CN101748153A - Tricholoma matsutake polysaccharose by utilizing high-yield selenium-rich function hybridization red rice bran self-fermentation broth - Google Patents
Tricholoma matsutake polysaccharose by utilizing high-yield selenium-rich function hybridization red rice bran self-fermentation broth Download PDFInfo
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Abstract
The invention relates to tricholoma matsutake polysaccharose by utilizing high-yield selenium-rich function hybridization red rice bran self-fermentation broth, belonging to the field of biology engineering technology; the tricholoma matsutake polysaccharose comprises the preparation of high-yield selenium-rich function hybridization red rice bran 611A/R319 self-fermentation broth, the culture of the tricholoma matsutake strains, fermentation process, mycelium and fermentation broth collection, extraction of tricholoma matsutake endoenzyme crude polysaccharides and ectoenzyme crude polysaccharides, and then the tricholoma matsutake endoenzyme crude polysaccharides and the ectoenzyme crude polysaccharides are combined to the tricholoma matsutake polysaccharose; wherein, the high-yield selenium-rich function hybridization red rice bran 611A/R319 is side product in the processing process of the hybridization red rice 611A/R319, but the high-yield selenium-rich function hybridization red rice bran 611A/R319 is rich in trace elements which are suitable for the growth of the tricholoma matsutake, by utilizing the enzymes of the high-yield selenium-rich function hybridization red rice bran 611A/R319, the rice bran self-fermentation broth can be prepared after hydrolysis is carried out, and then the rice bran self-fermentation broth is used for deep layer liquid fermentation culture of the tricholoma matsutake fungus, so as to lead the tricholoma matsutake fungus to utilize the nutrient elements in the red rice bran completely and cause a great amount of the tricholoma matsutake mycelium to grow rapidly; the method is utilized to produce the tricholoma matsutake polysaccharose, the period is short, the cost is low, the process is simple, the effective component content is high, the additional value is high, thereby being suitable for industrial production.
Description
One, technical field
Utilize high-yield selenium-rich function hybridization red rice bran self ferment liquid to produce blazei polysaccharide, belong to technical field of bioengineering.The preparation and the deep fermentation method that relate to high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid are cultivated tricholoma matsutake mycelium, wherein high-yield selenium-rich function hybridization red 611A/R319 rice bran is rich in the required trace element of suitable Tricholoma matsutake (lto et lmai) Singer growth, is prepared into high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid after utilizing himself enzyme hydrolysis; Murphy juice in the fermentation broth substitutes with high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid, make Tricholoma matsutake (lto et lmai) Singer make full use of nutritive element in the high-yield selenium-rich function hybridization red 611A/R319 rice bran, thereby raised growth go out tricholoma matsutake mycelium fast; Collect mycelium and fermented liquid then, and from mycelium and fermented liquid, extract Crude polysaccharides and crude extracellular polysaccharide in the matsutake born of the same parents with ultrasonic wave auxiliary heat water extract method and ethanol alcohol extracting method respectively, the two merging is obtained product matsutake Crude polysaccharides.
Two, background technology
Matsutake (Tricholoma matsutake Sing) has another name called loose bacterium, Song Qin, Trichotoma matsutake, belongs to Basidiomycota (Basidiomycota), Basidiomycetes (Basidiomycetes), Agaricales (Agaricales), Tricholomataceae (Tricholomataceae), Tricholoma (Tricholoma).This bacterium mushroom body hypertrophy, fine and tender taste, aromatic flavour, other tool local flavor has very high nutritive value and special medicinal effect.8 seed amino acids that its sporophore rich in proteins, fat and human body are necessary contain abundant VITMAIN B1, Wei ShengsuB2, vitamins C and vitamin PP, also contain rare B-G acid, nucleic acid derivative, peptide class, organic germanium, polysaccharide etc. in the mushroom.It is the famous and precious edible mushrooms of enjoying high reputation in the world.According to one's analysis, bright matsutake contains moisture 89.9% approximately, crude protein 17%, pure protein 8.7%, crude fat 5.8%, solubility do not have nitrogen compound total amount 61.5%, robust fibre 8.6%, ash content 7.1% in mushroom.In addition, containing a kind of pinane in the tricholoma matsutake mycelium, rare (antimicrobial substance of α-Pinene) can stop the breeding of virus.Trichotoma matsutake have keep fit, the effect of beneficial stomach, pain relieving, activating QI to eliminate phlegm.Modern scientific research shows, matsutake also has treatment diabetes, special role such as anticancer, and its main pharmacological component is a polysaccharose substance.
Matsutake generally adopts the solid state cultivation method to produce, but traditional solid state cultivation method has long, many drawbacks such as quality is wayward of cycle, and employing liquid submerged fermentation technology, bacterial classification enters fermentor tank through the repeatedly enlarged culturing of seed, mycelium is able to rapid amplification in the short period, than traditional Edible Fungi method obvious superiority is arranged.And studies show that often with the fungi of sporophore medication, also contain effective component identical with sporophore and similar active constituent content in their mycelium, this provides theoretical foundation just for liquid fermentative Production blazei polysaccharide.
Rice bran is a byproduct of rice processing, is the epidermal area of rice hulling postadhesion on brown rice, is made of the kind skin of paddy, ectoderm, aleurone layer, embryo and a small amount of endosperm, accounts for the 5%-7% of paddy total amount.Latest research proves, rice bran contains the nutritive ingredient and the 90% above needed by human body element of rice 64%, about 14%, the carbohydrate total amount about 50% of about 12%～18%, the fat 16%～20% of protein content, mineral substance 12%, food fibre wherein, also containing just like multiple functional mass such as food fibre, VITAMIN, thiaminogen, mineral substance, phytic acid and inositols in addition, is a kind of high added value resource of great exploitation potential for its.
Coloured rice is owing at pericarp with plant that the deposition pigment demonstrates particular color on the skin, and it is rich in special trace element and nutritive substance, and its content and pigment are closely related.High-yield selenium-rich function hybridization red 611A/R319 is the high-yield hybrid red rice of Sichuan University and Inst. of Paddy Rice, Sichuan Agriculture Univ.'s joint research, and this hybridisation rice is rich in healthy trace elements with household such as selenium, germanium; High-yield selenium-rich function hybridization red 611A/R319 rice bran is the by product of hybridization red 611A/R319 in the course of processing, but be rich in the required trace element of suitable macro fungi growth, therefore utilize high-yield selenium-rich function hybridization red 611A/R319 rice bran fermentation culture matsutake and extract the matsutake Crude polysaccharides to have very high using value.
Three, summary of the invention
The purpose of this invention is to provide a kind of method of utilizing high-yield selenium-rich function hybridization red rice bran self ferment liquid to produce blazei polysaccharide, adopt advanced biotechnology, prepare high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid, utilize high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid submerged fermentation to cultivate tricholoma matsutake mycelium again, make Tricholoma matsutake (lto et lmai) Singer make full use of nutritive element in the high-yield selenium-rich function hybridization red 611A/R319 rice bran, thereby raised growth goes out tricholoma matsutake mycelium fast, shortened the production cycle greatly, the production cycle of the present invention is 5-7 days (cycle of traditional solid culture sporophore is generally about 30-50 days).The present invention all adopts grain raw material, pollute little, with high content of technology, blazei polysaccharide yield height, added value of product height.
Technical scheme of the present invention: make high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid after utilizing in the high-yield selenium-rich function hybridization red 611A/R319 rice bran self enzyme hydrolysis, be used for the Tricholoma matsutake (lto et lmai) Singer liquid submerged fermentation and cultivate; The Tricholoma matsutake (lto et lmai) Singer spore inoculating is cultivated to fresh test tube slant, treated to be seeded to after the inclined-plane is grown well and carry out culture of seed liquid in the liquid nutrient medium; Again by three grades of cultivations, go at last and carry out the deep fermentation cultivation in the 3000L fermentor tank, wherein the murphy juice in the fermentation broth substitutes with high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid, make Tricholoma matsutake (lto et lmai) Singer make full use of nutritive element in the high-yield selenium-rich function hybridization red 611A/R319 rice bran, thereby raised growth go out tricholoma matsutake mycelium fast; After the fermentation ends, collect mycelium and fermented liquid, and from mycelium and fermented liquid, extract Crude polysaccharides and crude extracellular polysaccharide in the matsutake born of the same parents with ultrasonic wave auxiliary heat water extract method and ethanol alcohol extracting method respectively, the two merging is obtained product matsutake Crude polysaccharides.
Step is:
1) high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid preparation:
Get a certain amount of high-yield selenium-rich function hybridization red 611A/R319 rice bran, 60 orders sieve, and add water by 1: 15 solid-liquid ratio and mix, in 40 ℃ of water bath heat preservation 3h, utilize the enzyme hydrolyzed starch of rice bran self and protein etc., back filtered through gauze gets the red rice chaff from fermented liquid.
2) spawn culture:
Starting strain is matsutake (Tricholoma matsutake Sing.);
1. slant culture:
Culture medium prescription is in g/L: glucose 20, sal epsom 2.38, calcium chloride 1, potassium primary phosphate 0.5, primary ammonium phosphate 0.4, saltpetre 0.5, murphy juice 200, wort 120, vitamins B
10.01, agar 20, pH nature;
Cultural method: the matsutake strain inclined plane of preserving is inoculated on the fresh blank inclined-plane, cultivated 7-10 days down at 24 ℃;
2. 500mL one-level shake-flask seed is cultivated, liquid amount 200mL:
Culture medium prescription is in g/L: glucose 20, sal epsom 2.38, calcium chloride 1, potassium primary phosphate 0.5, primary ammonium phosphate 0.4, saltpetre 0.5, murphy juice 200, wort 120, vitamins B
10.01, the pH nature;
Cultural method: cultured strain inclined plane is seeded to sterilized shaking in the bottle, and at 24 ℃, rotating speed 120rpm cultivated 7 days down;
3. 5000mL secondary shake-flask seed is cultivated, liquid amount 2000mL:
Culture medium prescription is in g/L: glucose 20, sal epsom 2.38, calcium chloride 1, potassium primary phosphate 0.5, primary ammonium phosphate 0.4, saltpetre 0.5, murphy juice 200, wort 120, vitamins B
10.01, the pH nature;
Cultural method: cultured first order seed is seeded to sterilized shaking in the bottle by 10% inoculum size, and at 24 ℃, enlarged culturing is 5 days under the rotating speed 120rpm, shake-flask seed liquid;
3) three grade fermemtation is cultivated:
1. 50L one grade fermemtation seed tank culture, liquid amount 25-30L:
One grade fermemtation seed tank culture based formulas is in g/L: high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid 200, glucose 10, potassium primary phosphate 1, sal epsom 0.5, soya bean 20, get juice, Semen Maydis powder 5, wheat bran 50 with the colloidal mill defibrination after centrifugal after the soybean soaking, water intaking dissolubility wheat bran juice behind the wheat bran poach, yeast powder 3, soya-bean oil 2, pH nature; The 30min that under 0.1Mpa, sterilizes, shake-flask seed, 24 ℃ of temperature, tank pressure 0.05Mpa are inserted by 10% inoculum size in the cooling back, air flow 1: 0.3 (V/V), mixing speed 120rpm changes a jar standard: fermentation time 45-48h, mycelium pellet come-up 1/3rd, microscopy has clamp connexion, does not have assorted bacterium;
2. 500L second order fermentation seed tank culture, liquid amount 250-300L:
Second order fermentation seed tank culture based formulas is in g/L: high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid 200, glucose 10, potassium primary phosphate 1, sal epsom 0.5, soya bean 20, get juice, Semen Maydis powder 5, wheat bran 50 with the colloidal mill defibrination after centrifugal after the soybean soaking, water intaking dissolubility wheat bran juice behind the wheat bran poach, yeast powder 3, soya-bean oil 2, pH nature; The 30min that under 0.1Mpa, sterilizes, first order seed, 24 ℃ of temperature, tank pressure 0.05Mpa are inserted by 15% inoculum size in the cooling back, air flow 1: 0.3 (V/V), mixing speed 120rpm changes a jar standard: fermentation time 35-40h, mycelium pellet come-up 1/3rd, microscopy has clamp connexion, does not have assorted bacterium;
3. 3000L fermentor cultivation, liquid amount 1800L:
Fermentative medium formula is in g/L: high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid 200, glucose 10, potassium primary phosphate 1, sal epsom 0.5, soya bean 20, get juice, Semen Maydis powder 5, wheat bran 50 with the colloidal mill defibrination after centrifugal after the soybean soaking, water intaking dissolubility wheat bran juice behind the wheat bran poach, yeast powder 3, soya-bean oil 2, pH nature; 30min sterilizes under 0.1Mpa, secondary seed, 24 ℃ of temperature, tank pressure 0.05Mpa are inserted by 15% inoculum size in the cooling back, air flow 1: 0.3 (V/V), mixing speed 120rpm changes a jar standard: fermentation time 55-60h, pH5.0, the assorted bacterium of dull and stereotyped nothing, mycelium pellet come-up 1/2nd, microscopy has clamp connexion, and mycelium pellet can not self-dissolving;
4) extraction of blazei polysaccharide: with cultured fermented liquid centrifugal treating under 5000-6000rpm, collected fermented liquid and mycelium respectively, the mycelium freeze-drying is preserved;
1. the obtaining of Crude polysaccharides in the born of the same parents: the mycelium powder collected is added distilled water by 1: 30 solid-liquid ratio, use frequency 25Hz, the ultrasonication 30min of power 400W, back lixiviate polysaccharide 1.5h in 90 ℃ of water-baths, then in the centrifugal 15min of 4800rpm, abandon precipitation, supernatant liquor is at 0.095MPa, be concentrated into 1/2 of original volume under 50 ℃, again concentrated solution is mixed by 1: 3 (v/v) with 95% ethanol, put 4 ℃ of precipitation 12h down, at the centrifugal 15min of 4800rpm, abandon supernatant liquor, add 50mL distilled water in the precipitation it is dissolved again, adding Sevag reagent by 5: 1 (is chloroform: deproteinated primary isoamyl alcohol=4: 1), add 3 times of volume of ethanol then and precipitate 12h down at 4 ℃, the centrifugal 15min of 4800rpm, precipitation gets Crude polysaccharides in the matsutake born of the same parents after vacuum lyophilization;
2. obtaining of crude extracellular polysaccharide: will collect centrifugal after the fermented liquid concentrating under reduced pressure be original volume 1/5th after, 95% ethanol alcohol extracting 12-16h with 3 times of volumes, use the centrifugal 20-30min of 5000-6000rpm then, collecting precipitation, to precipitate with a small amount of distilled water redissolution postlyophilization, promptly get the matsutake crude extracellular polysaccharide;
3. the acquisition of product: the two merging of Crude polysaccharides in Crude polysaccharides and the matsutake born of the same parents in the matsutake born of the same parents that extraction is obtained promptly gets product matsutake Crude polysaccharides.
5) check: inspecting standard is as follows:
1. polysaccharide content 〉=20%
2. protein content 〉=4%
3. moisture≤8%
4. total number of bacterial colony≤1000cfu/g, intestinal bacteria≤40npn/100g, yeast≤25cfu/g, mould≤25cfu/g, pathogenic bacterium must not detect, and LD50 is negative.
5. lead≤1.5mg/kg, arsenic≤1.0mg/kg.
Beneficial effect of the present invention: the present invention adopts advanced biotechnology, utilize high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid submerged fermentation to cultivate tricholoma matsutake mycelium, make Tricholoma matsutake (lto et lmai) Singer make full use of nutritive element in the high-yield selenium-rich function hybridization red 611A/R319 rice bran, thereby raised growth goes out tricholoma matsutake mycelium fast, shortened the production cycle greatly, the production cycle of the present invention is 5-7 days (cycle of traditional solid culture sporophore is generally about 30-50 days).The present invention all adopts grain raw material, pollute little, with high content of technology, blazei polysaccharide yield height, added value of product height.
Four, embodiment
1. high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid preparation: get a certain amount of high-yield selenium-rich function hybridization red 611A/R319 rice bran, 60 orders sieve, adding water by 1: 15 solid-liquid ratio mixes, in 40 ℃ of water bath heat preservation 3h, utilize the enzyme hydrolyzed starch of rice bran self and protein etc., back filtered through gauze gets the red rice chaff from fermented liquid.
2. the preparation of seed liquor: the matsutake strain inclined plane of preserving is inoculated on the fresh blank inclined-plane, after 24 ℃ are cultivated 7-10 days down, the thalline on the inclined-plane is scraped in the triangular flask of an amount of sterilized water of being equipped with of the bacterium of having gone out in advance and granulated glass sphere, vibrated 10 minutes, and made bacteria suspension.Inhale this bacteria suspension of 4ml (shake the substratum of bottle formulated by above-mentioned one-level, liquid amount is 200mL) in 2 500mL triangular flasks of the bacterium of having gone out in advance, at 24 ℃, rotating speed 120rpm cultivates down and got the one-level shake-flask seed in 7 days; Under the aseptic condition, one bottle of cultured one-level shake-flask seed is poured in 5000mL (liquid amount the is 2000mL) triangular flask of the above-mentioned secondary shake-flask seed of usefulness formulated of the bacterium of having gone out in advance, at 24 ℃, rotating speed 120rpm cultivated 5 days down, long good after with two bottles of seed liquor merge shake-flask seed liquid.
3. first class seed pot is cultivated: by above-mentioned first class seed pot culture medium prescription, shake-flask seed is inserted in 30 minutes cooling backs of sterilization under 0.1Mpa.Cultivate by above-mentioned first class seed pot culture parameters.Adopt the 50L automatic fermenter, liquid amount is 25-30L.
4. the secondary seed jar is cultivated: by above-mentioned secondary seed jar culture medium prescription, first order seed is inserted in 30 minutes cooling backs of sterilization under 0.1Mpa.Cultivate by above-mentioned secondary seed jar culture parameters.Adopt the 500L automatic fermenter, liquid amount is 250-300L.
5. deep fermentation is cultivated: adopt the 3000L fermentor tank, liquid amount is 1800L.By above-mentioned fermentative medium formula, the secondary liquid seeds is inserted in the same terms sterilization back.Culture parameters by above-mentioned fermentor tank is cultivated.
6. the obtaining of Crude polysaccharides in the born of the same parents: with the extracting method of cultured fermented liquid, collect mycelium, obtain the dried mycelium of 25kg, obtain the interior Crude polysaccharides of 2.2kg born of the same parents after extracting polysaccharide with Crude polysaccharides in the above-mentioned born of the same parents.
7. obtaining of crude extracellular polysaccharide: the extracting method by above-mentioned crude extracellular polysaccharide, obtain the 260L concentrated solution, obtain the 5kg crude extracellular polysaccharide after extracting polysaccharide.
8. the two merging is obtained matsutake Crude polysaccharides 7.2kg.
Claims (1)
1. utilize high-yield selenium-rich function hybridization red rice bran self ferment liquid to produce blazei polysaccharide, the preparation and the deep fermentation method that it is characterized in that high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid are cultivated tricholoma matsutake mycelium, wherein high-yield selenium-rich function hybridization red 611A/R319 rice bran is rich in the required trace element of suitable Tricholoma matsutake (lto et lmai) Singer growth, is prepared into high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid after utilizing himself enzyme hydrolysis; The Tricholoma matsutake (lto et lmai) Singer spore inoculating is cultivated to fresh test tube slant, treated to be seeded to after the inclined-plane is grown well and carry out culture of seed liquid in the liquid nutrient medium; Again by three grades of cultivations, go at last and carry out the deep fermentation cultivation in the 3000L fermentor tank, wherein the murphy juice in the fermentation broth substitutes with high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid, make Tricholoma matsutake (lto et lmai) Singer make full use of nutritive element in the high-yield selenium-rich function hybridization red 611A/R319 rice bran, thereby raised growth go out tricholoma matsutake mycelium fast; After the fermentation ends, collect mycelium and fermented liquid, and from mycelium and fermented liquid, extract Crude polysaccharides and crude extracellular polysaccharide in the matsutake born of the same parents with ultrasonic wave auxiliary heat water extract method and ethanol alcohol extracting method respectively, the two merging is obtained product matsutake Crude polysaccharides; Step is:
1) high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid preparation:
Get a certain amount of high-yield selenium-rich function hybridization red 611A/R319 rice bran, 60 orders sieve, and add water by 1: 15 solid-liquid ratio and mix, in 40 ℃ of water bath heat preservation 3h, utilize the enzyme hydrolyzed starch of rice bran self and protein etc., back filtered through gauze gets the red rice chaff from fermented liquid;
2) spawn culture:
Starting strain is matsutake (Tricholoma matsutake Sing.);
1. slant culture:
Culture medium prescription is in g/L: glucose 20, sal epsom 2.38, calcium chloride 1, potassium primary phosphate 0.5, primary ammonium phosphate 0.4, saltpetre 0.5, murphy juice 200, wort 120, vitamins B
10.01, agar 20, pH nature;
Cultural method: the matsutake strain inclined plane of preserving is inoculated on the fresh blank inclined-plane, cultivated 7-10 days down at 24 ℃;
2. 500mL one-level shake-flask seed is cultivated, liquid amount 200mL:
Culture medium prescription is in g/L: glucose 20, sal epsom 2.38, calcium chloride 1, potassium primary phosphate 0.5, primary ammonium phosphate 0.4, saltpetre 0.5, murphy juice 200, wort 120, vitamins B
10.01, the pH nature;
Cultural method: cultured strain inclined plane is seeded to sterilized shaking in the bottle, and at 24 ℃, rotating speed 120rpm cultivated 7 days down;
3. 5000mL secondary shake-flask seed is cultivated, liquid amount 2000mL:
Culture medium prescription is in g/L: glucose 20, sal epsom 2.38, calcium chloride 1, potassium primary phosphate 0.5, primary ammonium phosphate 0.4, saltpetre 0.5, murphy juice 200, wort 120, vitamins B
10.01, the pH nature;
Cultural method: cultured first order seed is seeded to sterilized shaking in the bottle by 10% inoculum size, and at 24 ℃, enlarged culturing is 5 days under the rotating speed 120rpm, shake-flask seed liquid;
3) three grade fermemtation is cultivated:
1. 50L one grade fermemtation seed tank culture, liquid amount 25-30L:
One grade fermemtation seed tank culture based formulas is in g/L: high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid 200, glucose 10, potassium primary phosphate 1, sal epsom 0.5, soya bean 20, get juice, Semen Maydis powder 5, wheat bran 50 with the colloidal mill defibrination after centrifugal after the soybean soaking, water intaking dissolubility wheat bran juice behind the wheat bran poach, yeast powder 3, soya-bean oil 2, pH nature; The 30min that under 0.1Mpa, sterilizes, shake-flask seed, 24 ℃ of temperature, tank pressure 0.05Mpa are inserted by 10% inoculum size in the cooling back, air flow 1: 0.3 (V/V), mixing speed 120rpm changes a jar standard: fermentation time 45-48h, mycelium pellet come-up 1/3rd, microscopy has clamp connexion, does not have assorted bacterium;
2. 500L second order fermentation seed tank culture, liquid amount 250-300L:
Second order fermentation seed tank culture based formulas is in g/L: high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid 200, glucose 10, potassium primary phosphate 1, sal epsom 0.5, soya bean 20, get juice, Semen Maydis powder 5, wheat bran 50 with the colloidal mill defibrination after centrifugal after the soybean soaking, water intaking dissolubility wheat bran juice behind the wheat bran poach, yeast powder 3, soya-bean oil 2, pH nature; The 30min that under 0.1Mpa, sterilizes, first order seed, 24 ℃ of temperature, tank pressure 0.05Mpa are inserted by 15% inoculum size in the cooling back, air flow 1: 0.3 (V/V), mixing speed 120rpm changes a jar standard: fermentation time 35-40h, mycelium pellet come-up 1/3rd, microscopy has clamp connexion, does not have assorted bacterium;
3. 3000L fermentor cultivation, liquid amount 1800L:
Fermentative medium formula is in g/L: high-yield selenium-rich function hybridization red 611A/R319 rice bran self ferment liquid 200, glucose 10, potassium primary phosphate 1, sal epsom 0.5, soya bean 20, get juice, Semen Maydis powder 5, wheat bran 50 with the colloidal mill defibrination after centrifugal after the soybean soaking, water intaking dissolubility wheat bran juice behind the wheat bran poach, yeast powder 3, soya-bean oil 2, pH nature; 30min sterilizes under 0.1Mpa, secondary seed, 24 ℃ of temperature, tank pressure 0.05Mpa are inserted by 15% inoculum size in the cooling back, air flow .1: 0.3 (V/V), mixing speed 120rpm changes a jar standard: fermentation time 55-60h, pH5.0, the assorted bacterium of dull and stereotyped nothing, mycelium pellet come-up 1/2nd, microscopy has clamp connexion, and mycelium pellet can not self-dissolving;
4) extraction of blazei polysaccharide: with cultured fermented liquid centrifugal treating under 5000-6000rpm, collected fermented liquid and mycelium respectively, the mycelium freeze-drying is preserved;
1. the obtaining of Crude polysaccharides in the born of the same parents: the mycelium powder collected is added distilled water by 1: 30 solid-liquid ratio, use frequency 25Hz, the ultrasonication 30min of power 400W, back lixiviate polysaccharide 1.5h in 90 ℃ of water-baths, then in the centrifugal 15min of 4800rpm, abandon precipitation, supernatant liquor is at 0.095MPa, be concentrated into 1/2 of original volume under 50 ℃, again concentrated solution is mixed by 1: 3 (v/v) with 95% ethanol, put 4 ℃ of precipitation 12h down, at the centrifugal 15min of 4800rpm, abandon supernatant liquor, add 50mL distilled water in the precipitation it is dissolved again, adding Sevag reagent by 5: 1 (is chloroform: deproteinated primary isoamyl alcohol=4: 1), add 3 times of volume of ethanol then and precipitate 12h down at 4 ℃, the centrifugal 15min of 4800rpm, precipitation gets Crude polysaccharides in the matsutake born of the same parents after vacuum lyophilization;
2. obtaining of crude extracellular polysaccharide: will collect centrifugal after the fermented liquid concentrating under reduced pressure be original volume 1/5th after, 95% ethanol alcohol extracting 12-16h with 3 times of volumes, use the centrifugal 20-30min of 5000-6000rpm then, collecting precipitation, to precipitate with a small amount of distilled water redissolution postlyophilization, promptly get the matsutake crude extracellular polysaccharide;
3. the acquisition of product: the two merging of Crude polysaccharides in Crude polysaccharides and the matsutake born of the same parents in the matsutake born of the same parents that extraction is obtained promptly gets product matsutake Crude polysaccharides.
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| CN102805392A (en) * | 2011-05-31 | 2012-12-05 | 黑龙江省麒麟工贸公司 | Preparation method for tricholoma matsutake health care beverage |
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| CN1161839A (en) * | 1996-04-08 | 1997-10-15 | 张忠廉 | Technology for extracting effective component of matsutake |
| CN1286855C (en) * | 2005-02-21 | 2006-11-29 | 吉林农业大学 | Method for preparing Agaricus blazei Murrill active polysaccharide |
| KR100836262B1 (en) * | 2007-05-14 | 2008-06-10 | 일송영농조합법인 | Tricholoma matsutake granule tea and preparation method thereof |
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2010
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