CN109321473B - A kind of Liquid Culture Qaidam Agaricus bitorqui produces mycelial method - Google Patents
A kind of Liquid Culture Qaidam Agaricus bitorqui produces mycelial method Download PDFInfo
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Abstract
The invention discloses a kind of Liquid Culture Qaidam Agaricus bitorquis to produce mycelial method, cooperate deionized water using Qaidam Agaricus bitorqui slant strains, highland barley as raw material and as analytically pure glucose, agar powder, peptone, magnesium sulfate and calcium chloride, culture medium is got out, is then carried out by following below scheme: seed slant activation, the preparation of level liquid seed, secondary seed preparation, fermented and cultured.The present invention by design by seed slant activation, the preparation of level liquid seed, secondary seed preparation, fermented and cultured and etc. constitute mycelial method is produced by Liquid Culture Qaidam Agaricus bitorqui; it only needs to cultivate within one month or so to obtain Qaidam Agaricus bitorqui production mycelium in total; not only speed is fast; and method is simple, easy to operate; its mycelium can replace wild Qaidam Agaricus bitorqui fructification, so as to shield to wild Qaidam Agaricus bitorqui resource.
Description
Technical field
The present invention relates to technical field of biological culture, and in particular to a kind of mycelial side of culture Qaidam Agaricus bitorqui production
Method.
Background technique
Edible and medicinal fungi abbreviation edible mushroom, in China, oneself has long research history.Eastern Han Dynasty end before more than 2,000 years
Phase, the drug effect for just describing ganoderma lucidum in first drug monograph Shennong's Herbal in the world, obtaining the fungies such as an aromatic plant metioned in ancient books, pig an aromatic plant metioned in ancient books；The Ming Dynasty
Famous physician's Li Shizhen (1518-1593 A.D.) has recorded more than 20 kinds of fungies in Compendium of Material Medica.Existing result of study shows that oneself knows to sarcoma
Have nearly 300 kinds up to 60% one 100% fungi with ehrlich carcinoma inhibiting rate, adhere to 51 section more than 70 separately and belong to, most of fungies it is anti-swollen
Tumor activity is the polysaccharide and protein combination polysaccharide body with specific structure.Edible and medicinal fungi is abundant in china natural resources, is to find
The precious resources library of novel drugs, both at home and abroad have developed rapidly the development and utilization of edible and medicinal fungi polysaccharide, research of the past to polysaccharide
It is concentrated mainly on the exploitation to traditional resource, isolation and purification method is inquired into and physiological activity experiment aspect.At present so that in the future,
Its hot spot is concentrated mainly on the relationship probed between active polysaccharide and structure effect, and modification natural polysaccharide enhances its original activity；Research
The immunization mechanism of fungi polysaccharide expands clinical application field；The breeding and submerged fermentation of polysaccharide superior strain regulate and control.
Edible and medicinal fungi polysaccharide is a kind of life active material that can enhance immune function of human body, is referred to as in the world
Biological response modifiers (Biological Response Modifier, BRM).Early in nineteen fifty-seven, Benacerraf and
Sebestyn has found that zymosan (Zymosan) of the intravenous injection from yeast cell wall has an impact to phagocyte activity.
Subsequent people isolate and purify zymosan, and Riggi in 1961 has determined that this active constituent in zymosan is
Glucan has started the new era of glucan as immunologic active material.1969, Chihara et al. was true from edible medicinal respectively
Isolated bioactive substance in mushroom entity, and demonstrate the chief active that Q- (1-3)-D- glucan is tumor-inhibiting action
Ingredient.Hereafter, people successively from rainbow conk, obtain in a variety of edible and medicinal fungis such as an aromatic plant metioned in ancient books, pig an aromatic plant metioned in ancient books, ganoderma lucidum and isolate polysaccharide, and to them
Immunocompetence and anti-tumor capacity evaluated.
Since the 1970s, with the progress table of immune substance, biomembrane and various bioactivators
Bright, carbohydrate is not only the significant energy source of all living organisms and the necessary structural material that sustains life, more important
Be that it takes part in the various activities of cell in life science, such as participate in the adjusting of the immune function, identification of cell and cell, thin
The transport of intercellular substance, cancer Clinics and Practices, the division and differentiation of cell, the growth of cell and aging etc..Due to eating medicine
With fungi polysaccharide and its varied bioactive functions of compound and being widely used in functional food and clinically, make
It becomes one of hot spot of research fields such as life science, biology, medicine and Food Science net in recent years.
Qinghai-Tibet Platean is that macro fungi is distributed highest region in the world, is suitable for resist oxygen lack, cold-resistant macro fungi growth.According to
Rough estimates, Himalaya 3000~5800m of height above sea level just have more than macro fungi 300, needle mushroom, Armillaria luteo-virens, leady ash
Coccus equal distribution is more than height above sea level 4000m, rare for the world.So Qinghai-Tibet Platean summarizes the highest large size of distribution on global very
Bacterium, the referred to as world " high mountain fungi treasure-house ".Qaidam big fat mushroom Agaricus bitorquis (Qu é l.) Sacc. conduct
Representative species in Qinghai-Tibet fungus resource treasure-house is acknowledged as most having one of rare wild edible mushroom of Development volue, tool
There is the features such as underground is solid, fructification is huge, resistance is strong, full of nutrition, is that the special strain in plateau in big fat mushroom is (high refined
It is quick, 2010).Height above sea level is distributed mainly on from surrounding 4000m to the Caidamu Basin of middle part 2600m, is grown in basin Gobi Region
Under the salt deposit 50-60cm of surface, a variety of Substituted phenyl-lactic acid substances are rich in, are known as one of " basin four is precious ", it is all the time local
Herdsman eats extensively as the food materials of anti anoxia, raising resistance, and excavation leads to prairie soil vegetation and wild Qaidam year after year
Agaricus bitorqui growing environment is seriously destroyed.Some researches show that its mycelium to have and Agaricus bitorqui fructification (mushroom) consistent battalion
It forms point, using Liquid Culture Qaidam Agaricus bitorqui hyphal cell, produces mycelium and replace fructification, it not only can be with effective protection
This valuable source can also be further exploitation Qaidam Agaricus bitorqui intracellular polyse, triterpene isoreactivity ingredient, establish substance base
Plinth.Studies have shown that exhausting trip to mouse power by comparing Qaidam Agaricus bitorqui fruitbody polysaccharide, polysaccharide of fermentation broth and mycelium polysaccharides
Swimming time and power exhaust after swimming Hb in mouse blood, BUN, LDH and LA content in serum, in liver HG changes of contents difference
It is anisotropic, the results showed that Qaidam Agaricus bitorqui fruitbody polysaccharide, polysaccharide of fermentation broth and mycelium polysaccharides exhaust trip by extending mouse power
Swim the time, improve in mouse blood LDH content in Hb, HG and serum, reduce BUN and LA content in mice serum, to improve
The exercise tolerance of mouse alleviates sports fatigue.Illustrate that 3 kinds of polysaccharide have antifatigue effect, and mycelium polysaccharides is antifatigue
Effect is better than fruitbody polysaccharide and polysaccharide of fermentation broth.
Therefore, research can replace the mycelial side of artificial culture Qaidam Agaricus bitorqui of wild Qaidam Agaricus bitorqui fructification
Method becomes the current urgent problem to be solved of industry.
Summary of the invention
The technical problem to be solved by the present invention is to, provide a kind of method it is simple, it is easily operated, can quickly and effectively pass through
The mycelial method of Liquid Culture Qaidam Agaricus bitorqui.
In order to solve the above technical problems, the present invention adopts the following technical scheme: a kind of Liquid Culture Qaidam Agaricus bitorqui is raw
Produce mycelial method, it is characterised in that: it carries out according to the following steps,
1) raw material preparation: Qaidam Agaricus bitorqui slant strains, highland barley, deionized water and as analytically pure glucose,
Agar powder, peptone, magnesium sulfate and calcium chloride；
2) culture medium: solid slope culture medium, that is, PDA culture medium, level liquid seed culture medium i.e. improvement PDA culture medium,
Secondary seed tank culture medium and fermentation tank culture medium；
3) seed slant activation: take a ferfas body to solid slope culture medium i.e. PDA culture medium, from original seed with 22 ± 1
DEG C temperature be protected from light culture 12-15 days, cover with inclined-plane 2/3 and intensive to mycelia, refrigeration is for use in 4 DEG C of refrigerator；
4) prepared by level liquid seed: activated slant strains being placed in 48h at room temperature, prepare liquid by improvement PDA
Culture medium, it is spare after sterilizing, cooling；Several pieces of slant strains are taken with inoculation shovel, level liquid seed culture medium is inoculated into and changes
In good PDA culture medium, with 23 ± 1 DEG C of temperature stationary culture 48h, 6-10 then is shaken in dark in the shaking table of 120r/min
It, until growing intensive mycelium pellet；It is forwarded in triangular flask after sterilization and cooling with 10% inoculum concentration, with 23 ± 1 DEG C, 120r/min
Continuously shake in dark 3-4 days, as level liquid seed；
5) prepared by secondary seed: seeding tank first being carried out slack tank sterilizing, after cooling, prepares liquid by seed tank culture based formulas
Body culture medium after adjusting pH to 7.3, carries out high-temp steam sterilizing, is cooled to 23 ± 1 DEG C, the level liquid kind that will have been prepared
Son accesses seeding tank with 15% inoculum concentration, using flame inoculation method, and On-line Control seeding tank temperature is 23 ± 1 DEG C, stirring turns
Speed is 100rpm, is cultivated 2-4 days；
6) fermented and cultured: first carrying out slack tank sterilizing for fermentor, after cooling, prepares liquid training by fermentation tank culture based formulas
Support base, adjust pH to 7.3 after, carry out high-temp steam sterilizing, be cooled to 23 ± 1 DEG C, by the secondary seed solution prepared with
15% inoculum concentration, using pressure differential method access fermentor, On-line Control fermentation jar temperature be 23 ± 1 DEG C, speed of agitator 110rpm,
Culture 5-7 days.
Further, in step 3), the thallus area taken from original seed is about 0.5cm2, and solid slope culture medium
I.e. PDA culture medium shifts to an earlier date 15 days and prepares.
Further, in step 4), the triangular flask with liquid amount for 80mL/250mL prepares improvement PDA Liquid Culture
Base；It is 0.5-1cm that shovel, which takes and takes 3-5 block area with inoculation shovel when slant strains,2Strain block.
Further, in step 5), seeding tank 15L, 121 DEG C of sterilizing 30min of slack tank after cooling, are trained by seeding tank
It supports based formulas and prepares 5L fluid nutrient medium, adjust 121 DEG C of steam sterilizing 30min of high temperature after pH to 7.3；Seed tank culture based formulas
Are as follows: highland barley filtrate is 400g/L, soybean protein 5g/L, MgSO4For 0.1g/L, CaCl2For 0.14g/L, compound VBFor 1g/L,
PH is 6.5-7；With spare after 121 DEG C of temperature sterilizing 30min.
Further, in step 6), fermentor 50L, 121 DEG C of sterilizing 30min of slack tank after cooling, are trained by fermentor
It supports based formulas and prepares 30L fluid nutrient medium, adjust 121 DEG C of steam sterilizing 30min of high temperature after pH to 7.3；Fermentation tank culture basigamy
Side are as follows: highland barley filtrate is 400g/L, soybean protein 5g/L, MgSO4For 0.1g/L, CaCl2For 0.14g/L, compound VBFor 1g/
L, pH 6.5-7；With spare after 121 DEG C of temperature sterilizing 30min.
Further, in highland barley: water=400g:1000mL ratio adds water to submerge after boiling 20min, blueness is obtained by filtration
Highland barley filtrate；Using highland barley filtrate as base fluid, each nutritional ingredient is added, is configured to seed tank culture base.
Further, in highland barley: water=400g:1000mL ratio adds water to submerge after boiling 20min, blueness is obtained by filtration
Highland barley filtrate；Using highland barley filtrate as base fluid, each nutritional ingredient is added, is configured to fermentation tank culture medium.
Although Qaidam Agaricus bitorqui fructification, fermentation liquid and mycelium polysaccharides can extend mouse swimming time,
There is anti-fatigue active, but mycelial anti-fatigue active is better than polysaccharide of fermentation broth and fruitbody polysaccharide, therefore with bacterium
Filament replace Qaidam Agaricus bitorqui fructification be it is feasible (please refer to paper " Qaidam Agaricus bitorqui Polysaccharides on Mice it is anti-
Fatigue effect ", modern food science and technology, 2018, Vol.34, No.8, an article piece number: 1673-9078 (2018) 08-24-30, Jiao Ying
Spring, spacious intelligent, Wu Jianan, He Cheng, Chen Qihe).
The present invention is by design by seed slant activation, the preparation of level liquid seed, secondary seed preparation, fermented and cultured etc.
What step was constituted produces mycelial method by Liquid Culture Qaidam Agaricus bitorqui, only needs one month or so to cultivate in total
Mycelium is produced to Qaidam Agaricus bitorqui, not only speed is fast, but also method is simple, easy to operate, and mycelium can replace open country
Raw Qaidam Agaricus bitorqui fructification, so as to shield to wild Qaidam Agaricus bitorqui resource.
Detailed description of the invention
Fig. 1 is the change curve of biomass and incubation time in seeding tank；
Fig. 2 is the change curve of biomass and incubation time in seeding tank；
Fig. 3 is Qaidam Agaricus bitorqui 50L tank fermentation diagram curve graph.
Specific embodiment
The present invention will be further described combined with specific embodiments below, but this does not imply that any limit of the invention
System.
1. materials and methods
1.1 raw material and culture medium
Qaidam Agaricus bitorqui (Agaricusbitorquis (Qu é l.) Sacc.) slant strains: real by Qinghai University's food
Room offer is provided.
Highland barley (Hordeum vulgare Linn.Var.nudum Hook.F.): Xining, Qinghai purchase.
Reagent: glucose, agar powder, peptone, magnesium sulfate (MgSO4), calcium chloride (CaCl2) etc. be analysis it is pure；Water is
Deionized water.
Solid slope culture medium (PDA culture medium): potato (peeling) 200g/L, glucose 20g/L, agar powder 20g/L,
Peptone 5g/L, pH are natural；121 DEG C, 30min is spare after sterilizing.
Level liquid seed culture medium (improvement PDA culture medium): potato (peeling) 200g/L, fructose 40g/L, peptone
5g/L, MgSO4For 0.1g/L, CaCl2For 0.14g/L, compound VBFor 1g/L, pH nature；115 DEG C, 15min is spare after sterilizing.
Secondary seed tank culture medium: highland barley (filtrate) 400g/L, soybean protein 5g/L, MgSO4For 0.1g/L, CaCl2For
0.14g/L, compound VBFor 1g/L, pH 6.5-7；121 DEG C, 30min is spare after sterilizing.
Fermentation tank culture medium: ibid.
By highland barley: water=400:1000 (g:mL) adds water to submerge after boiling 20min, highland barley filtrate is obtained by filtration；With highland barley
Filtrate is liquid, and above-mentioned nutritional ingredient is added, and is configured to seed tank culture base and fermentation tank culture medium.
1.2 test apparatus
FA1004 type electronic balance (upper Nereid section balance), HWY-111 constant-temperature shaking incubator (Shanghai intelligence city analysis instrument system
Make Co., Ltd, vacuum freeze drier (U.S.'s Svant Products), Portable pressure steam sterilizing device (win fast reality in Shanghai
Medical Equipment Plant, industry Co., Ltd), low LXJ-IIB speed large capacity Multi-pipe centrifugal machine (Town in Shanghai pavilion scientific instrument head factory), 101-
(U.S.'s Varian has the purple visible spectrophotometer of the xeothermic case of 3B type electric heating air blast (Shanghai City laboratory apparatus head factory), CARY50
Limit company), GUJS 20L mechanical agitating fermentation tank (Zhenjiang east biology New Equipment Engineering Co., Ltd).
2. method
2.1 test method
2.1.1 seed slant activation
A fritter thallus (area about 0.5cm is taken from original seed2Size) it (is prepared within 15 days in advance) to PDA slant medium,
22 ± 1 DEG C are protected from light culture 12-15 days, cover with inclined-plane 2/3 and intensive to mycelia, and refrigeration is for use in 4 DEG C of refrigerators.
2.1.2 prepared by level-one (liquid) seed
Activated slant strains are placed in 48h at room temperature, prepare fluid nutrient medium, liquid amount 80mL/ by improvement PDA
250mL triangular flask, it is spare after sterilizing, cooling.0.5-1cm is taken with inoculation shovel2(3-5 block), is inoculated into aforesaid liquid culture medium,
23 ± 1 DEG C, after stationary culture 48h, in 120r/min shaking table shaken cultivation 6-10 days (being protected from light), until growing more mycelium pellet；With
10% inoculum concentration is forwarded in 300mL/1000mL triangular flask after sterilization and cooling, and 23 ± 1 DEG C, 120r/min continuous oscillation culture 3-
4 days (being protected from light), as first order seed.
2.1.3 prepared by second level (seeding tank) seed
Seed fermentation tank (15L) is first subjected to slack tank sterilizing (121 DEG C, 30min), after cooling, by seed tank culture basigamy
5L fluid nutrient medium is prepared by side, after adjusting pH to 7.3, carries out high-temp steam sterilizing (121 DEG C, 30min), is cooled to 23 ± 1 DEG C,
By the primary seed solution prepared with 15% inoculum concentration, seeding tank, On-line Control seeding tank are accessed using flame inoculation method
23 ± 1 DEG C, speed of agitator 100rpm are cultivated 2-4 days.
2.1.4 fermentation (fermentor) culture
Fermentor (50L) is first subjected to slack tank sterilizing (121 DEG C, 30min), after cooling, is matched by fermentation tank culture based formulas
30L fluid nutrient medium processed after adjusting pH to 7.3, carries out high-temp steam sterilizing (121 DEG C, 30min), is cooled to 23 ± 1 DEG C, will
The secondary seed solution prepared accesses fermentor, On-line Control fermentor 23 ± 1 with 15% inoculum concentration, using pressure differential method
DEG C, speed of agitator 110rpm is cultivated 5-7 days.
2.2 measuring method
Biomass estimation --- dry weight method；Determined amino acid --- ninhydrin method；Reducing sugar test --- 3,5- dinitro
Salicylic acid method；Exocellular polysaccharide measurement --- alcohol precipitation.
3 results and analysis
3.1 seeding tanks fermentation situation
Using above-mentioned culture medium and cultural method, Qaidam Agaricus bitorqui mycelial biomass reaches on day 3 in seeding tank
To highest, therefore determine that the best incubation time of seeding tank is 3 days, as depicted in figs. 1 and 2, at this point, in seeding tank pH variation also compared with
It is suitable for, and the fermentation tank culture time is as illustrated in the graph of fig. 3.
The above has been described in detail, described above, is only a preferred embodiment of the present invention, when cannot
It limit the scope of implementation of the present invention, i.e., it is all according to the made equivalent changes and modifications of the application range, it should still belong to covering scope of the present invention
It is interior.
Claims (7)
1. a kind of Liquid Culture Qaidam Agaricus bitorqui produces mycelial method, it is characterised in that: it carries out according to the following steps,
1) raw material preparation: Qaidam Agaricus bitorqui slant strains, highland barley, deionized water and as analytically pure glucose, agar
Powder, peptone, magnesium sulfate and calcium chloride；
2) culture medium: solid slope culture medium, that is, PDA culture medium, level liquid seed culture medium improve PDA culture medium, second level
Seed tank culture base and fermentation tank culture medium；
3) seed slant activation: take a ferfas body to solid slope culture medium i.e. PDA culture medium, from original seed with 22 ± 1 DEG C
Temperature is protected from light culture 12-15 days, covers with inclined-plane 2/3 and intensive to mycelia, and refrigeration is for use in 4 DEG C of refrigerator；
4) prepared by level liquid seed: activated slant strains being placed in 48h at room temperature, prepare Liquid Culture by improvement PDA
Base, it is spare after sterilizing, cooling；Several pieces of slant strains are taken with inoculation shovel, level liquid seed culture medium is inoculated into and improves PDA
In culture medium, with 23 ± 1 DEG C of temperature stationary culture 48h, then shaken in dark 6-10 days in the shaking table of 120r/min, until
Grow intensive mycelium pellet；Be forwarded in triangular flask after sterilization and cooling with 10% inoculum concentration, with 23 ± 1 DEG C, 120r/min it is continuous
Shake in dark 3-4 days, as level liquid seed；
5) prepared by secondary seed: seeding tank first being carried out slack tank sterilizing, after cooling, prepares liquid training by seed tank culture based formulas
Support base, adjust pH to 7.3 after, carry out high-temp steam sterilizing, be cooled to 23 ± 1 DEG C, by the level liquid seed prepared with
15% inoculum concentration accesses seeding tank using flame inoculation method, and On-line Control seeding tank temperature is 23 ± 1 DEG C, speed of agitator is
100rpm is cultivated 2-4 days；
6) fermented and cultured: first carrying out slack tank sterilizing for fermentor, after cooling, prepares Liquid Culture by fermentation tank culture based formulas
Base after adjusting pH to 7.3, carries out high-temp steam sterilizing, 23 ± 1 DEG C is cooled to, by the secondary seed solution prepared with 15%
Inoculum concentration, using pressure differential method access fermentor, On-line Control fermentation jar temperature be 23 ± 1 DEG C, speed of agitator 110rpm, culture
5-7 days.
2. Liquid Culture Qaidam described in claim 1 Agaricus bitorqui produces mycelial method, it is characterised in that: in step 3)
In, the thallus area taken from original seed is 0.5cm2, and solid slope culture medium, that is, PDA culture medium shifts to an earlier date 15 days and prepares.
3. Liquid Culture Qaidam described in claim 1 Agaricus bitorqui produces mycelial method, it is characterised in that: in step 4)
In, the triangular flask with liquid amount for 80mL/250mL prepares improvement PDA liquid medium；Shovel takes and is taken when slant strains with inoculation shovel
3-5 block area is 0.5-1cm2Strain block.
4. Liquid Culture Qaidam described in claim 1 Agaricus bitorqui produces mycelial method, it is characterised in that: in step 5)
In, seeding tank 15L, 121 DEG C of sterilizing 30min of slack tank after cooling, prepare 5L fluid nutrient medium by seed tank culture based formulas,
121 DEG C of steam sterilizing 30min of high temperature after adjusting pH to 7.3；Seed tank culture based formulas are as follows: highland barley filtrate is 400g/L, soybean
Albumen is 5g/L, MgSO4For 0.1g/L, CaCl2For 0.14g/L, compound VBFor 1g/L, pH 6.5-7；It is gone out with 121 DEG C of temperature
It is spare after bacterium 30min.
5. Liquid Culture Qaidam described in claim 1 Agaricus bitorqui produces mycelial method, it is characterised in that: in step 6)
In, fermentor 50L, 121 DEG C of sterilizing 30min of slack tank after cooling, prepare 30L fluid nutrient medium by fermentation tank culture based formulas,
121 DEG C of steam sterilizing 30min of high temperature after adjusting pH to 7.3；Fermentation tank culture based formulas are as follows: highland barley filtrate is 400g/L, soybean
Albumen is 5g/L, MgSO4For 0.1g/L, CaCl2For 0.14g/L, compound VBFor 1g/L, pH 6.5-7；It is gone out with 121 DEG C of temperature
It is spare after bacterium 30min.
6. Liquid Culture Qaidam Agaricus bitorqui described in claim 5 produces mycelial method, it is characterised in that: press highland barley:
Water=400g:1000mL ratio, adds water to submerge after boiling 20min, and highland barley filtrate is obtained by filtration；Using highland barley filtrate as base fluid,
Each nutritional ingredient is added, is configured to seed tank culture base.
7. Liquid Culture Qaidam described in claim 1 Agaricus bitorqui produces mycelial method, it is characterised in that: press highland barley:
Water=400g:1000mL ratio, adds water to submerge after boiling 20min, and highland barley filtrate is obtained by filtration；Using highland barley filtrate as base fluid,
Each nutritional ingredient is added, is configured to fermentation tank culture medium.
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| NL2011277C2 (en) * | 2013-08-07 | 2015-02-10 | Stichting Eco Consult | Meat substitute composition and method for providing thereof. |
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| CN109321473B (en) * | 2018-10-31 | 2019-09-27 | 青海伊纳维康生物科技有限公司 | A kind of Liquid Culture Qaidam Agaricus bitorqui produces mycelial method |
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| CN102766663A (en) * | 2012-05-30 | 2012-11-07 | 陕西师范大学 | Preparation method of active polysaccharides from phellinus linteus |
| CN105695530A (en) * | 2016-03-14 | 2016-06-22 | 青海大学 | Liquid fermentation medium for high yielding of Qaidam agaricus bitorguis polysaccharide and application |
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| 柴达木盆地野生大肥菇营养生理研究;徐鸿雁等;《食用菌》;20170123(第1期);第12-15页 * |
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