CN103156052A - Submerged fermentation technology of producing immunological enhancement active materials of inonotus obliquus - Google Patents
Submerged fermentation technology of producing immunological enhancement active materials of inonotus obliquus Download PDFInfo
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Abstract
The invention relates to the technical field of fermentation engineering, and discloses a submerged fermentation technology of producing immunological enhancement active materials of inonotus obliquus. The submerged fermentation technology of producing the immunological enhancement active materials of the inonotus obliquus comprises the main steps of culture activation, culture liquid cultivation, submerged fermentation and preparation of the immunological enhancement active materials of the inonotus obliquus and the like. The optimized submerged fermentation technology of producing the immunological enhancement active materials of the inonotus obliquus is high in content of the obtained immunological enhancement active materials, and good in activity of the obtained immunological enhancement active materials, and the obtained immunological enhancement active materials are beneficial for industrial application.
Description
Technical field
The present invention relates to the fermentation engineering field, particularly a kind of liquid deep layer fermenting is produced the technique of Inonotus obliquus immune-enhancing activity material.
Background technology
China is the country that utilizes the earliest fungi to prevent and cure diseases, and fungi is the important component part of China's traditional Chinese medicine.Recent study shows, fungi polysaccharide is the main active substances of medicinal fungi, has significant immunoloregulation function, simultaneously also antiviral, antitumor, anti-oxidant, protect liver, reducing blood lipid, the aspect such as hypoglycemic has biological effectiveness widely.Fungi polysaccharide is mainly isolated metabolite from basidiomycetes and sac fungus fructification, mycelium and zymotic fluid.Up till now for this reason, from nearly 300 kinds of fungies, got more than 200 both at home and abroad and planted the bioactive polysaccharide material of tool, the fungi polysaccharide that wherein China finds that there is important value as: coriolan, Cordyceps sinensis polysaccharide, hericium erinaceum polysaccharide, mushroom polysaccharide, grifola polysaccharide, grifolan, Methods of Polysaccharide From Agrocybe Chaxingu, Inonotue obliquus, Ji Songrong, GL-B etc. are kind more than 30 approximately.Fungi polysaccharide is the immunopotentiator that has more efficient to answer of generally acknowledging, polysaccharide has been done to a large amount of research both at home and abroad, found that, fungi polysaccharide can be brought into play regulating action to immune system by many levels, many approach.A large amount of immunization experiments confirm, polysaccharide can activate the activity of the immunocytes such as T, bone-marrow-derived lymphocyte, macrophage and NK (NK), and it promotes the effect of the each side such as the generation of cell factor and complement activation in addition in addition.Fungi polysaccharide is that the immune system by strengthening Immune Organs of Body, immunocyte and 3 levels of immune molecule realizes immunological enhancement
[1].Derive from the β-1,3/1 of medicinal fungi and yeast, the 6-glucan is a kind of effective immunopotentiator, and people have known their modes of action in immune system.In addition, β-1,3/1, the 6-glucan is avirulent, and their mechanism of action in the immune system of all animals (from the invertebrate to people) are identical.β-1,3/1, the 6-glucan singly can not work by injection, also can work by sneaking into feedstuff feeding.
Inonotus obliquus, have another name called CHAGA, formal name used at school
inonotus obliquus, Fuscoporia oblique, the European is referred to as " chaga ", is that the brown transverse hole fungus of a kind of Polyporaceae belongs to edible and medicinal fungi.Inonotus obliquus mainly grows in white birch, silvery birch, elm, alder etc. and lives under the bark of standing tree or trees dried-up upper after felling, forms sterile domestomycetes, its sclerotium can be after felling dried-up on existence for 6 years.It mainly is distributed in the north latitude 45-50 ° of extremely cold areas such as Russia the north, Northern Europe, China Dark Longjiang, Changbai mountain, Jilin, Hokkaido, Japan.Inonotus obliquus sporophore generally is warty, diameter 25-40 cm, and appearance is hard, and dark slight crack is arranged, harder, and crisp when dry, color is yellowish-brown or darker.
Since 16th century, Inonotus obliquus is widely used in malignant tumour in Russia, some countries of Northern Europe
[2], diabetes, angiocardiopathy, hepatopathy
[3]and AIDS
[4]treatment etc. disease.In Russia, Inonotus obliquus is widely used as daily health products.The researcher of Japan claims that Inonotus obliquus is a kind of " catholicon ".In Japan, the Powdered electuary of Inonotus obliquus is often drunk as health protection tea.Nineteen fifty-five Moscow medical courses in general institute (The Medical Academy of Science in Moscow) announces to classify Inonotus obliquus as cancer-resisting substance, and the government permission Inonotus obliquus can be used for the pharmaceuticals exploitation.The U.S. lists Inonotus obliquus in " special natural materials ".
Be rich in callose and superoxide dismutase in Inonotus obliquus sporophore, its content is respectively 30 and 23 times of famous and precious mushroom-red camphor tree bacterium, and the content of superoxide dismutase is 55 times of glossy ganoderma.For many years, Inonotus obliquus always as pure Chinese medicine in the disease that is used for the treatment of among the people, Inonotus obliquus also is considered to the functional health-care food of 21 century.Clinical trial shows: Inonotus obliquus is without any toxic and side effect, and in the treatment diabetes, prevent AIDS, strengthen the aspect such as immunity and there is obvious effect
[3].Current research report shows, the main active ingredient of Inonotus obliquus has polysaccharide, polyphenol, steroidal, inonotus obliquus, melanin, triterpene, sheath amine ester type compound, alkaloid, folic acid derivatives, vanillic acid, syringic acid and gamma-hydroxy formic acid etc., also has recently report to point out that the scholar has isolated tannin compound, steroids, low molecule Polyphenols and lignin compound
[5].
Fuscoporia obliqua polysaccharide is one of valuable pharmacological active component of Inonotus obliquus, and mycelia and the polysaccharide in sclerotium of Inonotus obliquus mainly are comprised of beta glucan, heteroglycan and albumen composition
[6].Research shows, the Inonotus obliquus sporophore polysaccharide mainly contains the monose such as glucose, galactolipin, mannose, rhamnose, wood sugar and pectinose and forms.And glucose is main monose, accounted for more than 70% of all monose compositions
[7].Between monose, be to be formed by connecting by β-(1,3) glycosidic bond or β-(Isosorbide-5-Nitrae) glycosidic bond.Between main chain and side chain, with β-(1,6) glycosidic bond, connect.Wood sugar and rhamnose are that the Fuscoporia obliqua polysaccharide biologically active is contributed maximum monose
[8].The functional characteristic of Fuscoporia obliqua polysaccharide is mainly manifested in the growth that its fruitbody polysaccharide can suppress the S180 sarcoma
[9], the secretion of its mycelium polysaccharides energy effective stimulus macrophage IL21, IL26, the cell factors such as TNF2A, NO, the immune state of cell factor by adjusting body be killing tumor cell indirectly
[10].In addition, the water-soluble and water-insoluble polysaccharide of Inonotus obliquus has the crude extract that falls hypoglycemic effect, especially Fuscoporia obliqua polysaccharide to diabetic mice, the sustainable 48h of its hypoglycemic activity.
The Inonotus obliquus polyphenol mainly is comprised of melanin class material and milk tree alkali homologue
[11]mainly contain 4-hydroxyl-3,5 dimethoxybenzoic acid-2 '-hydroxyl-1 '-methylol ethyl acetate in Inonotus obliquus sporophore, protocatechuic acid (PCA), caffeic acid (CA), 0412 (DB), 2,5-dihydroxy-1,3-phthalic acid (DTA), syringic acid (SA) and 3,4-dihydroxy benzalacetone (DBL)
[11]etc. little molecular phenolics, Lee
[12]by after Inonotus obliquus sporophore is extracted to purifying, having obtained six kinds of large molecule polyphenol compounds Deng the people, is respectively Phelligridin D, Phelligridin E, Phelligridin G, Inonoblin A, Inonoblin B, Inonoblin C.Zheng
[13]with fermentation tank, the Inonotus obliquus mycelium is carried out to liquid deep layer fermenting Deng the people, find after extraction and analysis, mainly contain the 4 large class materials such as little molecular phenolics, glucosides flavones, flavone aglycone, polyphenol in the Inonotus obliquus mycelium.
The Inonotus obliquus polyphenol has anti-oxidant
[13,14], antitumor
[16], protect liver
[3], immunity promotes
[11]etc. function.Report points out that the catechol of extracting from Inonotus obliquus sporophore has significant gene protection and anti-oxidation function in recent years
[17], to some free radicals especially DPPH free radical, O
2 -free radical and OH free radical all have stronger removing activity, the clearance rate that the Inonotus obliquus polyphenol is removed oxygen radical reaches more than 80%, compare the antioxidants commonly used such as D-VC sodium and n-propyl gallate and there is obvious advantage, have broad application prospects as a kind of natural.
Inonotus obliquus parasitizes on birch, is the bacterial classification extremely resisted cold, and can under the environment of subzero 69 ℃, grow, and reaches 15 years growth period.The adult bacterium of wild Inonotus obliquus can blot the elite of birch, causes trees withered, so its natural resources is very rare, its price far wins precious Medicinal Fungus Phellinus igniarius in the international market.And still there is no at present effective method artificial culture sclerotium and fructification, therefore adopting the liquid deep layer fermenting cultured mycelia to obtain active component is a feasible replacement method.Adopt the method for liquid deep layer fermenting, compare with traditional fungi breeding method, there is the superiority such as with short production cycle, that cost is low, output and quality is stable, product can be controlled.
Summary of the invention
The technique that provides a kind of liquid deep layer fermenting of optimization to produce Inonotus obliquus immune-enhancing activity material on disclosed technology CN102108371A basis early stage the inventor is provided, the immune-enhancing activity content of material obtained is higher, active better, product is more conducive to industrial applications.
The technical solution adopted for the present invention to solve the technical problems is:
A kind of liquid deep layer fermenting is produced the technique of Inonotus obliquus immune-enhancing activity material, and described processing step is as follows:
(1) actication of culture: take Inonotus obliquus as bacterial classification, be seeded on the slant strains culture medium and cultivate, obtain activated spawn;
(2) strain cultivation: activated spawn is proceeded in liquid seed culture medium, and shaking table is cultured to logarithmic phase and obtains liquid spawn;
(3) liquid deep layer fermenting: liquid spawn is accessed to fermented and cultured in the liquid deep layer fermenting culture medium, the volume ratio of liquid spawn and liquid deep layer fermenting culture medium is 1:8-10, the formula of described liquid deep layer fermenting culture medium is: glucose 30-32g/L, lignocellulosic 32-38g/L, promoter 0.9-1g/L, derivant 3 * 10
-5g/L, peptone 3g/L, KH
2pO
41g/L, ZnSO
4 .2H
2o 0.01g/L, K
2hPO
40.4g/L, FeSO
4 .7H
2o 0.05 g/L, MgSO
4 .7H
2o 0.5 g/L, CuSO
4 .5H
2o 0.02 g/L, CoCl
20.01 g/L, MnSO
4 .h
2o 0.08 g/L, pH value 6.0;
The technological parameter of liquid deep layer fermenting is: fermentation temperature 27-28 ℃, fermentation tank air mass flow 0.5-1.0 vvm, fermentation tank internal pressure 0.1-0.25 kg/cm
3, speed of agitator 70-150 rev/min, fermentation period 12-14 days;
(4) Inonotus obliquus immune-enhancing activity material preparation: tunning is filtered, obtain mycelium and zymotic fluid, by the mycelium supersonic wave wall breaking, obtain mycelium powder after drying and crushing, zymotic fluid is concentrated into after the 30%-40% of original volume to obtain to Fermented Condensed liquid, the ratio that adds 30-40 ml Fermented Condensed liquid in 1 g mycelium powder, mix mycelium powder and Fermented Condensed liquid, obtains the Inonotus obliquus immune-enhancing activity material of powdery after drying.
Carrying out along with research, the inventor finds, inventor's disclosed technology (CN102108371A) Inonotus obliquus in early stage immune-enhancing activity material is that output and the activity thereof of polysaccharide, polyphenol is not very desirable, simultaneously neither be very thorough to the decomposition of lignocellulosic, therefore, the inventor has carried out optimizing and creating again to technique, is mainly reflected in the innovation of culture medium:
Add promoter in the liquid deep layer fermenting culture medium, permeability by increasing cell and/or directly affect polysaccharide and the generation of polyphenol synthase, reach the purpose that improves Inonotus obliquus liquid fermentation production exocellular polysaccharide, polyphenol, melanin output, improved the output of Inonotus obliquus immune-enhancing activity material.
Add promoter and lignocellulosic in the liquid deep layer fermenting culture medium simultaneously, on the basis produced at the increase cell permeability of promoter and polysaccharide, polyphenol synthase, promoted the secretion of lignocellulose degradation enzyme (laccase, lignin peroxidase, manganese peroxidase) synthetic in born of the same parents, thereby strengthen the decomposition to lignocellulosic in culture medium, lignocellulose degradation is more thorough, reaches the purpose of further raising polysaccharide and polyphenol (Inonotus obliquus immune-enhancing activity material) output.Particularly make the productive rate of Epigallo-catechin gallate (EGCG) (EGCG) in polyphenol compound, L-Epicatechin gallate (ECG), phelligridin G, davallialactone, inoscavin B significantly improve.
Add derivant in the liquid deep layer fermenting culture medium, promoted the biosynthesis of Inonotus obliquus polyphenols (Inonotus obliquus immune-enhancing activity material a kind of).The present invention utilizes derivant to improve Inonotus obliquus fermentation polyphenol, the particularly high activity polyphenol output as Epigallo-catechin gallate (EGCG) (EGCG), L-Epicatechin gallate (ECG).
Add promoter and derivant in the liquid deep layer fermenting culture medium simultaneously, and coordinate with lignocellulosic, on the basis produced at the increase cell permeability of promoter and polyphenol synthase, add the biosynthetic derivant of polyphenol, collaborative promotion mutually, thereby greatly promoted Inonotus obliquus fermentation polysaccharide, the biosynthesis of polyphenols, lignocellulose degradation more thoroughly reaches further raising polysaccharide, polyphenol, particularly the high activity polyphenol is as Epigallo-catechin gallate (EGCG) (EGCG), the purpose of L-Epicatechin gallate (ECG) output, make the activity of Inonotus obliquus immune-enhancing activity material of acquisition better.
Mycelium is rich in polyphenol in intracellular polyse and born of the same parents, zymotic fluid is rich in exocellular polysaccharide and the outer polyphenol of born of the same parents, inventor's disclosed technology in early stage (CN102108371A) is to mycelium, zymotic fluid is extraction process respectively, thereby obtain different active materials, so not only process complicated, and cost is high, be not suitable for industrial applications, and can not bring into play the cooperative effect of the interior polyphenol of intracellular polyse and born of the same parents and exocellular polysaccharide and the outer polyphenol of born of the same parents, the present invention does drying and crushing after to the mycelium broken wall and processes, zymotic fluid is cooked to concentration, and the proportioning of control mycelium powder and Fermented Condensed liquid, mycelium powder and Fermented Condensed liquid mixture homogeneity are good like this, the dry Inonotus obliquus immune-enhancing activity material that obtains, best proportioning with the outer polyphenol of polyphenol and exocellular polysaccharide and born of the same parents in arrival intracellular polyse and born of the same parents, and mixture homogeneity is good, bring into play best effect, the activity of product so not only, effect is good, and process simple, production cost is low, be applicable to suitability for industrialized production, the additive that can directly be used as all kinds of animal feeds is used.
As preferably, described promoter is oleic acid, methyl alcohol or Tween-80.
As preferably, described derivant is diazosulfide or methyl jasmonate.Diazosulfide is the salicylic functional analogue of plant endogenous sex hormone analog, can promote the generation of polyphenols, and methyl jasmonate is naturally occurring plant growth regulator, can promote the biosynthesis of anthocyanidin.
As preferably, described lignocellulosic is selected from a kind of in agricultural crop straw, wood chip, peanut shell, bagasse.Most preferred scheme is at peanut shell, in bagasse, select a kind of as lignocellulosic material, the inventor is by large quantity research invention, agricultural crop straw, the effect as lignocellulosic material time the such as wood chip is unsatisfactory, this is because the restriction of the speciality of material itself, and then find after research and probe, peanut shell, bagasse is as lignocellulosic material, in its tunning, the activity of polysaccharide and polyphenol can be higher, the degraded of lignocellulosic is also more thorough, push away its reason: be mainly peanut shell, 3 of lignocellulosic component fibre elements in bagasse, the composition of proportions of hemicellulose and lignin is more suitable for the degraded of Inonotus obliquus.
As preferably, the formula of described slant strains culture medium is: malt extract 40 g/100mL, and peptone 4 g/100mL, agar 10 g/100mL, the pH value is 5.4-5.6.
As preferably, the parameter of cultivating on the slant strains culture medium is: temperature 27-28 ℃, cultivate 200-250 hour.
As preferably, the formula of described liquid seed culture medium is: glucose 5g/100mL, peptone 0.5g/100mL, yeast extract 0.1g/100mL, KH
2pO
40.1g/100mL, MgSO
40.15 g/100mL, CaCl
20.01 g/100mL.
As preferably, the parameter of strain cultivation is: temperature 27-28 ℃, cultivate 3.5-4.5d, shaking speed 80-140 rev/min.
The invention has the beneficial effects as follows:
1, add promoter in the liquid deep layer fermenting culture medium, permeability by increasing cell and/or directly affect polysaccharide and the generation of polyphenol synthase, reach the purpose that improves Inonotus obliquus liquid fermentation production exocellular polysaccharide, polyphenol, melanin output, improved the output of Inonotus obliquus immune-enhancing activity material.
2, add promoter and lignocellulosic in the liquid deep layer fermenting culture medium simultaneously, on the basis produced at the increase cell permeability of promoter and polysaccharide, polyphenol synthase, promoted the secretion of lignocellulose degradation enzyme (laccase, lignin peroxidase, manganese peroxidase) synthetic in born of the same parents, thereby strengthen the decomposition to lignocellulosic in culture medium, lignocellulose degradation is more thorough, reaches the purpose of further raising polysaccharide and polyphenol (Inonotus obliquus immune-enhancing activity material) output.Particularly make the productive rate of Epigallo-catechin gallate (EGCG) (EGCG) in polyphenol compound, L-Epicatechin gallate (ECG), phelligridin G, davallialactone, inoscavin B significantly improve.
3, add derivant in the liquid deep layer fermenting culture medium, promoted the biosynthesis of Inonotus obliquus polyphenols (Inonotus obliquus immune-enhancing activity material a kind of).The present invention utilizes derivant to improve Inonotus obliquus fermentation polyphenol, the particularly high activity polyphenol output as Epigallo-catechin gallate (EGCG) (EGCG), L-Epicatechin gallate (ECG).
4, the activity of product, effect are good, and process simply, and production cost is low, is applicable to suitability for industrialized production, can be directly as the additive of all kinds of animal feeds, use.
The accompanying drawing explanation
Fig. 1 is the growth-promoting Cytokine figure of the polysaccharide of tunning extraction of the present invention, wherein Fig. 1 (a) TNF: TNF, Fig. 1 (b) IFN: interferon, Fig. 1 (c) IL-2: interleukin 2, Fig. 1 (d) IL-1 β: interleukin-1 ' beta '.
Fig. 2 is the promotion macrophage proliferation design sketch of the polysaccharide of tunning extraction of the present invention.
The specific embodiment
Below by specific embodiment, and by reference to the accompanying drawings, technical scheme of the present invention is described in further detail.
In the present invention, if not refer in particular to, the raw material adopted and equipment etc. all can be buied from market or this area is commonly used.Method in following embodiment, if no special instructions, be the conventional method of this area.
Inonotus obliquus is commercially available, purchased from Xuzhou Normal University.
Embodiment 1
(1) actication of culture: take Inonotus obliquus as bacterial classification, streak inoculation, on the slant strains culture medium 27 ℃, is cultivated 250 hours, obtains activated spawn; The formula of slant strains culture medium is: malt extract 40 g/100mL, and peptone 4 g/100mL, agar 10 g/100mL, the pH value is 5.4-5.6.
(2) strain cultivation: activated spawn is proceeded to 27 ℃ of liquid seed culture mediums, cultivate 4.5d on shaking table and obtain liquid spawn to logarithmic phase, 80 rev/mins of shaking speed.The formula of liquid seed culture medium is: glucose 5g/100mL, peptone 0.5g/100mL, yeast extract 0.1g/100mL, KH
2pO
40.1 g/100mL, MgSO
40.15 g/100mL, CaCl
20.01 g/100mL.
(3) liquid deep layer fermenting: the fermentation cylinder for fermentation that the liquid spawn access is equipped with to the liquid deep layer fermenting culture medium is cultivated, the volume ratio of liquid spawn and liquid deep layer fermenting culture medium is 1:8, the technological parameter of liquid deep layer fermenting is: 27 ℃ of fermentation temperatures, fermentation tank air mass flow 0.5vvm, fermentation tank internal pressure 0.1 kg/cm
3, 70 rev/mins of speeds of agitator, fermentation period 14 days; The formula of described liquid deep layer fermenting culture medium is: glucose 30g/L, maize straw 32g/L, oleic acid 1g/L, diazosulfide 3 * 10
-5g/L, peptone 3g/L, KH
2pO
41g/L, ZnSO
4 .2H
2o 0.01g/L, K
2hPO
40.4g/L, FeSO
4 .7H
2o 0.05 g/L, MgSO
4 .7H
2o 0.5 g/L, CuSO
4 .5H
2o 0.02 g/L, CoCl
20.01 g/L, MnSO
4 .h
2o 0.08 g/L, pH value 6.0; The standard that fermentation termination is established be content of reducing sugar lower than 2%, microscopy finds that mycelium senesces.
(4) Inonotus obliquus immune-enhancing activity material preparation: tunning is filtered, obtain mycelium and zymotic fluid, by the mycelium supersonic wave wall breaking, obtain mycelium powder after drying and crushing, by zymotic fluid be concentrated into original volume 30% after Fermented Condensed liquid, the ratio that adds 30ml Fermented Condensed liquid in the 1g mycelium powder, mix mycelium powder and Fermented Condensed liquid, obtains the Inonotus obliquus immune-enhancing activity material of powdery after vacuum drying.
(1) actication of culture: take Inonotus obliquus as bacterial classification, streak inoculation, on the slant strains culture medium 28 ℃, is cultivated 200 hours, obtains activated spawn; The formula of slant strains culture medium is: malt extract 40 g/100mL, and peptone 4 g/100mL, agar 10 g/100mL, the pH value is 5.4-5.6.
(2) strain cultivation: activated spawn is proceeded to 28 ℃ of liquid seed culture mediums, cultivate 3.5d on shaking table and obtain liquid spawn to logarithmic phase, 140 rev/mins of shaking speed.The formula of liquid seed culture medium is: glucose 5g/100mL, peptone 0.5g/100mL, yeast extract 0.1g/100mL, KH
2pO
40.1 g/100mL, MgSO
40.15 g/100mL, CaCl
20.01 g/100mL.
(3) liquid deep layer fermenting: the fermentation cylinder for fermentation that the liquid spawn access is equipped with to the liquid deep layer fermenting culture medium is cultivated, the volume ratio of liquid spawn and liquid deep layer fermenting culture medium is 1:10, the technological parameter of liquid deep layer fermenting is: 28 ℃ of fermentation temperatures, fermentation tank air mass flow 1.0vvm, fermentation tank internal pressure 0.25 kg/cm
3, 150 rev/mins of speeds of agitator, fermentation period 12 days; The formula of described liquid deep layer fermenting culture medium is: glucose 32g/L, peanut shell 38g/L, methyl alcohol 0.9g/L, methyl jasmonate 3 * 10
-5g/L, peptone 3g/L, KH
2pO
41g/L, ZnSO
4 .2H
2o 0.01g/L, K
2hPO
40.4g/L, FeSO
4 .7H
2o 0.05 g/L, MgSO
4 .7H
2o 0.5 g/L, CuSO
4 .5H
2o 0.02 g/L, CoCl
20.01 g/L, MnSO
4 .h
2o 0.08 g/L, pH value 6.0; The standard that fermentation termination is established be content of reducing sugar lower than 2%, microscopy finds that mycelium senesces.
(4) Inonotus obliquus immune-enhancing activity material preparation: tunning is filtered, obtain mycelium and zymotic fluid, by the mycelium supersonic wave wall breaking, obtain mycelium powder after drying and crushing, by zymotic fluid be concentrated into original volume 40% after Fermented Condensed liquid, the ratio that adds 40ml Fermented Condensed liquid in the 1g mycelium powder, mix mycelium powder and Fermented Condensed liquid, obtains the Inonotus obliquus immune-enhancing activity material of powdery after freeze drying.
Embodiment 3
(1) actication of culture: take Inonotus obliquus as bacterial classification, streak inoculation, on the slant strains culture medium 28 ℃, is cultivated 220 hours, obtains activated spawn; The formula of slant strains culture medium is: malt extract 40 g/100mL, and peptone 4 g/100mL, agar 10 g/100mL, the pH value is 5.4-5.6.
(2) strain cultivation: activated spawn is proceeded to 28 ℃ of liquid seed culture mediums, cultivate 4d on shaking table and obtain liquid spawn to logarithmic phase, 100 rev/mins of shaking speed.The formula of liquid seed culture medium is: glucose 5g/100mL, peptone 0.5g/100mL, yeast extract 0.1g/100mL, KH
2pO
40.1 g/100mL, MgSO
40.15 g/100mL, CaCl
20.01 g/100mL.
(3) liquid deep layer fermenting: the fermentation cylinder for fermentation that the liquid spawn access is equipped with to the liquid deep layer fermenting culture medium is cultivated, the volume ratio of liquid spawn and liquid deep layer fermenting culture medium is 1:9, the technological parameter of liquid deep layer fermenting is: 28 ℃ of fermentation temperatures, fermentation tank air mass flow 0.8vvm, fermentation tank internal pressure 0.2 kg/cm
3, 100 rev/mins of speeds of agitator, fermentation period 13 days; The formula of described liquid deep layer fermenting culture medium is: glucose 31g/L, bagasse 35g/L, Tween-80 1g/L, diazosulfide 3 * 10
-5g/L, peptone 3g/L, KH
2pO
41g/L, ZnSO
4 .2H
2o 0.01g/L, K
2hPO
40.4g/L, FeSO
4 .7H
2o 0.05 g/L, MgSO
4 .7H
2o 0.5 g/L, CuSO
4 .5H
2o 0.02 g/L, CoCl
20.01 g/L, MnSO
4 .h
2o 0.08 g/L, pH value 6.0, the standard that fermentation termination is established be content of reducing sugar lower than 2%, microscopy finds that mycelium senesces.
(4) Inonotus obliquus immune-enhancing activity material preparation: tunning is filtered, obtain mycelium and zymotic fluid, by the mycelium supersonic wave wall breaking, obtain mycelium powder after drying and crushing, by zymotic fluid be concentrated into original volume 35% after Fermented Condensed liquid, the ratio that adds 35ml Fermented Condensed liquid in the 1g mycelium powder, mix mycelium powder and Fermented Condensed liquid, obtains the Inonotus obliquus immune-enhancing activity material of powdery after freeze drying.
According to inventor's extracting method for polyphenol (extracting from zymotic fluid) outside exocellular polysaccharide (extracting from zymotic fluid), intracellular polyse (extracting from mycelium), born of the same parents that the embodiment 1 of disclosed technology CN102108371A puts down in writing in earlier stage, in born of the same parents, polyphenol extracting method is: mycelium adds 70% acetone, uses the ultrasonic cell disruption instrument broken wall.By the filtration of the slurries after broken wall, centrifugal, supernatant concentration, dried product are polyphenol in born of the same parents.
Zymotic fluid and mycelium that technique of the present invention is obtained are analyzed, and with the CN102108371A(Comparative Examples) Data Comparison, the results are shown in following table.
As seen from the above table, polysaccharide, polyphenol content by the Inonotus obliquus liquid fermentation production of improving one's methods of the present invention are significantly increased, and have improved the output of Inonotus obliquus immune-enhancing activity material.Lignocellulose degradation is more thorough, reaches the purpose of further raising polysaccharide and polyphenol (Inonotus obliquus immune-enhancing activity material) output.Particularly make the productive rate of Epigallo-catechin gallate (EGCG) (EGCG) in polyphenol compound, L-Epicatechin gallate (ECG), phelligridin G, davallialactone, inoscavin B significantly improve.
The activity of product, effect are good, and process simply, and production cost is low, is applicable to suitability for industrialized production, can be directly as the additive of all kinds of animal feeds, use.
The polysaccharide extracted with tunning of the present invention discloses the immunocompetence of product of the present invention, as seen from Figure 1, exocellular polysaccharide, intracellular polyse that tunning of the present invention extracts all show as the growth-promoting successful, and, along with the increase of concentration, this growth-promoting functions is strengthened.Tunning of the present invention extracts as shown in Figure 2 exocellular polysaccharide, intracellular polyse all show as the effect that improves the macrophage proliferation ability.
Product Inonotus obliquus immune-enhancing activity material of the present invention: calculate with 100 gram Inonotus obliquus immune-enhancing activity materials, the composition polysaccharide mainly comprised (outside born of the same parents+born of the same parents are interior) the 8-10 gram, protein 15-20 gram, and polyphenol (outside born of the same parents+born of the same parents are interior) the 2-3 gram, melanin 1-2 gram.
Above-described embodiment is a kind of preferably scheme of the present invention, not the present invention is done to any pro forma restriction, also has other variant and remodeling under the prerequisite that does not exceed the technical scheme that claim puts down in writing.
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Claims (8)
1. a liquid deep layer fermenting is produced the technique of Inonotus obliquus immune-enhancing activity material, and it is characterized in that: described processing step is as follows:
(1) actication of culture: take Inonotus obliquus as bacterial classification, be seeded on the slant strains culture medium and cultivate, obtain activated spawn;
(2) strain cultivation: activated spawn is proceeded in liquid seed culture medium, and shaking table is cultured to logarithmic phase and obtains liquid spawn;
(3) liquid deep layer fermenting: liquid spawn is accessed to fermented and cultured in the liquid deep layer fermenting culture medium, the volume ratio of liquid spawn and liquid deep layer fermenting culture medium is 1:8-10, the formula of described liquid deep layer fermenting culture medium is: glucose 30-32g/L, lignocellulosic 32-38g/L, promoter 0.9-1g/L, derivant 3 * 10
-5g/L, peptone 3g/L, KH
2pO
41g/L, ZnSO
4 .2H
2o 0.01g/L, K
2hPO
40.4g/L, FeSO
4 .7H
2o 0.05 g/L, MgSO
4 .7H
2o 0.5 g/L, CuSO
4 .5H
2o 0.02 g/L, CoCl
20.01 g/L, MnSO
4 .h
2o 0.08 g/L, pH value 6.0;
The technological parameter of liquid deep layer fermenting is: fermentation temperature 27-28 ℃, fermentation tank air mass flow 0.5-1.0vvm, fermentation tank internal pressure 0.1-0.25 kg/cm
3, speed of agitator 70-150 rev/min, fermentation period 12-14 days;
(4) Inonotus obliquus immune-enhancing activity material preparation: tunning is filtered, obtain mycelium and zymotic fluid, by the mycelium supersonic wave wall breaking, obtain mycelium powder after drying and crushing, zymotic fluid is concentrated into after the 30%-40% of original volume to obtain to Fermented Condensed liquid, the ratio that adds 30-40ml Fermented Condensed liquid in the 1g mycelium powder, mix mycelium powder and Fermented Condensed liquid, obtains the Inonotus obliquus immune-enhancing activity material of powdery after drying.
2. technique according to claim 1, it is characterized in that: described promoter is oleic acid, methyl alcohol or Tween-80.
3. technique according to claim 1, it is characterized in that: described derivant is diazosulfide or methyl jasmonate.
4. technique according to claim 1 is characterized in that: described lignocellulosic is selected from a kind of in agricultural crop straw, wood chip, peanut shell, bagasse.
5. according to claim 1 or 2 or 3 or 4 described techniques, it is characterized in that: the formula of described slant strains culture medium is: malt extract 40 g/100mL, and peptone 4 g/100mL, agar 10 g/100mL, the pH value is 5.4-5.6.
6. technique according to claim 5, it is characterized in that: the parameter of cultivating on the slant strains culture medium is: temperature 27-28 ℃, cultivate 200-250 hour.
7. according to claim 1 or 2 or 3 or 4 described techniques, it is characterized in that: the formula of described liquid seed culture medium is: glucose 5g/100mL, peptone 0.5g/100mL, yeast extract 0.1g/100mL, KH
2pO
40.1 g/100mL, MgSO
40.15 g/100mL, CaCl
20.01 g/100mL.
8. technique according to claim 7, it is characterized in that: the parameter of strain cultivation is: temperature 27-28 ℃, cultivate 3.5-4.5d, shaking speed 80-140 rev/min.
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