CN104099385B - A kind of deep layer liquid state fermentation produces the method for Inonotus obliquus exocellular polysaccharide - Google Patents
A kind of deep layer liquid state fermentation produces the method for Inonotus obliquus exocellular polysaccharide Download PDFInfo
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Abstract
The invention belongs to microbial technology field, be specifically related to a kind of method that deep layer liquid state fermentation produces Inonotus obliquus exocellular polysaccharide.The specifically production method of the Inonotus obliquus exocellular polysaccharide of a kind of industrially scalable, the method is that the Inonotus obliquus bacterial classification activated is carried out expansion joined by being linked into seeding tank, again the liquid seeds that expansion prepares is linked in the special liquid culture medium producing polysaccharide and ferments, then zymotic fluid is separated, extract exocellular polysaccharide.The present invention is directed to above-mentioned the deficiencies in the prior art study, on the basis of lab scale and pilot scale continue to optimize fermentation medium (especially carbon source), promote biomass and exocellular polysaccharide, solve be amplified to industrialized production runs into by lab scale Fermentation Process of Parameter control, extraction equipment type selecting, the difficult problem such as optimization, finally being successfully grafted onto 10 20t fermentation tanks carries out industrialization production, and produce the Fuscoporia obliqua polysaccharide of high-quality smoothly, be truly realized continuously, automation, the production of industrialization.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of method that deep layer liquid state fermentation produces Inonotus obliquus exocellular polysaccharide.
Background technology
Inonotus obliquus has another name called CHAGA, belongs to Basidiomycotina, Hymenomycetes, non-brown Zoopagales, Polyporaceae, brown transverse hole fungus genus, is one
Plant the large-scale medicinal fungi of parasitics being born on birch.Its fructification presents the black block-shape morphology being similar to carbon, is distributed mainly on north
The rare medical fungus in the areas such as U.S. the north, Russia, Finland, China Dark Longjiang, Hokkaido, Japan
Inonotus obliquus main parasitic is under the bark of other birch classes such as white birch, elm, silvery birch, and the i.e. sclerotium surface black of fructification is deep
Splitting, tube leading portion ftractures, bacterial context suberin and have ring grain, and spore ellipticity or ovum shape, be that a kind of life is the domestomycetes in frigid zone.
Inonotus obliquus is widely used on Russia and other places, is a kind of Russian common drug among the people, the applicating history of existing at least centuries,
Because it is notable, in Russia, Central European, China northeast in aspect effects such as treatment gastrointestinal disease, alleviation cancer symptoms, prevention diseases
It is widely used in area, is widely accepted, be locally known as " catholicon ".
Along with the development of modern biotechnology, researcher finds that the polysaccharide of Inonotus obliquus is probably its main pharmacodynamics composition, the most
The Fuscoporia obliqua polysaccharide pharmacological action of report mainly includes antitumor, antiviral, hypoglycemic, reducing blood lipid, removing free radical, suppression
Lipid peroxide generation, anti-inflammatory, enhancing immunity, anticoagulation, anti-aging, pain relieving etc..It is reported, Inonotus obliquus antitumor
The main immune system being activated body by polysaccharose substances such as glucans of effect, plays the work of the antineoplastic immune strengthening body
With.In terms for the treatment of tumour, in serum, the content of nitric oxide of higher concentration can affect conduction and the intra-tumor of tumour cell signal
The formation of blood vessel, promotes tumor growth.Fuscoporia obliqua polysaccharide has obvious inhibitory action to the growth of murine sarcoma.Inonotus obliquus
Polysaccharide for reducing blood sugar, the research of effect for reducing blood fat show, Fuscoporia obliqua polysaccharide can promote that the reverse metabolism of cholesterol is converted to cholesteryl ester,
Suppression enteron aisle inner cholesterol absorbs, and can improve function of vascular endothelium simultaneously, reduces blood fat.In Inonotus obliquus, polysaccharose substance has
Immune enhancing function, activity and function to immunity related lymphocytes have facilitation, it is possible to remove interior free yl, extend thin
Born of the same parents divide, and extend cell survival.
Increasing scholar begins one's study the effective component of Inonotus obliquus and pharmacological action in the recent decade, and Inonotus obliquus has become medicine
With one of bacterial classification having DEVELOPMENT PROSPECT most in fungi.But current domestic wild Inonotus obliquus resource scarcity, the Planting of fructification
Success rate is low and poor repeatability in training, and solid state cultivation cycle length, pollution rate are high, are easily limited by surrounding environment, it is impossible to meet growing
The market demand.And deep layer liquid state fermentation technology has the advantages such as fermentation period is short, high yield, pollution rate are low, not by surrounding environment
Limit, resource apparatus utilization rate advantages of higher, thus favored by increasing researchers.Also there is a small amount of junior college both at home and abroad
The researcher of universities and colleges carried out research to the submerged fermentation of Inonotus obliquus, but often its main emphasis is how to obtain higher biology
Amount, only small part researcher pay close attention to the research of the functional component polysaccharide of Inonotus obliquus, but it is many mainly to extract acquisition by mycelium
Sugar, and the part postgraduate of only Institutes Of Technology Of Zhejiang and University Of Science and Technology Of Tianjin carried out liquid state fermentation and produced outside Inonotus obliquus born of the same parents many
The research of sugar, but its deficiency mainly has: the most all it is limited only to the shake flask test of lab scale, belongs to phase of basic research；2. from theirs
It can be seen that cultivation cycle about 9 days in data, biomass is 1.0-1.1g/100ml, and yield of extracellular polysaccharide is 0.11-0.14
G/100ml, biomass and activated product born of the same parents outer sugar yield is the lowest, and production cost is prohibitively expensive, and its technique need to promote further, if
The production Fuscoporia obliqua polysaccharide of scale to be reached and industrialization also needs to carry out substantial amounts of research and experimental work.
Summary of the invention
The invention aims to solve the technical bottleneck of industrial-scale production Fuscoporia obliqua polysaccharide, use liquid state fermentation to produce birch
Obliquus polysaccharide overcomes solid state fermentation cycle length, investment is big, be subject to seasonal restrictions, be difficult to the drawbacks such as industrialization, uses this invention energy
The most continuously, automation, the Fuscoporia obliqua polysaccharide producing high-quality of industrialization.
To achieve these goals, the present invention is carried out essentially according to following step:
The industrialization deep layer liquid state fermentation of the present invention produces the method for Inonotus obliquus exocellular polysaccharide and comprises the steps:
A kind of method preparing Inonotus obliquus zymotic fluid, accesses the fermentation medium after sterilizing, inoculation by Inonotus obliquus seed culture fluid
Amount is 10～20%, and ventilating ratio is 1:0.3 1.0vvm, tank pressure 0.02～0.05Mpa, cultivates 3～4 days at 22～30 DEG C of bottom fermentations,
Obtain Inonotus obliquus zymotic fluid；Wherein said fermentative medium formula is: glucose 1-3%, starch 1-3%, dried silkworm chrysalis meal
0.1-0.5%, peptone 0.1-0.5%, potassium dihydrogen phosphate 0.1-0.5%, epsom salt 0.1-0.5%, defoamer 0.01-0.05%,
The Thermostable α-Amylase 0.001-0.007% (v/v) of 20000u/ml, regulates pH value 6.5-7.5.In above-mentioned culture medium composition
" % " in addition to amylase, all represents g/100ml.
Described defoamer can be vegetable oil (soya-bean oil, the rapeseed oil) material such as class, organic ethers.
Wherein, described Inonotus obliquus seed culture fluid is preferably prepared by the following method and obtains:
1) first order seed is cultivated: activated shaking flask bacterial classification accesses the liquid seed culture medium after sterilizing, and inoculum concentration is 0.1～2%,
Ventilating ratio is 1:0.3 1.0vvm, tank pressure 0.02～0.05Mpa, cultivates 5～6 days, obtain Inonotus obliquus one-level at 22～30 DEG C
Seed culture fluid；
2) secondary seed is cultivated: by step 1) the first order seed nutrient solution of gained accesses the liquid seed culture medium after sterilizing, connects
The amount of kind is 10～20%, and ventilating ratio is 1:0.3 1.0vvm, tank pressure 0.02～0.05Mpa, cultivates 3～4 days at 22～30 DEG C,
Obtain described Inonotus obliquus seed culture fluid.
Described liquid seed culture medium formula is preferably: glucose 2-4%, dried silkworm chrysalis meal 0.1-0.5%, soybean cake powder 1-3%, phosphorus
Acid dihydride potassium 0.1-0.5%, epsom salt 0.1-0.5%, defoamer 0.01-0.05%, regulate pH value 6.5-7.5.Above-mentioned training
" % " that support in base composition all represents g/100ml.
Described fermented and cultured terminal is: bacterium ball becomes broken, filtrate is muddy；Reduced sugar is down to minimum and slightly fluctuates, and reduced sugar controls
0.3% (g/100g) is below；Microscopy mycelia is in small, broken bits, without living contaminants.
A kind of method that Inonotus obliquus exocellular polysaccharide extracts, the method comprises the steps:
1) separate: by separated for the Inonotus obliquus fermentation culture of claim 1 gained, obtain Inonotus obliquus supernatant；
2) concentrate: by step 1) filtrate of gained carries out using two grade low-temps to concentrate, obtain concentrating proportion be 1.25～1.40 dense
Contracting slurry；
3) alcohol precipitation, drying: add 95% alcohol of thickened pulp weight 3-5 times amount, detects mixed liquor alcoholic strength about 65-80% with alcohol meter
(v/v), standing 8-16h, sediment carries out vacuum drying, temperature 50-75 DEG C, vacuum is not less than 0.085MPa；Dry
It is 3～8% (g/100g) to dried object moisture, obtains the outer sugar of born of the same parents.
Wherein, described step 1) described in separation use horizontal screw centrifuge to carry out, rotating speed controls at 2500 3500r/min.
Described step 2) employed in secondary concentration be: one-level use 1 3t double effect evaporator concentrate, zymotic fluid is concentrated to ratio
Being heavily 1.15 1.20 (50 DEG C of heat are surveyed), two grades use 1 3t haplo-effect concentrator to concentrate, and will proportion be 1.1 1.20 (50 DEG C of heat are surveyed)
Concentrate continue to be concentrated to the thickened pulp that proportion is 1.25～1.40 (50 DEG C of heat are surveyed)；Concentration process is required to control temperature not
Higher than 75 DEG C, vacuum is not less than 0.085MPa.
In the present invention, alcohol precipitation is to use 1 3t Alcohol-settling tank, and alcohol precipitation number of times is the most, and the content of the outer sugar of born of the same parents is the highest.Vacuum drying uses
Vacuum drying chamber, the born of the same parents that the present invention extracts outer sugar content is 20 50%.
The present invention has a following prominent advantage:
The present invention is directed to above-mentioned the deficiencies in the prior art study, continue to optimize fermentation medium (especially carbon at lab scale and pilot scale
Source), promote biomass and exocellular polysaccharide on the basis of, solve and be amplified to, by lab scale, the sweat ginseng that runs in industrialized production
The difficult problems such as numerical control system, extraction equipment type selecting, optimization, being the most successfully grafted onto 10 20t fermentation tanks carries out industrialization production, and suitable
Profit produces the Fuscoporia obliqua polysaccharide of high-quality, be truly realized continuously, automation, the production of industrialization.
In order to improve the yield of purpose product exocellular polysaccharide, the present invention is optimized in the carbon source of fermentation medium and combines: use
Fugitive glucose and the compound prescription of long-acting starch.Earlier fermentation is to grow mycelium, and it is main for improving biological content, therefore culture medium
In with the addition of the glucose carbon source that thalline is preferentially selected, breed rapidly；The content of fermentation later stage purpose to be improved product, therefore carbon source
There is provided thalline relatively to utilize slower starch, and add appropriate amount of starch enzyme in the medium, control thalline quantity at proper size, and
Constantly produce the outer sugar of metabolin born of the same parents.
In order to provide born of the same parents the yield of outer sugar, the present invention optimizes the formula of fermentation medium, have employed compound prescription for carbon source part,
Both ensure that the biomass of early stage thalline, ensure again the yield of later stage metabolite.
Using the present invention according to the market demand and product standard, can prepare the polysaccharide of high-quality of different content, polyoses content can be
20 50%.
Detailed description of the invention
The present invention is further illustrated below by way of specific embodiment.But the detail of embodiment is only used for explaining the present invention, should not
It is interpreted as limited overall technical solution.
The Inonotus obliquus that following example use is bought in China Forest Microbiological Culture Collection administrative center, bacterial classification coding cfcc
60260, as a example by this strain bacterium, describe the concrete scheme of the present invention in detail.But the inventive method is common to all of Inonotus obliquus,
It is not limited in this bacterial strain.
Embodiment 1
Industrialization deep layer liquid state fermentation of the present invention produces the method for Inonotus obliquus exocellular polysaccharide and carries out in the steps below:
1, the preparation of Inonotus obliquus zymotic fluid: the Inonotus obliquus shaking flask bacterial strain activated is inoculated in seed culture medium by inoculum concentration 1%
(300L), wherein seed culture medium consist of glucose 3%, dried silkworm chrysalis meal 0.3%, soybean cake powder 1.8%, potassium dihydrogen phosphate 0.3%,
Epsom salt 0.15%, defoamer 0.02%, regulate pH value 7.0, ventilating ratio is 1:0.3vvm, tank pressure 0.03Mpa, 26～
28 DEG C of shaken cultivation i.e. obtain Inonotus obliquus primary seed solution in 5 days；Primary seed solution is accessed secondary seed medium by inoculum concentration 15%
(3000L), wherein secondary seed medium consist of glucose 3%, dried silkworm chrysalis meal 0.3%, soybean cake powder 1.8%, potassium dihydrogen phosphate 0.3%,
Epsom salt 0.15%, defoamer 0.02%, regulate pH value 7.0, ventilating ratio is 1:0.3vvm, tank pressure 0.03Mpa, 26～
28 DEG C of cultivations i.e. obtain Inonotus obliquus secondary seed solution in 3 days；Secondary seed solution is accessed fermentation medium (7500L) by inoculum concentration 18%,
Wherein fermentation medium consist of glucose 2%, starch 2%, dried silkworm chrysalis meal 0.5%, peptone 0.3%, potassium dihydrogen phosphate 0.2%,
Epsom salt 0.2%, defoamer soya-bean oil 0.03%, thermostable α-amylase (Zaozhuang outstanding person promise enzyme biology Co., Ltd, 20000u/ml)
0.002% (v/v), regulates pH value 7.0；Ventilating ratio is 1:0.5vvm, tank pressure 0.03Mpa, cultivates 3 days for 26～28 DEG C, sends out
Ferment to bacterium ball becomes broken, filtrate is muddy；Reduced sugar is down to minimum 0.2% and slightly fluctuates；Microscopy mycelia is in small, broken bits, without living contaminants.
Obtain Inonotus obliquus zymotic fluid 8850Kg.
2, Inonotus obliquus separation of fermentative broth: above-mentioned Inonotus obliquus zymotic fluid 8850Kg is separated by horizontal screw centrifuge, rotating speed
Controlling at 3000r/min, disengaging time, at 2h, obtains the wet mycelium (water content of Inonotus obliquus filtrate 8150Kg and 575Kg
It is 80%), wet mycelium directly low temperature drying can pulverize prepared fermentation Inonotus obliquus bacterium powder.
4, low temperature concentrates: the Inonotus obliquus filtrate 8150Kg vacuum of step 2 gained is transferred to 3t double effect evaporator, in vacuum
Degree is 0.085MPa, and thickening temperature is concentrated into proportion under the conditions of being 65 DEG C be 1.20 (50 DEG C of heat are surveyed) concentrate, then by concentrate
Vacuum is transferred to 1.5t single-action ball-type inspissator, is 0.09MPa in vacuum, and thickening temperature continues to be concentrated under the conditions of being 60 DEG C
Proportion is thickened pulp 310kg of 1.30 (50 DEG C of heat are surveyed).
5, alcohol precipitation, drying: add 95% alcohol of weight 1200kg in the Alcohol-settling tank filling 310kg thickened pulp, use alcohol meter
Detection mixed liquor alcoholic strength 73% (v/v), stands 12h, is dried by bottom sediment separation vacuum drying chamber, temperature 60
DEG C, vacuum 0.09MPa；Dry 12h and obtain born of the same parents outer sugar 85kg.Detection moisture is 4%, and polyoses content is 30%.
Embodiment 2
1, the preparation of Inonotus obliquus zymotic fluid: the Inonotus obliquus shaking flask bacterial strain activated is inoculated in seed culture by inoculum concentration 1.5%
Base (300L), wherein seed culture medium consist of glucose 3%, dried silkworm chrysalis meal 0.3%, soybean cake powder 1.8%, potassium dihydrogen phosphate 0.3%,
Epsom salt 0.15%, defoamer 0.02%, regulate pH value 7.0, ventilating ratio is 1:0.3vvm, tank pressure 0.03Mpa, 25～
27 DEG C of shaken cultivation i.e. obtain Inonotus obliquus primary seed solution in 6 days；Primary seed solution is accessed secondary seed medium by inoculum concentration 15%
(3000L), wherein secondary seed medium consist of glucose 3%, dried silkworm chrysalis meal 0.3%, soybean cake powder 1.8%, potassium dihydrogen phosphate 0.3%,
Epsom salt 0.15%, defoamer 0.02%, regulate pH value 7.0, ventilating ratio is 1:0.3vvm, tank pressure 0.03Mpa, 25～
27 DEG C of cultivations i.e. obtain Inonotus obliquus secondary seed solution in 3 days；Secondary seed solution is accessed fermentation medium (15000L) by inoculum concentration 18%,
Wherein fermentation medium consist of glucose 2%, starch 2%, dried silkworm chrysalis meal 0.5%, peptone 0.3%, potassium dihydrogen phosphate 0.2%,
Epsom salt 0.2%, defoamer rapeseed oil 0.03%, thermostable α-amylase (Zaozhuang outstanding person promise enzyme biology Co., Ltd,
20000u/ml) 0.002% (v/v), regulates pH value 7.0；Ventilating ratio is 1:0.5vvm, tank pressure 0.04Mpa, 25～27 DEG C of trainings
Support 7 days, ferment to the change of bacterium ball is broken, filtrate is muddy；Reduced sugar is down to minimum 0.2% and slightly fluctuates；Microscopy mycelia is in small, broken bits, nothing
Living contaminants, had both obtained Inonotus obliquus zymotic fluid 17700Kg.
2, the separation of Inonotus obliquus zymotic fluid: above-mentioned Inonotus obliquus zymotic fluid 17700Kg is separated by horizontal screw centrifuge,
Obtaining the wet mycelium (water content is 75%) of Inonotus obliquus filtrate 16550Kg and 950Kg, wet mycelium can directly low temperature drying
Pulverize and prepare fermentation Inonotus obliquus bacterium powder.Concrete technology condition is: rotating speed controls at 3200r/min, and disengaging time is at 4.5h.
4, low temperature concentrates: the Inonotus obliquus filtrate 16750Kg vacuum of step 2 gained is transferred to 3t double effect evaporator, in vacuum
Degree is 0.09MPa, and thickening temperature is concentrated into proportion under the conditions of being 63 DEG C be 1.18 (50 DEG C of heat are surveyed) concentrate, then by concentrate
Vacuum is transferred to 1.5t single-action ball-type inspissator, is 0.09MPa in vacuum, and thickening temperature is concentrated into proportion under the conditions of being 65 DEG C
It it is thickened pulp 580kg of 1.35 (50 DEG C of heat are surveyed).
5, alcohol precipitation, drying: add 95% alcohol of weight 2200kg in the Alcohol-settling tank filling 580kg thickened pulp, use alcohol meter
Detection mixed liquor alcoholic strength 70% (v/v), stands 10h, is dried by bottom sediment separation vacuum drying chamber, temperature 65
DEG C, vacuum 0.09MPa；Dry 18h and obtain born of the same parents outer sugar 175kg.Detection moisture is 4.5%, and polyoses content is 32%.
Embodiment 3
The excellent of Fuscoporia obliqua polysaccharide is produced in industrialization for the compound prescription of fugitive glucose and long-acting starch in the more preferably checking present invention
More property, has done again several contrast test.Test only have adjusted carbon source glucose in liquid fermentation medium or (with) use of starch
Amount, does not contains starch in comparative example 1, concentration of glucose is 1.5%；Not containing glucose in comparative example 2, starch concentration is 2.5%,
In comparative example 3, glucose content is 2.5%, and content of starch is 0.5%；Remaining condition is all with embodiment 1, and result of the test is shown in Table 1:
Table 1
Claims (7)
1. the method preparing Inonotus obliquus zymotic fluid, it is characterised in that Inonotus obliquus seed culture fluid is accessed sending out after sterilizing
Ferment culture medium, inoculum concentration is 10～20% (v/v), and ventilating ratio is 1:0.3 1.0vvm, tank pressure 0.02～0.05Mpa, at 22～30 DEG C
Bottom fermentation is cultivated 3～4 days, obtains Inonotus obliquus zymotic fluid；Wherein said fermentative medium formula is: glucose 1-3%, starch
1-3%, dried silkworm chrysalis meal 0.1-0.5%, peptone 0.1-0.5%, potassium dihydrogen phosphate 0.1-0.5%, epsom salt 0.1-0.5%,
The thermostableα-amylase 0.001-0.007% (v/v) of defoamer 0.01-0.05%, 20000u/ml, regulates pH value 6.5-7.5.
The method preparing Inonotus obliquus zymotic fluid the most according to claim 1, it is characterised in that described Inonotus obliquus seed
Nutrient solution is prepared by the following method and obtains:
1) first order seed is cultivated: activated shaking flask bacterial classification accesses the liquid seed culture medium after sterilizing, and inoculum concentration is 0.1～2%
(v/v), ventilating ratio is 1:0.3 1.0vvm, tank pressure 0.02～0.05Mpa, cultivates 5～6 days, obtain birch brown at 22～30 DEG C
Pore fungi first order seed nutrient solution；
2) secondary seed is cultivated: by step 1) the first order seed nutrient solution of gained accesses the liquid seed culture medium after sterilizing, connects
The amount of kind is 10～20% (v/v), and ventilating ratio is 1:0.3 1.0vvm, tank pressure 0.02～0.05Mpa, cultivates 3～4 at 22～30 DEG C
My god, obtain described Inonotus obliquus seed culture fluid.
A kind of method preparing Inonotus obliquus zymotic fluid the most according to claim 2, it is characterised in that described liquid seeds training
Foster based formulas is: glucose 2-4%, dried silkworm chrysalis meal 0.1-0.5%, soybean cake powder 1-3%, potassium dihydrogen phosphate 0.1-0.5%, seven water sulphur
Acid magnesium 0.1-0.5%, defoamer 0.01-0.05%, regulate pH value 6.5-7.5.
A kind of method preparing Inonotus obliquus zymotic fluid the most according to claim 1, it is characterised in that described fermented and cultured is eventually
Point is: bacterium ball becomes broken, filtrate is muddy；Reduced sugar is down to minimum and slightly fluctuates, and reduced sugar controls less than 0.3%；Microscopy mycelia
In small, broken bits, without living contaminants.
5. the method that an Inonotus obliquus exocellular polysaccharide extracts, it is characterised in that the method comprises the steps:
1) separate: the Inonotus obliquus fermentation liquor of claim 1 gained is separated, obtains Inonotus obliquus supernatant；
2) concentrating: by step 1) filtrate of gained carries out using two grade low-temps to concentrate, and obtaining concentrating proportion is 1.25～1.40
Thickened pulp；
3) alcohol precipitation, drying: add 95% alcohol of thickened pulp weight 3-5 times amount, detects mixed liquor alcoholic strength 65-80% with alcohol meter
(v/v), standing 8-16h, sediment carries out vacuum drying, temperature 50 75 DEG C, vacuum is not less than 0.085MPa；Dry
It is 3～8% to dried object moisture, obtains exocellular polysaccharide；Wherein moisture is in terms of the quality of water in 100g dried object.
The method that Inonotus obliquus exocellular polysaccharide the most according to claim 5 extracts, it is characterised in that described step 1) described in
Separation use horizontal screw centrifuge carry out, rotating speed controls at 2500 3500r/min.
Inonotus obliquus exocellular polysaccharide the most according to claim 5 extract method, it is characterised in that described step 2) in adopted
Two grade low-temps concentrate be: one-level use double effect evaporator concentrate, it is 1.15 1.20 that zymotic fluid is concentrated to proportion, adopts for two grades
With haplo-effect concentrator concentrate, will proportion be 1.1 1.20 concentrate continue to be concentrated to the thickened pulp that proportion is 1.25～1.40；Dense
Compression process is required to control temperature and is not higher than 75 DEG C, and vacuum is not less than 0.085MPa.
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