CN103156052B - 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 fermentation engineering field, particularly a kind of liquid submerged fermentation is produced the technique of Phaeopoms 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, fungus polysaccharide is the main active substances of medicinal fungi, has significant immunoloregulation function, simultaneously also antiviral, antitumor, anti-oxidant, protect liver, reducing blood-fat, the aspect such as hypoglycemic has biological effectiveness widely.Fungus polysaccharide is mainly isolated meta-bolites from basidiomycetes and ascomycetes sporophore, mycelium and fermented liquid.Up till now for this reason, from nearly 300 kinds of fungies, get more than 200 both at home and abroad and plant the bioactive polysaccharide material of tool, fungus polysaccharide that wherein China finds that there is important value is as the approximately kind more than 30 such as krestin, Cordyceps polysaccharide, hericium erinaceum polysaccharide, mushroom polysaccharide, polyporusum bellatus, grifolan, Methods of Polysaccharide From Agrocybe Chaxingu, Inonotue obliquus, Agaricus blazei Murrill, ganoderan.Fungus polysaccharide is the immunostimulant 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, fungus polysaccharide can be brought into play regulating effect to immunity system by many levels, many approach.A large amount of immunization experiments confirmations, polysaccharide can activate the activity of the immunocytes such as T, bone-marrow-derived lymphocyte, scavenger cell and natural killer cell (NK), and it promotes effect of the each side such as generation and complement activation of cytokine in addition in addition.Fungus polysaccharide is to realize immuno-potentiation by the immunity system of enhancing body immune organ, immunocyte and 3 levels of immune molecule
[1].Derive from β-1 of medicinal fungi and yeast, 3/1,6-dextran is a kind of effectively immunostimulant, and people have known their modes of action in immunity system.In addition, β-1,3/1,6-dextran is avirulent, and their mechanism of action in the immunity system of all animals (from invertebrates to people) are identical.β-1,3/1,6-dextran singly can not work by injection, also can work by sneaking into feedstuff feeding.
Phaeopoms obliquus, has another name called Chaga, formal name used at school
inonotus obliquus, Fuscoporia oblique, European is referred to as " chaga ", is that the brown transverse hole fungus of a kind of polyporaceae belongs to edible and medicinal fungi.Phaeopoms obliquus mainly grows in the dried-up upper of trees under the bark of standing tree or after felling of living such as white birch, silvery birch, elm, alder, forms sterile wood-decay fungi, its sclerotium can be after felling dried-up on existence for 6 years.It is mainly 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 is generally warty, diameter 25-40 cm, and appearance is hard, has dark slight crack, harder, and crisp when dry, color is tawny or darker.
Since 16th century, Phaeopoms obliquus is widely used in malignant tumour in Russia, some countries of Northern Europe
[2], diabetes, cardiovascular disorder, hepatopathy
[3]and acquired immune deficiency syndrome (AIDS)
[4]etc. the treatment of disease.In Russia, Phaeopoms obliquus is widely used as daily health products.The researchist of Japan claims that Phaeopoms obliquus is a kind of " panacea ".In Japan, the Powdered electuary of Phaeopoms obliquus is often drunk as health tea.Nineteen fifty-five Moscow medical courses in general institute (The Medical Academy of Science in Moscow) announces to classify Phaeopoms obliquus as cancer-resisting substance, and government permission Phaeopoms obliquus can be used for pharmaceuticals exploitation.The U.S. lists Phaeopoms obliquus in " special crude substance ".
In Inonotus obliquus sporophore, be rich in callose and superoxide dismutase, 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, Phaeopoms obliquus always as pure Chinese medicine in the disease that is used for the treatment of among the people, Phaeopoms obliquus is also considered to the functional health-care food of 21 century.Clinical experiment shows: Phaeopoms obliquus is without any toxic side effect, and in treatment diabetes, prevent AIDS, the aspect such as strengthening immunity has obvious effect
[3].Current research report shows, the main active ingredient of Phaeopoms obliquus has polysaccharide, polyphenol, steroidal, inonotus obliquus, melanochrome, triterpene, sheath amine ester compound, alkaloid, folic acid derivatives, vanillic acid, syringic acid and gamma-hydroxy formic acid etc., also has recently report to point out that scholar has isolated tannin compound, steroid, low molecule Polyphenols and lignin compound
[5].
Fuscoporia obliqua polysaccharide is one of valuable pharmacological activeconstituents of Phaeopoms obliquus, and the polysaccharide in mycelia and the sclerotium of Phaeopoms obliquus is mainly made up of beta-glucan, mixed polysaccharide and albumen composition
[6].Research shows, Inonotus obliquus sporophore polysaccharide mainly contains the monose compositions such as glucose, semi-lactosi, seminose, rhamnosyl, wood sugar and pectinose.And glucose is main monose, account for the more than 70% of all monose compositions
[7].Between monose, be to be formed by connecting by β-(1,3) glycosidic link or β-(Isosorbide-5-Nitrae) glycosidic link.Between main chain and side chain, connect with β-(1,6) glycosidic link.Wood sugar and rhamnosyl are that Fuscoporia obliqua polysaccharide biological activity is contributed maximum monose
[8].The functional performance of Fuscoporia obliqua polysaccharide is mainly manifested in the growth that its fruitbody polysaccharide can suppress S180 sarcoma
[9], the cytokines such as its mycelium polysaccharides energy effective stimulus scavenger cell secretion IL21, IL26, TNF2A, NO, cytokine is by adjusting the immunological status killing tumor cell indirectly of body
[10].In addition, the water-soluble and water-insoluble polysaccharide of Phaeopoms obliquus has the crude extract that falls hypoglycemic effect, especially Fuscoporia obliqua polysaccharide to diabetic mice, its sustainable 48h of hypoglycemic activity.
Phaeopoms obliquus polyphenol is mainly made up of melanochrome class material and oppositeleaf fig root and leaf alkali homologue
[11]in Inonotus obliquus sporophore, mainly contain 4-hydroxyl-3,5 dimethoxybenzoic acid-2 '-hydroxyls-1 '-methylol ethyl acetate, Protocatechuic Acid (PCA), coffic acid (CA), 3,4-Dihydroxy benzaldehyde (DB), 2,5-dihydroxyl-1,3-phthalic acid (DTA), Syringic acid (SA) and 3,4-dihydroxyl benzalacetone (DBL)
[11]deng small molecules phenols, Lee
[12]by Inonotus obliquus sporophore is extracted after purifying, having obtained six kinds of macromole polyphenolic compounds Deng people, is respectively Phelligridin D, Phelligridin E, Phelligridin G, Inonoblin A, Inonoblin B, Inonoblin C.Zheng
[13]with fermentor tank, Phaeopoms obliquus mycelium is carried out to liquid submerged fermentation Deng people, after extraction and analysis, find, in Phaeopoms obliquus mycelium, mainly contain the 4 large class materials such as small molecules phenols, glucosides flavones, Flavone aglycone, polyphenol.
Phaeopoms obliquus polyphenol has anti-oxidant
[13,14], antitumor
[16], protect liver
[3], immunological enhancement
[11]etc. function.Report points out that the catechol of extracting from Inonotus obliquus sporophore has significant gene protection and anti-oxidant 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 Phaeopoms obliquus polyphenol is removed oxyradical reaches more than 80%, compare the conventional antioxidants such as D-VC sodium and Tenox PG and there is obvious advantage, have broad application prospects as a kind of natural antioxidants.
Phaeopoms obliquus parasitizes on birch, is the bacterial classification extremely resisting cold, and can under the environment of subzero 69 DEG C, grow, and reaches 15 years vegetative period.The adult bacterium of wild Phaeopoms 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 means artificial culture sclerotium and sporophore, therefore adopting liquid submerged fermentation cultured mycelia to obtain activeconstituents is a feasible replacement method.Adopt the method for liquid submerged fermentation, compare with traditional fungi method of cultivation, there is the superiority such as with short production cycle, cost is low, output and quality is stable, product can be controlled.
Summary of the invention
The object of the invention is provides a kind of liquid submerged fermentation of optimization to produce the technique of Phaeopoms obliquus immune-enhancing activity material contriver early stage on disclosed technology CN102108371A basis, the immune-enhancing activity substances content obtaining 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:
Liquid submerged fermentation is produced a technique for Phaeopoms obliquus immune-enhancing activity material, and described processing step is as follows:
(1) actication of culture: taking Phaeopoms obliquus as bacterial classification, be seeded on slant strains substratum 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 submerged fermentation: liquid spawn is accessed to fermentation culture in liquid submerged fermentation substratum, the volume ratio of liquid spawn and liquid submerged fermentation substratum is 1:8-10, the formula of described liquid submerged fermentation substratum is: glucose 30-32g/L, lignocellulose 32-38g/L, promotor 0.9-1g/L, inductor 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 processing parameter of liquid submerged fermentation is: leavening temperature 27-28 DEG C, fermentor tank air flow quantity 0.5-1.0 vvm, fermentor tank internal pressure 0.1-0.25 kg/cm
3, mixing speed 70-150 rev/min, fermentation period 12-14 days;
(4) Phaeopoms obliquus immune-enhancing activity material preparation: tunning is filtered, obtain mycelium and fermented liquid, by mycelium supersonic wave wall breaking, after drying and crushing, obtain mycelium powder, fermented liquid is concentrated into after the 30%-40% of original volume to obtain to Fermented Condensed liquid, add the ratio of 30-40 ml Fermented Condensed liquid in 1 g mycelium powder, mycelium powder and Fermented Condensed liquid are mixed, after being dried, obtain the Phaeopoms obliquus immune-enhancing activity material of powdery.
Along with the carrying out of research, contriver finds, contriver's disclosed technology (CN102108371A) Phaeopoms 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 lignocellulose, therefore, contriver has carried out optimizing and creating again to technique, is mainly reflected in the innovation of substratum:
In liquid submerged fermentation substratum, add promotor, by increasing the permeability of cell and/or directly affecting polysaccharide and the generation of polyphenol synthase, reach the object that improves Phaeopoms obliquus liquid fermentation production exocellular polysaccharide, polyphenol, melanochrome output, improved the output of Phaeopoms obliquus immune-enhancing activity material.
In liquid submerged fermentation substratum, add promotor and lignocellulose simultaneously, on the basis producing at the increase cell permeability of promotor and polysaccharide, polyphenol synthase, promote the secretion of lignocellulose degradation enzyme (laccase, lignin peroxidase, manganese peroxidase) synthetic in born of the same parents, thereby strengthen the decomposition to lignocellulose in substratum, lignocellulose degradation is more thorough, reaches the object of further raising polysaccharide and polyphenol (Phaeopoms obliquus immune-enhancing activity material) output.Particularly make the productive rate of NVP-XAA 723 (EGCG) in polyphenolic compound, L-Epicatechin gallate (ECG), phelligridin G, davallialactone, inoscavin B significantly improve.
In liquid submerged fermentation substratum, add inductor, promoted the biosynthesizing of Phaeopoms obliquus polyphenols (one of Phaeopoms obliquus immune-enhancing activity material).The present invention utilizes inductor to improve Phaeopoms obliquus fermentation polyphenol, particularly high reactivity polyphenol as the output of NVP-XAA 723 (EGCG), L-Epicatechin gallate (ECG).
In liquid submerged fermentation substratum, add promotor and inductor simultaneously, and coordinate with lignocellulose, on the basis producing at increase cell permeability and the polyphenol synthase of promotor, add the biosynthetic inductor of polyphenol, collaborative promotion mutually, thereby greatly promote Phaeopoms obliquus fermentation polysaccharide, the biosynthesizing of polyphenols, lignocellulose degradation more thoroughly reaches further raising polysaccharide, polyphenol, particularly high reactivity polyphenol is as NVP-XAA 723 (EGCG), the object of L-Epicatechin gallate (ECG) output, make the activity of the Phaeopoms obliquus immune-enhancing activity material obtaining better.
Mycelium is rich in polyphenol in intracellular polyse and born of the same parents, fermented liquid is rich in exocellular polysaccharide and the outer polyphenol of born of the same parents, contriver's disclosed technology in early stage (CN102108371A) is to mycelium, fermented liquid is extraction process respectively, thereby obtain different active substances, 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 processing after to mycelium broken wall, fermented liquid is cooked to concentration, and control the proportioning of mycelium powder and Fermented Condensed liquid, mycelium powder and Fermented Condensed liquid mixture homogeneity are good like this, the dry Phaeopoms obliquus immune-enhancing activity material that obtains, with the best proportioning of 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 so not only activity of product, effect is good, and process simple, production cost is low, be applicable to suitability for industrialized production, can directly use as the additive of all kinds of animal-feeds.
As preferably, described promotor is oleic acid, methyl alcohol or Tween-80.
As preferably, described inductor is diazosulfide or methyl jasmonate.Diazosulfide is the salicylic functional analogue of plant endogenous sexual hormoue analogue, can promote the generation of polyphenols, and methyl jasmonate is naturally occurring plant-growth regulator, can promote the biosynthesizing of anthocyanidin.
As preferably, described lignocellulose is selected from the one in agricultural crop straw, wood chip, Pericarppium arachidis hypogaeae, bagasse.Most preferred scheme is at Pericarppium arachidis hypogaeae, in bagasse, select a kind of as lignocellulosic material, contriver invents by large quantity research, agricultural crop straw, the effect when as lignocellulosic material such as wood chip is unsatisfactory, this is because the restriction of the speciality of material itself, and then find after research and probe, Pericarppium arachidis hypogaeae, bagasse is as lignocellulosic material, in its tunning, the activity of polysaccharide and polyphenol can be higher, the degraded of lignocellulose is also more thorough, push away its reason: be mainly Pericarppium arachidis hypogaeae, 3 of lignocellulose component fibre elements in bagasse, the composition of proportions of hemicellulose and xylogen is more suitable for the degraded of Phaeopoms obliquus.
As preferably, the formula of described slant strains substratum is: malt extract 40 g/100mL, and peptone 4 g/100mL, agar 10 g/100mL, pH value is 5.4-5.6.
As preferably, the parameter of cultivating on slant strains substratum is: temperature 27-28 DEG C, cultivates 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 DEG C, cultivates 3.5-4.5d, shaking speed 80-140 rev/min.
The invention has the beneficial effects as follows:
1, in liquid submerged fermentation substratum, add promotor, by increasing the permeability of cell and/or directly affecting polysaccharide and the generation of polyphenol synthase, reach the object that improves Phaeopoms obliquus liquid fermentation production exocellular polysaccharide, polyphenol, melanochrome output, improved the output of Phaeopoms obliquus immune-enhancing activity material.
2, in liquid submerged fermentation substratum, add promotor and lignocellulose simultaneously, on the basis producing at the increase cell permeability of promotor and polysaccharide, polyphenol synthase, promote the secretion of lignocellulose degradation enzyme (laccase, lignin peroxidase, manganese peroxidase) synthetic in born of the same parents, thereby strengthen the decomposition to lignocellulose in substratum, lignocellulose degradation is more thorough, reaches the object of further raising polysaccharide and polyphenol (Phaeopoms obliquus immune-enhancing activity material) output.Particularly make the productive rate of NVP-XAA 723 (EGCG) in polyphenolic compound, L-Epicatechin gallate (ECG), phelligridin G, davallialactone, inoscavin B significantly improve.
3, in liquid submerged fermentation substratum, add inductor, promoted the biosynthesizing of Phaeopoms obliquus polyphenols (one of Phaeopoms obliquus immune-enhancing activity material).The present invention utilizes inductor to improve Phaeopoms obliquus fermentation polyphenol, particularly high reactivity polyphenol as the output of NVP-XAA 723 (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 directly use as the additive of all kinds of animal-feeds.
Brief description of the drawings
Fig. 1 is the growth-promoting Cytokine figure of the polysaccharide of tunning extraction of the present invention, wherein Fig. 1 (a) TNF: tumour necrosis factor, Fig. 1 (b) IFN: Interferon, rabbit, Fig. 1 (c) IL-2: interleukin II, 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.
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, raw material and the equipment etc. adopting all can be buied from market or this area is conventional.Method in following embodiment, if no special instructions, is the ordinary method of this area.
Phaeopoms obliquus is commercially available, purchased from Xuzhou Normal University.
Embodiment 1
(1) actication of culture: taking Phaeopoms obliquus as bacterial classification, streak inoculation, on slant strains substratum 27 DEG C, is cultivated 250 hours, obtains activated spawn; The formula of slant strains substratum is: malt extract 40 g/100mL, and peptone 4 g/100mL, agar 10 g/100mL, pH value is 5.4-5.6.
(2) strain cultivation: activated spawn is proceeded to 27 DEG C of liquid seed culture mediums, cultivate 4.5d on shaking table and obtain liquid spawn to logarithmic phase, 80 revs/min 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 submerged fermentation: the fermentation cylinder for fermentation that liquid spawn access is equipped with to liquid submerged fermentation substratum is cultivated, the volume ratio of liquid spawn and liquid submerged fermentation substratum is 1:8, the processing parameter of liquid submerged fermentation is: 27 DEG C of leavening temperatures, fermentor tank air flow quantity 0.5vvm, fermentor tank internal pressure 0.1 kg/cm
3, 70 revs/min of mixing speed, fermentation period 14 days; The formula of described liquid submerged fermentation substratum 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; Fermentation termination establish standard be reducing sugar content lower than 2%, microscopy find mycelium senesce.
(4) Phaeopoms obliquus immune-enhancing activity material preparation: tunning is filtered, obtain mycelium and fermented liquid, by mycelium supersonic wave wall breaking, after drying and crushing, obtain mycelium powder, by fermented liquid be concentrated into original volume 30% after Fermented Condensed liquid, add the ratio of 30ml Fermented Condensed liquid in 1g mycelium powder, mycelium powder and Fermented Condensed liquid are mixed, after vacuum-drying, obtain the Phaeopoms obliquus immune-enhancing activity material of powdery.
(1) actication of culture: taking Phaeopoms obliquus as bacterial classification, streak inoculation, on slant strains substratum 28 DEG C, is cultivated 200 hours, obtains activated spawn; The formula of slant strains substratum is: malt extract 40 g/100mL, and peptone 4 g/100mL, agar 10 g/100mL, pH value is 5.4-5.6.
(2) strain cultivation: activated spawn is proceeded to 28 DEG C of liquid seed culture mediums, cultivate 3.5d on shaking table and obtain liquid spawn to logarithmic phase, 140 revs/min 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 submerged fermentation: the fermentation cylinder for fermentation that liquid spawn access is equipped with to liquid submerged fermentation substratum is cultivated, the volume ratio of liquid spawn and liquid submerged fermentation substratum is 1:10, the processing parameter of liquid submerged fermentation is: 28 DEG C of leavening temperatures, fermentor tank air flow quantity 1.0vvm, fermentor tank internal pressure 0.25 kg/cm
3, 150 revs/min of mixing speed, fermentation period 12 days; The formula of described liquid submerged fermentation substratum is: glucose 32g/L, Pericarppium arachidis hypogaeae 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; Fermentation termination establish standard be reducing sugar content lower than 2%, microscopy find mycelium senesce.
(4) Phaeopoms obliquus immune-enhancing activity material preparation: tunning is filtered, obtain mycelium and fermented liquid, by mycelium supersonic wave wall breaking, after drying and crushing, obtain mycelium powder, by fermented liquid be concentrated into original volume 40% after Fermented Condensed liquid, add the ratio of 40ml Fermented Condensed liquid in 1g mycelium powder, mycelium powder and Fermented Condensed liquid are mixed, after lyophilize, obtain the Phaeopoms obliquus immune-enhancing activity material of powdery.
Embodiment 3
(1) actication of culture: taking Phaeopoms obliquus as bacterial classification, streak inoculation, on slant strains substratum 28 DEG C, is cultivated 220 hours, obtains activated spawn; The formula of slant strains substratum is: malt extract 40 g/100mL, and peptone 4 g/100mL, agar 10 g/100mL, pH value is 5.4-5.6.
(2) strain cultivation: activated spawn is proceeded to 28 DEG C of liquid seed culture mediums, cultivate 4d on shaking table and obtain liquid spawn to logarithmic phase, 100 revs/min 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 submerged fermentation: the fermentation cylinder for fermentation that liquid spawn access is equipped with to liquid submerged fermentation substratum is cultivated, the volume ratio of liquid spawn and liquid submerged fermentation substratum is 1:9, the processing parameter of liquid submerged fermentation is: 28 DEG C of leavening temperatures, fermentor tank air flow quantity 0.8vvm, fermentor tank internal pressure 0.2 kg/cm
3, 100 revs/min of mixing speed, fermentation period 13 days; The formula of described liquid submerged fermentation substratum 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, fermentation termination establish standard be reducing sugar content lower than 2%, microscopy find mycelium senesce.
(4) Phaeopoms obliquus immune-enhancing activity material preparation: tunning is filtered, obtain mycelium and fermented liquid, by mycelium supersonic wave wall breaking, after drying and crushing, obtain mycelium powder, by fermented liquid be concentrated into original volume 35% after Fermented Condensed liquid, add the ratio of 35ml Fermented Condensed liquid in 1g mycelium powder, mycelium powder and Fermented Condensed liquid are mixed, after lyophilize, obtain the Phaeopoms obliquus immune-enhancing activity material of powdery.
According to contriver's extracting method for polyphenol (extracting from fermented liquid) outside exocellular polysaccharide (extracting from fermented liquid), intracellular polyse (extracting from mycelium), born of the same parents that the embodiment 1 of disclosed technology CN102108371A records in earlier stage, in born of the same parents, polyphenol extracting method is: mycelium adds 70% acetone, uses ultrasonic cell disruption instrument broken wall.By the slurries filtration after broken wall, centrifugal, supernatant concentration, dried product are polyphenol in born of the same parents.
Fermented liquid and mycelium that technique of the present invention is obtained are analyzed, and with CN102108371A(comparative example) Data Comparison, the results are shown in following table.
As seen from the above table, be significantly increased by polysaccharide, the polyphenol content of the Phaeopoms obliquus liquid fermentation production of improving one's methods of the present invention, improved the output of Phaeopoms obliquus immune-enhancing activity material.Lignocellulose degradation is more thorough, reaches the object of further raising polysaccharide and polyphenol (Phaeopoms obliquus immune-enhancing activity material) output.Particularly make the productive rate of NVP-XAA 723 (EGCG) in polyphenolic compound, L-Epicatechin gallate (ECG), phelligridin G, davallialactone, inoscavin B significantly improve.
Activity, effect of product are good, and process simply, and production cost is low, are applicable to suitability for industrialized production, can directly use as the additive of all kinds of animal-feeds.
The polysaccharide extracting 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 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 macrophage proliferation ability.
Product Phaeopoms obliquus immune-enhancing activity material of the present invention: calculate with 100 grams of Phaeopoms obliquus immune-enhancing activity materials, the composition polysaccharide that mainly comprises (born of the same parents outer+born of the same parents in) 8-10 gram, protein 15-20 gram, and polyphenol (outside born of the same parents+born of the same parents are interior) 2-3 gram, melanochrome 1-2 gram.
Above-described embodiment is preferably scheme of one 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 records.
Reference
[1]?Wasser?S?P.?Medicinal?mushrooms?as?a?source?of?antitumor?and?immune-?modulating?polysaccharides?[J].?Appl?Microbiol?Biotechnol,?2002,?60:258–274.
[2]?Glowacki?H,?Tomaszewski?J.?Attempted?therapy?of?inoperable?cancer?of?the?female?genitalia?with?
Poria?oblique?[J].?Ginekol?Pol.?1962,?33:?445-450.
[3]?Wasser?SP,?Weis?AL.?Therapeutic?effects?of?substances?occurring?in?higher?Basidiomycetes?mushrooms:?a?modern?perspective?[J].?Crit.?Rev?Immunol,?1999,?19:?65-96.
[4]?Sakuma?K.?Aids?virus?multiplication?inhibitor?and?method?of?culturing?active?ingredient?thereof:?JP,?93/159,?946,?[P].?1993-07-07.
[5]?Babitskaia?V?G,?et?al.?Melanin?complex?of?the?fungus?
Inonotus?obliquus?[J].?Prikl?Biokhim?Mikrobiol,?2000,?36(4):?439-444.
[6]?Mizuno?T,?Zhuang?C.?Antiumor?and?hypoglycemic?activities?of?polysaccharides?from?the?scleritia?and?mycelia?of?Inonotus?obliquas?[J].?Int?J?of?Med?Mushrooms,?1999,?l?(4):?301-316.
[7]?Leung?M.?Y.?K,?Liu?C.?Polysaccharides?biological?response?modifiers?[J].?Immunol?Lett,?2006,?105:?101-114.
[8]?Jin?G,?et?al.?Antitumor?activities?of?polysaccharides?from?
Fuscoporia?obique?[J].?J?MedSci?YanbianUniv,?2004,?27:?257-250.
[9]?Kim?Y?O,?et?al.?Immuno-stimulating?effect?of?the?endo-polysaccharide?produced?by?submerged?culture?of?
Inonotus?obliquus?[J].?Life?Sci,?2005,?77(19):?2438-2456.
[10]?Zheng?W?F,?Zhao?Y?X,?Zhang?M?M?,?Yin?Z?J,?Chen?C?F,?Wei?Z?W.?Phenolic?compounds?from?
Inonotus?obliquus?and?their?immune-stimulating?effects?[J].?Mycosystema,?2008,?27(4):?574-581.
[11]?Nakajima?Y,?et?al.?Antioxidant?Small?Phenolic?Ingredients?in?
Inonotus?obliquus?[J].?Chem.?Pharm.?Bull,?2007,?55(8):?1222-1226.
[12]?Lee?I?K,?et?al.?New?antioxidant?polyphenols?from?the?medicinal?mushroom?
Inonotus?obliquus?[J].?Bioorganic?&?Medicinal?Chemistry?Letters,?2007,?17:?6678-6681.
[13]?Zheng?W,?et?al.?Accumulation?of?antioxidant?phenolic?constituentsin?submerged?cultures?of?
Inonotus?obliquus?[J].?Bioresource?Technol,?2009,?100:1327-1335.
[14]?Xu?X,?Zhu?J.?Enhanced?phenolic?antioxidants?production?in?submerged?cultures?of?
Inonotus?obliquus?in?a?ground?corn?stover?medium.?Biochem?Eng?J,?2011,?58-59:103-109.
[15]?Cui?Y,?Kim?D?S,?Park?K?C.?Antioxidant?effect?of?
Inonotus?obliquus?[J].?Ethnopharmacol,?2005,?96(1-2):?79-85.
[16]?Zhang?X,?Song?Y,?Huang?H.?Extracellular?hydroxyl?radical?scavenger?produced?by?white?rot?fungus?[J].?J?Huazhong?Univ?Sci?Technol?(Nature?Science?Edition),?2005,?35:129-132.
[17]?Babitskaia?V?G,?Shcherba?VV.?The?nature?of?melanin?pigments?from?some?micro-?and?macromycetes?[J].?Prikl?Biokhlm?Mikrobiol,?2002,?38(3):?286-291。
Claims (5)
1. liquid submerged fermentation is produced a technique for Phaeopoms obliquus immune-enhancing activity material, it is characterized in that: described processing step is as follows:
(1) actication of culture: taking Phaeopoms obliquus as bacterial classification, be seeded on slant strains substratum 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 submerged fermentation: liquid spawn is accessed to fermentation culture in liquid submerged fermentation substratum, the volume ratio of liquid spawn and liquid submerged fermentation substratum is 1:8-10, the formula of described liquid submerged fermentation substratum is: glucose 30-32g/L, lignocellulose 32-38g/L, promotor 0.9-1g/L, inductor 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; Described promotor is oleic acid, methyl alcohol or Tween-80; Described inductor is diazosulfide or methyl jasmonate; Described lignocellulose is selected from the one in Pericarppium arachidis hypogaeae, bagasse;
The processing parameter of liquid submerged fermentation is: leavening temperature 27-28 DEG C, fermentor tank air flow quantity 0.5-1.0vvm, fermentor tank internal pressure 0.1-0.25 kg/cm
3, mixing speed 70-150 rev/min, fermentation period 12-14 days;
(4) Phaeopoms obliquus immune-enhancing activity material preparation: tunning is filtered, obtain mycelium and fermented liquid, by mycelium supersonic wave wall breaking, after drying and crushing, obtain mycelium powder, fermented liquid is concentrated into after the 30%-40% of original volume to obtain to Fermented Condensed liquid, add the ratio of 30-40ml Fermented Condensed liquid in 1g mycelium powder, mycelium powder and Fermented Condensed liquid are mixed, after being dried, obtain the Phaeopoms obliquus immune-enhancing activity material of powdery.
2. technique according to claim 1, is characterized in that: the formula of described slant strains substratum is: malt extract 40 g/100mL, and peptone 4 g/100mL, agar 10 g/100mL, pH value is 5.4-5.6.
3. technique according to claim 2, is characterized in that: the parameter of cultivating on slant strains substratum is: temperature 27-28 DEG C, cultivates 200-250 hour.
4. technique according to claim 1, 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.
5. technique according to claim 4, is characterized in that: the parameter of strain cultivation is: temperature 27-28 DEG C, cultivates 3.5-4.5d, shaking speed 80-140 rev/min.
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