272 | 12.735 | 3.237 |
| 3 | 0.244 | 1.547 | 0.287 | 13.134 | 3.343 |
| 4 | 0.236 | 1.716 | 0.278 | 12.888 | 3.423 |
| 5 | 0.184 | 4.814 | 0.198 | 13.067 | 2.463 |
| 6 | 0.237 | 1.528 | 0.288 | 13.037 | 3.297 |
| 7 | 0.220 | 1.721 | 0.311 | 12.515 | 3.020 |
| 8 | 0.222 | 1.641 | 0.280 | 12.895 | 3.537 |
| 9 | 0.201 | 3.733 | 0.192 | 12.164 | 2.820 |
It is analyzed using SPSS softwares, it is as a result as follows：
| Biological indicators | Adenosine | Polysaccharide | Ergosterol | Mannitol | Mycelia yield |
| Primary and secondary sequence | DBCA | DBAC | DBCA | ACDB | DCAB |
| Optimum organization | A1B3C2D3 | A1B1C1D1 | A3B3C3D2 | A1B2C1D3 | A1B3C1D3 |
Wherein CaCl2Yield is influenced significantly, other are without conspicuousness.Primary and secondary sequence and required life are influenced according to factor
Object index determines inorganic salts optimal combination：KH2PO4 0g/L、MgSO4·7H2O 0-1g/L、(NH4)3C6H5O7 0g/L、
CaCl20-2g/L.Optimal combination is subjected to experimental verification, three parallel, repeats three times, as a result as follows：
Every Biological indicators content (%) in the lower bacterium powder of 4.6 small-scale inorganic salt of table verification
| Biological indicators | Adenosine | Polysaccharide | Ergosterol | Mannitol | Mycelia yield |
| 0.188 | 1.666 | 0.212 | 9.045 | 3.415 |
Every Biological indicators content (%) in the lower bacterium powder of 4.7 vitamin of table screening
It is analyzed using software, it is as a result as follows：
Wherein VB1 influences conspicuousness height to ergosterol, other are without conspicuousness.According to factor influence primary and secondary sequence and
Required Biological indicators obtain vitamin optimal combination：A3B1C1, i.e. VB1 0-0.5g/L, VB6 0g/L, VC 0g/L.It will be best
Combination carries out experimental verification, and three parallel, repeats three times, as a result as follows：
Every Biological indicators content (%) in the lower bacterium powder of 4.8 vitamin of table verification
Influence of the wheatfeed and yeast powder of 5 different proportion of embodiment to Phellinus liquid fermentation
5.1 experimental method
It is found in yeast powder screening test, addition wheatfeed can inhibit the aging of mycelia, continue to grow, therefore set
The additive amount (the two additive amount is 0-15g/L) of fixed experiment, more different wheatfeeds and yeast powder is to the shadow of Phellinus liquid fermentation
It rings.This experiment is combined as basal medium with carbon nitrogen optimum proportioning, then adds the wheatfeed of different proportion：Yeast powder=0:1,1:
0,1:1,1:2,2:1.Culture medium sterilizes 30min at 121 DEG C, and inoculation is placed under 26-28 DEG C of constant-temperature table, rotating speed 140-
160r/min is cultivated to fermenting-ripening.Every group three parallel, in triplicate, finally determines suitable combination.
5.2 experimental result
Influence of the wheatfeed and yeast powder of 5.2 different proportion of table to Phellinus items Biological indicators content (%)
By result it is found that by homogeneity test of variance, meet condition.Yield, polysaccharide, adenosine are differentiated.It is comprehensive
It closes and considers each factor, it is wheat bran 5-10g/L, yeast powder 0-10g/L to choose optimal combination.
Claims (3)
1. application of the wheatfeed in inhibiting Phellinus fermentation mycelium aging.
2. application of the wheatfeed in delay Phellinus fermentation mycelium aging according to claim 1, wherein the application process
For：Wheatfeed is added in Phellinus fermentation medium.
3. application of the wheatfeed in delay Phellinus fermentation mycelium aging according to claim 2, it is characterised in that：Wheatfeed
A concentration of 5-10g/L in Phellinus fermentation medium.
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