CN101643759B - Method for preparing Schizophyllum commune Fr polysaccharides and dedicated culture medium thereof - Google Patents
Method for preparing Schizophyllum commune Fr polysaccharides and dedicated culture medium thereof Download PDFInfo
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Abstract
The invention discloses a method for preparing Schizophyllum commune Fr polysaccharides and a dedicated culture medium thereof. The culture medium comprises the following components per kilogram: 400g to 450g of corncobs, cornstalks or cornhusks, 50g to 100g of wheat bran, rice bran or cane trash, 0.05g to 0.1g of growth-promoting factor V<B1>, 0.05g to 0.1g of L-glutamic acid and the balance being water. The method of the invention for preparing Schizophyllum commune Fr polysaccharides through solid fermentation using agricultural produce resources (such as cornstalks, corncobs, cornhusks, wheat bran, cane trash and the like) rich in fiber matrixes as raw materials, thereby greatly reducing the material cost, achieving the effective transformation of the agricultural produce resources, ensuring the environmental protection and acquiring the economic value; the yield of the method is high; and the content of polysaccharides in the fermentation materials reaches 4.5% to 5.8% (by mass percentage).
Description
Technical field
The present invention relates to a kind of method and special culture media for preparing Schizophyllum commune Fr polysaccharides.
Background technology
Split-gill (Schizophyllum commune Fr.) has another name called white ginseng bacterium, tree flower bacterium, is a kind of white saprophytic load guiding principle fungi with very high edible and pharmaceutical use.Split-gill distributes quite extensive at occurring in nature, it is xylogen, semicellulose and the part Mierocrystalline cellulose in the degradation of fibers matrix effectively, can be a kind of medicinal fungi that is easy to cultivate well growing under the harsh conditions relatively.Schizophyllum commune Fr polysaccharides is a kind of water-soluble exocellular polysaccharide that is present in schizophyllum sporophores, mycelium or the fermented liquid, has the typical β of medicinal fungi polysaccharide-(1,3)-D-VISOSE main chain, β-(1,6)-D-glucose branched structure.Multiple physiologically actives such as antitumor, the Azelaic Acid of relevant Schizophyllum commune Fr polysaccharides, radioprotective, enhance immunity power have obtained domestic and international investigator's confirmation.In addition, the HV of Schizophyllum commune Fr polysaccharides, high retentiveness make that also it maybe be as thickening material, stablizer and be widely used in food, daily necessities and field of petrochemical industry, and development potentiality is huge.
The production technique of Schizophyllum commune Fr polysaccharides is main with solution fermentation at present, and through liquid fermentation producing technology in batches or continuously, existing both at home and abroad indivedual companies have realized commercially producing of Schizophyllum commune Fr polysaccharides, and product is mainly used in the pharmacy and the cosmetic industry of high added value.Liquid fermentation technology is the good approach that realizes mikrobe and product industriallization thereof, large-scale production.But because Split-gill is a kind of large-scale filamentous fungus, and this polysaccharide has the hyperviscosity characteristic, exists problems such as mass transfer difficulty, energy consumption are big in the solution fermentation production process.This also is to cause the major reason that its suitability for industrialized production is small, the products production cost is high at present.Solid (attitude) fermentation technique is an ancient biotechnology, is mainly used in traditional fermentation industry (wine, soy sauce, vinegar etc.) in the past.Solid-fermented technique has that equipment is simple, material cost and advantages such as energy consumption is low, pollute less, also is one of developing direction of modern biofermentation technique.Domestic existing indivedual investigators and manufacturer utilize solid fermentation to produce fungus polysaccharides such as ganoderan, tea tree mushroom polysaccharide, grifolan; But do not see the report for preparing Schizophyllum commune Fr polysaccharides with solid fermentation at present, wherein reason maybe be relevant with the research and development deficiency of suitable bacterial classification and culture condition.
As agriculture prodn big country, there are agricultural byproducts resources such as abundant agricultural crop straw, corn cob, wheat bran, rice bran, bagasse in China.And these resources are not fully used at present, serious waste of resources.How above-mentioned agricultural byproducts resource being carried out the deep processing high added value and transform, is the emphasis of processing of farm products field concern in recent years.
Summary of the invention
An object of the present invention is to provide a kind of substratum that solid fermentation prepares Schizophyllum commune Fr polysaccharides that is used for.
Substratum provided by the present invention, it consists of: contain corn cob, corn straw and/or maize peel 400-450g in every kilogram of substratum, Testa Tritici, rice bran and/or bagasse 50-100g, somatomedin V
B10.05-0.1g, L-L-glutamic acid 0.05-0.1g, all the other are water.
In the above-mentioned substratum, contain corn cob, corn straw and/or maize peel 450g in every kilogram of substratum, Testa Tritici, rice bran and/or bagasse 50g, somatomedin V
B10.1g, L-L-glutamic acid 0.1g, all the other are water.
In above-mentioned arbitrary said substratum, said corn cob, corn straw or maize peel can be pulverized and obtained behind the 20-40 mesh sieve excessively.
Another object of the present invention provides the method that a kind of solid fermentation prepares Schizophyllum commune Fr polysaccharides.
The method for preparing Schizophyllum commune Fr polysaccharides provided by the present invention comprises the steps: to obtain Schizophyllum commune Fr polysaccharides with above-mentioned arbitrary said substratum fermentation Split-gill (Schizophyllum commune Fr.).
Wherein, said Split-gill (Schizophyllum commune Fr.) specifically can be the Split-gill of following deposit number: CFCC NO.6812, CFCC NO.83457 or ACCC NO.50875.
In the above-mentioned fermenting process, temperature can be 26-30 ℃.
In the above-mentioned fermenting process, the time can be 7-10 days.
In the above-mentioned fermenting process, the liquid-spawn inoculation amount can be the 50ml-100ml liquid spawn: the 1000g substratum.
Among the above-mentioned preparation method, after said fermentation, also can comprise the step of extraction; The method of said extraction can comprise the steps: that the fermented product that said fermentation is obtained carries out fragmentation, adds entry then, under 60-80 ℃ temperature, stirs, and is centrifugal, collects supernatant.
In the said extracted process, the method for said fragmentation can be earlier fermented product to be poured in the stirrer, carries out fragmentation, carries out fragmentation to wherein adding entry with hollander again.
In the said extracted process, the ratio of the fermented product after said water and the said fragmentation is (15-25) L water: the fermented product after the 1kg fragmentation.
In the said extracted process, the time of said stirring is 40-60min.
In the said extracted process, the said centrifugal time is 15-25min, and centrifuge speed is 3000rpm.
Among the above-mentioned preparation method, after said extraction, also can comprise the step of Deproteinization; The method of said Deproteinization can comprise the steps: to regulate supernatant pH value that said extraction obtains to 4.5-5.0, leaves standstill 0.5-1.5h, and is centrifugal, collect supernatant.
Among the above-mentioned preparation method, behind said Deproteinization, also can comprise the step of decolouring; The method of said decolouring can comprise the steps: that the supernatant that said Deproteinization is obtained adsorbs with gac and macroporous resin successively, collects elutriant; The ratio of said gac and supernatant can be the 1kg gac: (80-120) L supernatant; Said D301 macroporous resin column with can be the 1kg macroporous resin with the ratio of the elutriant that obtains after the charcoal absorption: (120-150) L elutriant.
Solid fermentation of the present invention prepares the method for Schizophyllum commune Fr polysaccharides; Be that agricultural byproducts resource (corn straw, corn cob, maize peel, Testa Tritici, bagasse etc.) with fiber-enriched matrix is fermented as raw material; Greatly reduce raw materials cost; Realized the efficient conversion of agricultural byproducts resources again, not only environmental protection but also have higher economic worth; The yield that this method prepares Schizophyllum commune Fr polysaccharides can reach 4.5-5.8% (quality percentage composition); The polysaccharide that obtains is except that the enhancing immunity that possesses typical medicinal fungi polysaccharide, anti-tumor activity; Also have ultra high molecular weight (average molecular mass reaches 20000KDa), HV physicochemical characteristics such as (apparent characteristic viscosity surpass the food grade XG 550); Can be developed as a kind of natural edible glue product innovation, be widely used in industries such as medicine, daily use chemicals, food with good physiological function and application function.
Summary of the invention
Fig. 1 is the influence of polysaccharide concentration to soltion viscosity.
Fig. 2 is the influence of system pH to soltion viscosity.
Fig. 3 is the influence of temperature to soltion viscosity.
Fig. 4 is Split-gill solid fermentation and polysaccharide extracting process flow process.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Split-gill solid fermentation of the present invention and polysaccharide extracting process flow process are as shown in Figure 4.
Employed Split-gill among the following embodiment (Schizophyllum commune Fr.) specifically can be the Split-gill of following deposit number: CFCC NO.6812, CFCC NO.83457 or ACCC NO.50875.Split-gill (Schizophyllum commune Fr.) CFCC NO.6812 and Split-gill (Schizophyllum commune Fr.) CFCC NO.83457 are available from China Forest microbial strains preservation administrative center, and Split-gill (Schizophyllum commune Fr.) ACCC NO.50875 is available from Chinese agriculture microbial strains preservation administrative center.
One, preparation method
(1) actication of culture
CFCC NO.6812 is inoculated into the PDA slant medium with Split-gill (Schizophyllum commune Fr.), carries out the rejuvenation activation culture.
Consisting of of PDA slant medium: contain yam 20g, glucose 2g, yeast extract paste 0.5g, sal epsom 0.15g, potassium hydrogen phosphate 0.3g and agar 2g in every liter of substratum, all the other are water.
(2) enlarged culturing of bacterial classification
Get cultured slant strains, be inoculated in the liquid PDA substratum, 25-29 ℃, shaking speed 150-180 rev/min following the cultivation 48-52 hour, obtain the liquid spawn that mycelium pellet is evenly distributed, fermented liquid is as clear as crystal, this is the level liquid bacterial classification.
Get the level liquid bacterial classification inoculation in liquid PDA substratum,, obtain second-class liquid isolate 25-29 ℃, shaking speed 150-180 rev/min following the cultivation 48-52 hour.Culture vessel can adopt liquid fermentation tank (50-100L) or big triangular flask (5L) in this process.
Consisting of of liquid PDA substratum: contain yam 20g, glucose 2g, yeast extract paste 0.5g, sal epsom 0.15g and potassium hydrogen phosphate 0.3g in every liter of substratum, all the other are water.
(3) solid fermentation of bacterial classification
In the aseptic technique environment, the second-class liquid isolate that primary fermentation is good is inoculated in the fermention medium, and inoculative proportion is second-class liquid isolate: fermention medium=70mL: 1000g; Place 28 ℃ to cultivate 8 days down, when treating that mycelia is covered with bacterium bag (or bacterium bottle), solid fermentation finishes, and the mixture after this fermentation is known as fermentation raw material.
Consisting of of fermention medium: contain corn cob 450g, Testa Tritici 50g, somatomedin V in every kilogram of substratum
B10.1g, L-L-glutamic acid 0.1g, all the other are water.
The preparation method of fermention medium is following: at first dry corn cob is pulverized, crossed the 20-40 mesh sieve, mix in proportion with Testa Tritici; Somatomedin V
B1Soluble in water after weighing; With somatomedin-V
B1Solution and corn cob, Testa Tritici mix, and add water, again to wherein adding L-L-glutamic acid, add water and complement to 1 kilogram, stir, and obtain the solid fermentation substratum.The fermention medium branch is packed in bacterium bag or the bacterium bottle, placed sterilization 121 ℃ of following wet heat treatment 2.5-4.0 hours; Take out, it is for use to be cooled to room temperature.
(4) extraction of Schizophyllum commune Fr polysaccharides and purifying
1, slightly carry: fermentation raw material is poured in the stirrer, broken earlier; Add less water again, the water of adding accounts for the 10-15% of fermentation raw material and water TV, crosses hollander fine grinding, homogenate; The raw material after benefit adds water to making beating in the raw material after making beating then and the ratio of water reach 1kg: 20L, stir extraction 50min down at 70 ℃; It is centrifugal that (3000rpm 20min), collects supernatant, and this supernatant is the Schizophyllum commune Fr polysaccharides crude extract.
2, Deproteinization: adopt isoelectric point precipitation to remove the foreign protein in the crude extract, regulate crude extract pH value to 4.8, leave standstill 1h, centrifugal, collection supernatant.
3, decolouring: (ratio of gac and supernatant is 1kg to the supernatant that step 2 is obtained: 90L), (ratio of elutriant is 1kg to the D301 macroporous resin column after resin and the charcoal absorption: 150L) doing the processing of decolouring through activated carbon column successively.Comprehensive percent of decolourization reaches 93.5%, polysaccharide retention rate 84.2%.At last the pH value of solution value is transferred to 7.0, obtain the Schizophyllum commune Fr polysaccharides refined liquid.
Percent of decolourization: the difference of absorbancy multiply by 100% again divided by the preceding absorbancy of decolouring and promptly get (absorbancy is measured) under the 426nm wavelength before and after the solution decolouring.
Polysaccharide retention rate: the ratio of polysaccharide total amount (determination of polysaccharide adopts the phenolsulfuric acid method) in the solution before and after the decolouring.
(5) preparation of liquid product and solid phase prod
1, the preparation of liquid product
Adjust concentration: the polysaccharide in the Schizophyllum commune Fr polysaccharides refined liquid is adjusted to 1.0% concentration.
Sterilization: adopt the packing of 5-25kg plastic tank, carry out 60Co-gamma-ray irradiation germicidal treatment (irradiation dose 3-5kGy) then; Product places cool place, lucifuge place to preserve 1 year storage period.
Gained liquid Schizophyllum commune Fr polysaccharides product of the present invention be a kind of colourless, be clear to the oyster white thick liquid.
2, solid phase prod preparation
Concentration adjustment: the Schizophyllum commune Fr polysaccharides refined liquid is carried out vacuum concentration to solid content reach 2.5%;
Adopt separating out alcohol method to obtain the deposition polysaccharide, in enriched material, progressively add 95% edible ethanol to solution alcohol concn and reach 60-65%, obtain the polysaccharide flocks; Left standstill under the room temperature 8-12 hour, polysaccharide precipitation is collected in press filtration; Precipitate 2-3 time with 95% washing with alcohol again, remove unnecessary liquid and obtain half-dried material; Should place oven drying at low temperature under the vacuum condition by half-dried material, and pulverize, pack airtight preservation, 2 years storage periods.This is the Schizophyllum commune Fr polysaccharides solid phase prod, and this product is that a kind of white is to pale powder.
(6) calculating of yield
Quality * 100% of the quality/fermentation raw material of VISOSE in yield (%)=solid phase prod
The experiment (being that step () is to (six)) of above-mentioned fermentation and solid phase prod preparation all repeats 3 times, and average yield is 5.8%.
Two, the evaluation of Schizophyllum commune Fr polysaccharides
1, product is identified
1) essentially consist analysis
The Schizophyllum commune Fr polysaccharides solid phase prod that experiment one is obtained carries out compositional analysis.Survey wherein polysaccharide content with the phenolsulfuric acid method, survey wherein protein contnt with nitrogen determination.
3 repetitions are established in experiment.Result: total sugar content: 86.8%; Protein contnt 8.0%; Weight loss on drying 10.0%.Show that the principal constituent in the product of the present invention is a sugar.
2) monose compositional analysis: performance liquid chromatography (HPLC) method
Get 10mg gained solid phase prod of the present invention, at 20ml, 1M H
2SO
4100 ℃ of hydrolysis are 3 hours in the solution; Cooling adds BaCO
3Be neutralized to pH7.0; Filter, collect filtrating; Micro-filtration (φ 0.45 μ m), filtrate for later use.
HPLC condition: Sugar pak1,6.8 * 300mm chromatographic column (Waters company); Moving phase is ultrapure water; Flow velocity 0.5ml/min; 85 ℃ of column temperatures; 30 ℃ of pond temperature; Sample size 10 μ l; Detector is a differential refraction detector.
The glucose standard substance are available from Sigma company, and catalog number is G5400; The wood sugar standard substance are purchased Sigma company, and catalog number is 95729; The pectinose standard substance are available from Sigma company, and catalog number is 10860.
Under as above chromatographic condition, the RT of glucose standard substance is 13.023min; The RT of wood sugar standard substance is 15.008min; The RT of pectinose standard substance is 16.285min.
3 repetitions are established in experiment.Hydrolysate through measuring total reducing sugar in the products obtained therefrom of the present invention is: glucose 85.92%, wood sugar 8.52%, pectinose 5.56%.Show, be mainly glucose after the syrup in the products obtained therefrom of the present invention is separated, wherein contain the polysaccharide material that constitutes with wood sugar and pectinose on a small quantity, but do not influence its practical application.
3) molecular weight of product measure of spread: high performance gel filtration chromatography (HPSEC) method
Products obtained therefrom of the present invention is mixed with the solution that polysaccharide concentration is 5mg/ml, and solvent is 0.1M NaNO
3Solution; Micro-filtration (φ 0.45 μ m), filtrate for later use.
The HPLC condition: Ultrahydrogel Linear * 2,7.8 * 300mm gel chromatographic columns (Waters company, WAT011545); Moving phase is 0.1M NaNO
3Solution; Flow velocity 0.9ml/min; 45 ℃ of column temperatures; 37 ℃ of pond temperature; Sample size 20 μ l; Detector is a differential refraction detector.This chromatographic column is demarcated through series standard VISOSE (Dextran, Fluka company), and the relation between RT (T) and the average molecular mass (MW) is LogMW=1.37e-5.33e
-1T.
Two polysaccharide peaks that molecular weight is relatively large occur through measuring products obtained therefrom of the present invention, wherein average molecular mass (Mw) is 23,140, and the polysaccharide fraction of 680Da accounts for 92.72% of total polysaccharides content; Other has an average molecular mass is 44, and the polysaccharide fraction of 578Da accounts for 7.28% of total polysaccharides content.Show that the glucose in the product of the present invention exists with the polymer form.
To sum up, qualification result shows that the principal constituent of product of the present invention is VISOSE (being Schizophyllum commune Fr polysaccharides).
2, product viscosity specificity analysis
Measure relative viscosity, specific viscosity and the apparent characteristic viscosity of Schizophyllum commune Fr polysaccharides solution by Ubbelohde viscometer (φ 0.57mm).In the solvent system of pH2.0-10.0, the polysaccharide series solution of preparation 1.5-3.5mg/ml is at 25-75 ℃ of flowing time (t that measures solvent and solution down
0, t).Solvent is a deionized water, prepares with the Schizophyllum commune Fr polysaccharides solid sample.
The viscosity calculations formula:
Relative viscosity (η rel)=t
0/ t;
Specific viscosity (η sp)=η rel-1;
Apparent characteristic viscosity ([η] app)=[2 (η sp-ln η rel)]
0.5* 1000/C, unit: ml/g; Wherein C is strength of solution (mg/ml).
3 repetitions are established in experiment, and the result is shown in Fig. 1-3.The result shows that Schizophyllum commune Fr polysaccharides viscosity is the index ascendant trend with the increase of concentration; Polysaccharide viscosity slightly descends with the increase of system pH, and overall variation trend is not obvious; Rising viscosity with temperature is linear decline.
Other has studied the apparent characteristic viscosity ([η] app) of gained polysaccharide of the present invention under the same concentration conditions and the difference of edible gum-XG 550 commonly used.The result shows that under 2.5mg/ml concentration, [η] app value of product of the present invention and XG 550 is respectively 1500 ± 32ml/g, 2250 ± 120ml/g, is 1.5 times of the latter approximately, shows that this product has better thickening property.
Three, the physiological function of Schizophyllum commune Fr polysaccharides experiment
1, immunoloregulation function experiment
Experimental arrangement carries out immunoloregulation function with reference to the method for " protective foods check and evaluation technique standard " (Ministry of Health 2003) to Kunming mouse and measures.Detect index comprise 1. immune organ dirty/body weight ratio; 2. sheep red blood cell (SRBC) (SRBC) inducing mouse DTH (the sufficient sole of the foot thickens method) delayed allergy (DTH); 3. the blood clotting method is measured serum hemolysin antibody product (blood clotting method); 4. Turnover of Mouse Peritoneal Macrophages is engulfed chicken red blood cell experiment (half intracorporal method) mensuration phagocytic rate and phagocytic index.The visible above-mentioned standard of concrete grammar and calculation formula.
Experimentize with the Schizophyllum commune Fr polysaccharides refined liquid.50,100,150mg Schizophyllum commune Fr polysaccharides/kg tests daily ration the Schizophyllum commune Fr polysaccharides refined liquid is added in the basal diet, be prepared into the test daily ration of various dose polysaccharide:.
Mouse is divided into 4 groups, and 10 every group, as control group, low dose group (50mg/kg), middle dose groups (100mg/kg), high dose group (150mg/kg), feed the respectively basal diet and the test daily ration of homopolysaccharide dosage not.2 repetitions are established in experiment.
Table 1 Schizophyllum commune Fr polysaccharides is to the influence of mouse thymus and spleen/body weight
Annotate: the significance of difference of comparing with control group,
*P＜0.05,
*P＜0.01.
Table 2 Schizophyllum commune Fr polysaccharides is to mouse delayed allergy (DTH)
Annotate: the significance of difference of comparing with control group,
*P＜0.05,
*P＜0.01.
Table 3 Schizophyllum commune Fr polysaccharides is to the influence of mice serum hemolytic antibody product
Each dose groups has the trend of increasing, but difference is not remarkable.
Table 4 Schizophyllum commune Fr polysaccharides is to the influence of mouse monokaryon macrophage phagocytic function
Annotate: the significance of difference of comparing with control group,
*P＜0.05,
*P＜0.01.
Show by The above results; Compare with control group; The middle and high dose groups of Schizophyllum commune Fr polysaccharides can significantly improve thymus index and index and spleen index, enhancing delayed allergy (DTH) and mononuclear-macrophage phagocytic function (p＜0.05 of mouse; P＜0.01), judges that thus gained Schizophyllum commune Fr polysaccharides of the present invention has immunoloregulation function.
2, anti tumor activity in vitro experiment
Investigated the extracorporeal inhibiting rate of Schizophyllum commune Fr polysaccharides solution to people's lung cancer A549 cell, the former leukemia cell K562 of the chronic marrow of people and human liver cancer cell HepG2.Above-mentioned three types of people's lung cancer A549 cells, the former leukemia cell K562 of the chronic marrow of people and human liver cancer cell HepG2 are all available from Tumour Inst., Chinese Medical Academy.The Schizophyllum commune Fr polysaccharides refined liquid is added in the cell culture medium, make its concentration be controlled at 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml, 0.03125mg/ml; Then to inoculated tumour cell wherein, cultivate for some time after, measure light absorption value at 570nm, calculate the tumour inhibiting rate of Schizophyllum commune Fr polysaccharides.
The external tumour inhibiting rate (%) of Schizophyllum commune Fr polysaccharides=(1-drug group absorbance/control group absorbance) * 100%.
2 repetitions are established in experiment.The result shows: when the concentration of Schizophyllum commune Fr polysaccharides in the substratum reaches 1mg/ml; To A549 cell inhibiting rate is 33.5% (p＜0.01); To K562 cell inhibiting rate is 73.7% (p＜0.01); To HepG2 cell inhibiting rate is 59.4% (p＜0.01), infers that thus the external tumour inhibiting rate of Schizophyllum commune Fr polysaccharides shows as utmost point level of signification.
One, preparation method
(1) actication of culture
Adopt Split-gill (Schizophyllum commune Fr.) CFCC NO.83457 bacterial strain, method is with consistent described in the embodiment 1.
(2) enlarged culturing of bacterial classification
Method is with consistent described in the embodiment 1.
(3) solid fermentation of bacterial classification
In the aseptic technique environment, the second-class liquid isolate that primary fermentation is good is inoculated in the fermention medium, and inoculative proportion is second-class liquid isolate: fermention medium=50mL: 1000g; Place 26 ℃ to cultivate 10 days down, when treating that mycelia is covered with bacterium bag (or bacterium bottle), solid fermentation finishes, and the mixture after this fermentation is promptly as fermentation raw material.
Consisting of of fermention medium: contain maize peel 400g, rice bran 100g, somatomedin-V in every liter of substratum
B10.08g, L-L-glutamic acid 0.05g, all the other are water.
The preparation method of fermention medium is with consistent described in the embodiment 1.
(4) extraction of Schizophyllum commune Fr polysaccharides and purifying
1, slightly carry: fermentation raw material is poured in the stirrer, broken earlier; Add less water again, the water of adding accounts for 13% of fermentation raw material and water TV, crosses hollander fine grinding, homogenate; The raw material after benefit adds water to making beating in the raw material after making beating then and the ratio of water reach 1kg: 15L, stir extraction 40min down at 60 ℃; It is centrifugal that (3000rpm 15min), collects supernatant, and this supernatant is the Schizophyllum commune Fr polysaccharides crude extract.
2, Deproteinization: adopt isoelectric point precipitation to remove the foreign protein in the liquid of extracting polysaccharide, regulate liquid of extracting polysaccharide pH value to 4.5, leave standstill 0.5h, centrifugal, collection supernatant.
3, decolouring: (ratio of gac and supernatant is 1kg to the supernatant that step 2 is obtained: 80L), (ratio of elutriant is 1kg to the D301 macroporous resin column after resin and the charcoal absorption: 120L) doing the processing of decolouring through activated carbon column successively.Comprehensive percent of decolourization reaches 92.5%, polysaccharide retention rate 85.4%.At last the pH value of solution value is transferred to 6.5, obtain the Schizophyllum commune Fr polysaccharides refined liquid.
(5) preparation of liquid product and solid phase prod
1, the preparation of liquid product
Adjust concentration: the polysaccharide in the Schizophyllum commune Fr polysaccharides refined liquid is adjusted to 1.0% concentration.
Sterilization: adopt the packing of 5-25kg plastic tank, carry out 60Co-gamma-ray irradiation germicidal treatment (irradiation dose 3-5kGy) then; Product places cool place, lucifuge place to preserve 1 year storage period.
Gained liquid Schizophyllum commune Fr polysaccharides product of the present invention be a kind of colourless, be clear to the oyster white thick liquid.
2, solid phase prod preparation
Concentration adjustment: the Schizophyllum commune Fr polysaccharides refined liquid is carried out vacuum concentration to solid content reach 2.5%;
Adopt separating out alcohol method to obtain the deposition polysaccharide, in enriched material, progressively add 95% edible ethanol to solution alcohol concn and reach 60-65%, obtain the polysaccharide flocks; Left standstill under the room temperature 8-12 hour, polysaccharide precipitation is collected in press filtration; Precipitate 2-3 time with 95% washing with alcohol again, remove unnecessary liquid and obtain half-dried material, should place oven drying at low temperature under the vacuum condition by half-dried material, pulverize, pack airtight preservation, 2 years storage periods.This is the Schizophyllum commune Fr polysaccharides solid phase prod, and this product is that a kind of white is to pale powder.
(6) calculating of yield
Method is with consistent described in the embodiment 1.
3 repetitions are established in experiment, and average yield is 4.5%.
Two, the evaluation of Schizophyllum commune Fr polysaccharides
1, product is identified
Method: with consistent described in the embodiment 1.
The result shows, polysaccharide content 86.5% in the Schizophyllum commune Fr polysaccharides solid phase prod that the present invention obtains, protein contnt 8.3%, weight loss on drying 8.0%.Monose consists of after the Schizophyllum commune Fr polysaccharides hydrolysis: glucose 84.28%, wood sugar 8.40%, pectinose 4.96%.The average molecular mass of polysaccharide (Mw) is distributed as: 23,240, and the polysaccharide fraction of 180Da accounts for 91.23% of total polysaccharides content, and other has an average molecular mass is 45, and the polysaccharide fraction of 508Da accounts for 8.77% of total polysaccharides content.Qualification result shows that the principal constituent of Schizophyllum commune Fr polysaccharides solid phase prod of the present invention is VISOSE (being Schizophyllum commune Fr polysaccharides).
2, analysis on Viscosity Character
Method: with consistent described in the embodiment 1.
3 repetitions are established in experiment.The result shows that Schizophyllum commune Fr polysaccharides viscosity is the index ascendant trend with the increase of concentration; Polysaccharide viscosity slightly descends with the increase of pH value, and overall variation trend is not obvious; Rising viscosity with temperature is linear decline; The viscosity number of gained polysaccharide of the present invention big than XG 550 under the same concentration conditions is 1.8 times of XG 550; [η] app on average reaches 2460ml/g.
Three, the physiological function of Schizophyllum commune Fr polysaccharides experiment
1, anti tumor activity in vitro experiment
Method: with consistent described in the embodiment 1.
3 repetitions are established in experiment.The result shows: when the concentration of Schizophyllum commune Fr polysaccharides in the substratum is 1mg/ml; To A549 cell inhibiting rate is 35.4% (p＜0.01); To K562 cell inhibiting rate is 72.1% (p＜0.01); To HepG2 cell inhibiting rate is 60.5% (p＜0.01), and the external tumour inhibiting rate of Schizophyllum commune Fr polysaccharides shows as utmost point level of signification.
The preparation method of embodiment 3, Schizophyllum commune Fr polysaccharides
One, actication of culture
Adopt Split-gill (Schizophyllum commune Fr.) ACCC NO.50875, method is with consistent described in the embodiment 1.
Two, the enlarged culturing of bacterial classification
Method is with consistent described in the embodiment 1.
Three, the solid fermentation of bacterial classification
In the aseptic technique environment, the second-class liquid isolate that primary fermentation is good is inoculated in the fermention medium, and inoculative proportion is second-class liquid isolate: fermention medium=100mL: 1000g; Place 29 ℃ to cultivate 7 days down, when treating that mycelia is covered with bacterium bag (or bacterium bottle), solid fermentation finishes, and the mixture after this fermentation is known as fermentation raw material.
Consisting of of fermention medium: contain corn straw 430g, bagasse 70g, somatomedin-V in every liter of substratum
B10.05g, L-L-glutamic acid 0.07g, all the other are water.
The preparation method of fermention medium is following: with consistent described in the embodiment 1.
Four, the extraction of Schizophyllum commune Fr polysaccharides and purifying
1, slightly carry: fermentation raw material is poured in the stirrer, broken earlier; Add less water again, the water of adding accounts for 15% of fermentation raw material and water TV, crosses hollander fine grinding, homogenate; The raw material after benefit adds water to making beating in the raw material after making beating then and the ratio of water reach 1kg: 25L, stir extraction 60min down at 80 ℃; It is centrifugal that (3000rpm 25min), collects supernatant, and this supernatant is the Schizophyllum commune Fr polysaccharides crude extract.
2, Deproteinization: adopt isoelectric point precipitation to remove the foreign protein in the liquid of extracting polysaccharide, regulate liquid of extracting polysaccharide pH value to 5.0, leave standstill 1.5h, centrifugal, collection supernatant.
3, decolouring: (ratio of gac and supernatant is 1kg to the supernatant that step 2 is obtained: 120L), (ratio of elutriant is 1kg to the D301 macroporous resin column after resin and the charcoal absorption: 150L) doing the processing of decolouring through activated carbon column successively.Comprehensive percent of decolourization reaches 95.0%, polysaccharide retention rate 81.5%.At last the pH value of solution value is transferred to 7.0, obtain the Schizophyllum commune Fr polysaccharides refined liquid.
Five, the preparation of liquid product and solid phase prod
1, the preparation of liquid product
Adjust concentration: the polysaccharide in the Schizophyllum commune Fr polysaccharides refined liquid is adjusted to 1.0% concentration.
Sterilization: adopt the packing of 5-25kg plastic tank, carry out 60Co-gamma-ray irradiation germicidal treatment (irradiation dose 3-5kGy) then; Product places cool place, lucifuge place to preserve 1 year storage period.Gained liquid Schizophyllum commune Fr polysaccharides product of the present invention be a kind of colourless, be clear to the oyster white thick liquid.
2, solid phase prod preparation
Concentration adjustment: the Schizophyllum commune Fr polysaccharides refined liquid is carried out vacuum concentration to solid content reach 3.0%;
Adopt separating out alcohol method to obtain the deposition polysaccharide, in enriched material, progressively add 95% edible ethanol to solution alcohol concn and reach 60-65%, obtain the polysaccharide flocks; Left standstill under the room temperature 8-12 hour, polysaccharide precipitation is collected in press filtration; Precipitate 2-3 time with 95% washing with alcohol again, remove unnecessary liquid and obtain half-dried material; The half-dried material of this polysaccharide is placed oven drying at low temperature under the vacuum condition, pulverize, pack airtight preservation, 2 years storage periods.Solid phase prod is a kind of pearl to a light grey powder.
(6) calculating of yield
With consistent described in the embodiment 1.
3 repetitions are established in experiment, and average yield is 5.5%.
Wherein polysaccharide content 85.0%, protein contnt 13.0%, weight loss on drying 9.0%.
Two, the evaluation of Schizophyllum commune Fr polysaccharides
1, product is identified
Method: with consistent described in the embodiment 1.
The result shows, polysaccharide content 85.0% in the Schizophyllum commune Fr polysaccharides solid phase prod that the present invention obtains, protein contnt 13.0%, weight loss on drying 9.0%.Monose consists of after the Schizophyllum commune Fr polysaccharides hydrolysis: glucose 84.08%, wood sugar 8.95%, pectinose 4.74%.The average molecular mass of polysaccharide (Mw) is distributed as: 23,256, and the polysaccharide fraction of 100Da accounts for 90.13% of total polysaccharides content, and other has an average molecular mass is 45, and the polysaccharide fraction of 400Da accounts for 8.42% of total polysaccharides content.Qualification result shows that the principal constituent of Schizophyllum commune Fr polysaccharides solid phase prod of the present invention is VISOSE (being Schizophyllum commune Fr polysaccharides).
2, analysis on Viscosity Character
Method: with consistent described in the embodiment 1.
3 repetitions are established in experiment.The result shows that Schizophyllum commune Fr polysaccharides viscosity is the index ascendant trend with the increase of concentration; Polysaccharide viscosity slightly descends with the increase of pH value, and overall variation trend is not obvious; Rising viscosity with temperature is linear decline; The viscosity number of gained polysaccharide of the present invention big than XG 550 under the same concentration conditions is 1.7 times of XG 550; [η] app on average reaches 2415ml/g.
Three, the physiological function of Schizophyllum commune Fr polysaccharides experiment
1, anti tumor activity in vitro experiment
Method: with consistent described in the embodiment 1.
3 repetitions are established in experiment.The result shows: when the concentration of Schizophyllum commune Fr polysaccharides in the substratum is 1mg/ml; To A549 cell inhibiting rate is 34.3% (p＜0.01); To K562 cell inhibiting rate is 70.9% (p＜0.01); To HepG2 cell inhibiting rate is 60.1% (p＜0.01), and the external tumour inhibiting rate of Schizophyllum commune Fr polysaccharides shows as utmost point level of signification.
Claims (13)
1. a method for preparing Schizophyllum commune Fr polysaccharides comprises the steps: with 1)-3) in any substratum fermentation Split-gill (Schizophyllum commune Fr.), obtain Schizophyllum commune Fr polysaccharides;
1) contains corn cob 450g, Testa Tritici 50g, V in every kilogram of substratum
B10.1g, L-L-glutamic acid 0.1g, all the other are water;
2) contain maize peel 400g, rice bran 100g, V in every liter of substratum
B10.08g, L-L-glutamic acid 0.05g, all the other are water;
3) contain corn straw 430g, bagasse 70g, V in every liter of substratum
B10.05g, L-L-glutamic acid 0.07g, all the other are water.
2. method according to claim 1 is characterized in that: said Split-gill (Schizophyllum commune Fr.) is the Split-gill of following deposit number: CFCC NO.6812, CFCC NO.83457 or ACCC NO.50875.
3. method according to claim 1 and 2 is characterized in that: the temperature of said fermentation is 26-30 ℃.
4. method according to claim 3 is characterized in that: the time of said fermentation is 7-10 days.
5. method according to claim 1 and 2 is characterized in that: in the said method, after said fermentation, comprise the step of extraction; The method of said extraction is: the fermented product that said fermentation is obtained carries out fragmentation, adds entry then, under 60-80 ℃ temperature, stirs, and is centrifugal, collects supernatant.
6. method according to claim 3 is characterized in that: in the said method, after said fermentation, comprise the step of extraction; The method of said extraction is: the fermented product that said fermentation is obtained carries out fragmentation, adds entry then, under 60-80 ℃ temperature, stirs, and is centrifugal, collects supernatant.
7. method according to claim 4 is characterized in that: in the said method, after said fermentation, comprise the step of extraction; The method of said extraction is: the fermented product that said fermentation is obtained carries out fragmentation, adds entry then, under 60-80 ℃ temperature, stirs, and is centrifugal, collects supernatant.
8. method according to claim 5 is characterized in that: in the said method, after said extraction, comprise the step of Deproteinization; The method of said Deproteinization is: the pH value of regulating the supernatant that said extraction obtains leaves standstill 0.5-1.5h to 4.5-5.0, and is centrifugal, collect supernatant.
9. method according to claim 6 is characterized in that: in the said method, after said extraction, comprise the step of Deproteinization; The method of said Deproteinization is: the pH value of regulating the supernatant that said extraction obtains leaves standstill 0.5-1.5h to 4.5-5.0, and is centrifugal, collect supernatant.
10. method according to claim 7 is characterized in that: in the said method, after said extraction, comprise the step of Deproteinization; The method of said Deproteinization is: the pH value of regulating the supernatant that said extraction obtains leaves standstill 0.5-1.5h to 4.5-5.0, and is centrifugal, collect supernatant.
11. method according to claim 8 is characterized in that: in the said method, behind said Deproteinization, comprise the step of decolouring; The method of said decolouring is: the supernatant that said Deproteinization is obtained adsorbs with gac and macroporous resin successively, collects elutriant; The ratio of said gac and supernatant is the 1kg gac: (80-120) L supernatant; Said D301 macroporous resin column with use the ratio of the elutriant that obtains after the charcoal absorption to be the 1kgD301 macroporous resin: (120-150) L elutriant.
12. method according to claim 9 is characterized in that: in the said method, behind said Deproteinization, comprise the step of decolouring; The method of said decolouring is: the supernatant that said Deproteinization is obtained adsorbs with gac and macroporous resin successively, collects elutriant; The ratio of said gac and supernatant is the 1kg gac: (80-120) L supernatant; Said D301 macroporous resin column with use the ratio of the elutriant that obtains after the charcoal absorption to be the 1kgD301 macroporous resin: (120-150) L elutriant.
13. method according to claim 10 is characterized in that: in the said method, behind said Deproteinization, comprise the step of decolouring; The method of said decolouring is: the supernatant that said Deproteinization is obtained adsorbs with gac and macroporous resin successively, collects elutriant; The ratio of said gac and supernatant is the 1kg gac: (80-120) L supernatant; Said D301 macroporous resin column with use the ratio of the elutriant that obtains after the charcoal absorption to be the 1kgD301 macroporous resin: (120-150) L elutriant.
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| CN107828664A (en) * | 2017-10-25 | 2018-03-23 | 湖南省农业生物技术研究中心 | Schizophyllum commune XT 1 and its cultural method and application |
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| CN107828664A (en) * | 2017-10-25 | 2018-03-23 | 湖南省农业生物技术研究中心 | Schizophyllum commune XT 1 and its cultural method and application |
| CN107828664B (en) * | 2017-10-25 | 2020-10-23 | 湖南省农业生物技术研究中心 | Schizophyllum commune XT-1 and cultivation method and application thereof |
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