CN1995322A - Culture medium for liquid deep fermentation for producing truffle polysaccharide - Google Patents
Culture medium for liquid deep fermentation for producing truffle polysaccharide Download PDFInfo
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Abstract
The invention discloses a culture medium of liquid deep-layer ferment manufacturing block granulose, which is characterized by the following: allocating the culture medium through lactin, peptone, magnesium sulfate and potassium dihydrogen phosphate according to certain proportion; making inclined bacteria; culturing liquid seed; fermenting liquid; considering different carbon sources, nitrogen sources and original pH values to produce the granulose.
Description
Technical field
The invention belongs to biotechnology and technical field, be specifically related to a kind of substratum of liquid deep fermentation for producing truffle polysaccharide, also relate to a kind of method of producing the liquid submerged fermentation of truffle polysaccharides, this substratum is applicable to liquid submerged fermentation and the large-scale industrialization fermentative production truffle polysaccharides of ferfas.
Technical background
Ferfas is one of most precious in the world medicine-food two-purpose fungi, and its ascoma is spherical in shape, semisphere or bulk, and diameter is generally at 1.5-12 centimetre, ripe back be brown, sorrel to Vandyke brown, base portion often caves in, surperficial cloth is with little wart not of uniform size.The place of production of ferfas is narrow, mainly is distributed in the north temperate zone and the refrigerant latitudes of Continent of Europe, and supply falls short of demand always on market for its output, is worth illustrious.
Ferfas (Truffle) is important ascomycetes monoid in the underground living fungus, sporophore is grown in soil, remove individual species and partly expose native off-balancesheet when maturation, most of kind is buried from start to finish and is born in undergroundly, is and the symbiotic ectomycorrhiza type of trees medicine-food two-purpose fungi.
Ferfas belongs to Ascomycotina (Ascomycotina), Tuberale (Tuberales), Tuberaceae (Tuberaceae), Tuber (Tuber) on taxology, common ferfas has black truffle (Tuber melanosporum Vittad), India truffle (Tuber indicum Cooke), Chinese ferfas (Tuber sinense Tao et Liu) etc.The India truffle that Yunnan Province of China is produced is with similar to black truffle in shape with the Chinese ferfas of Sichuan product, its quality also can compare favourably with the black truffle that produce in Europe, the price of the fresh black truffle of per kilogram is up to more than 1000 dollars on the world market at present, and price that it is expensive and black, verrucose profile are endowed the good reputation of " black diamond " in the forest.
The research of relevant truffle polysaccharides starts from the eighties of last century the nineties, and how research at that time extracts truffle polysaccharides and biological activity thereof from the sporophore of ferfas if laying particular emphasis on, as:
Document: Hu Huijuan, Li Peizhen, the soughing of the wind in forest trees, Hang Bingqian, Guo Yuewei.Truffle polysaccharides is to mouse tumor and immune influence.China Medicine University's journal, 1994; 25 (5): the 289-292 reported first from the sporophore of Chinese ferfas, extract a kind of new protein binding polysaccharide---truffle polysaccharides, the mouse that S-180 sarcoma, EAC sarcoma liquid are arranged by administration in 10 days inoculation is continuously found the truffle polysaccharides energy obvious suppression mouse S-180 sarcoma of 25 mg/kg, 50 mg/kg and the growth of EAC sarcoma.Find that by successive administration truffle polysaccharides has tangible increase effect to the weight of mouse spleen, leukocyte count and T lymphocyte percentage in the peripheral blood simultaneously; Can promote the T lymphocyte transformation, improve in the mice serum IgG level etc.Experimental result shows, truffle polysaccharides is as a new fungus polysaccharide, and toxicity is low, good water solubility, function of tumor inhibition are obvious, is expected to develop the medicine that becomes antitumor immunotherapy.
Ferfas has the property of cultivation as a kind of medicine-food two-purpose fungi, can obtain a large amount of ferfas mycelium and fermentating metabolism product thereof fast simply by the substratum of cheapness, simultaneously can design the suitable medium component targetedly by the principle of metabolic engineering, induce truffle polysaccharides efficiently to accumulate, fermenting process is carried out towards the direction that helps truffle polysaccharides production, and whole fermentation process is amplified to the scale of suitability for industrialized production easily.But present fermentation research mainly lays particular emphasis on the mycelium that how to obtain ferfas, as:
Document: Chen Huiqun, Liu Hongyu, plum is flat.China's ferfas major physiological characteristic preliminary study.The edible mushrooms journal, 1998,5 (4): 26-30 reported Chinese ferfas (Tuber sinense Tao et Liu) has been carried out pure culture on bright pine needle substratum (potato 20%, bright pine needle 3%, glucose 1%, maltose 1%, peptone 0.5%, sal epsom 0.05%, agar 2%), and the increment by mycelia relatively and colony diameter size have been investigated the influence to Chinese ferfas mycelial growth of temperature, carbon source, nitrogenous source, pH respectively.Conclusion shows that the optimum growth temperature of Chinese ferfas is that 23-25 ℃, optimal pH are 5-6, and the best carbon source of mycelial growth is maltose, sucrose, fructose, and the best nitrogenous source of mycelial growth is peptone and ammonium sulfate.
Truffle polysaccharides can directly extract from kames, can also obtain by the method for ferfas liquid submerged fermentation, but the former does not fit into large-scale industrialization production, and reason mainly is:
1. ferfas grows in subterranean medicine-food two-purpose fungi as a kind of, require the environment grow in alkaline soil, have a moderate climate at occurring in nature, simultaneously with the root system symbiosis of seeds such as Oak Tree, robur, harsh growth conditions has directly caused its natural output, and supply falls short of demand.
2. carried out the research of half manual simulation cultivation for the deficiency that remedies the natural output of ferfas once has many scholars, but generally needed the 7-9 year from being inoculated into results, the cycle is longer, need expend a large amount of man power and materials.
3. because ferfas grows in undergroundly, and the artificial difficulty of finding is bigger, is vulnerable to damage in the gatherer process, ferfas grows in nature and is subjected to the influence of environmental change bigger simultaneously, and quality be cannot say for sure card.
4. the price of every kilogram of fresh black truffle is up to 1000 dollars on the present American-European market, and the market price of the Chinese ferfas that produce in Yunnan is also up to 400 yuan/kilogram.Therefore if directly then can't avoid problems such as expensive raw material price, output be limited as the raw materials for production of truffle polysaccharides with kames.
Present bibliographical information only be at the physiological property research of Chinese ferfas carried out pure culture and in half manual simulation's cultivation process of black truffle the mycelial growth to ferfas carried out preliminary study, relevant report is not seen in the research that relates to the ferfas pure culture by liquid deep fermentation for producing truffle polysaccharide as yet, and relevant culture medium prescription research belongs to report for the first time in the present invention.In a word, the liquid submerged fermentation substratum of a kind of not only suitable ferfas growth but also suitable truffles exopolysaccharide production is most important for the technology and the cost of whole truffle polysaccharides production.
Summary of the invention
The object of the present invention is to provide a kind of substratum of liquid deep fermentation for producing truffle polysaccharide, this culture medium prescription is reasonable, and is with low cost, simple for production; The China ferfas grows in this liquid submerged fermentation substratum rapidly, truffles exopolysaccharide output height; Utilizing ferfas to ferment at this substratum mid-deep strata produces truffle polysaccharides and can solve problems such as kames is underproduce.
Another object of the present invention is to provide a kind of preparation method of liquid deep fermentation for producing truffle polysaccharide, and is easy to implement the method, easy and simple to handle, is fit to the suitability for industrialized production of truffle polysaccharides.
Technical scheme of the present invention: (Tuber sinense Tao et Liu) is starting strain with Chinese ferfas, the content of relevant this bacterial strain see for details reference (Chen Hui group Liu Hong jade. Chinese ferfas major physiological characteristic preliminary study, the edible mushrooms journal, 1998,5 (4): 26-30).Adopt the Production Flow Chart of slant strains cultivation, level liquid seed culture, the cultivation of secondary liquid seeds and liquid submerged fermentation to produce truffles exopolysaccharide.In the liquid submerged fermentation stage, best liquid submerged fermentation substratum is optimized in the influence of truffles exopolysaccharide output by investigating different carbon sources, nitrogenous source and initial pH value.
Realize that concrete steps of the present invention are as follows:
One, slant strains is cultivated: slant medium of the present invention is: maltose 30-150 grams per liter, sal epsom 1.0-10 grams per liter, potassium primary phosphate 1.0-10 grams per liter, agar 10-30 grams per liter, 1.0 liters of potato extracting solutions, pH value 5-8.Sterilising conditions is: 121 ℃, 20 minutes.Chinese ferfas is inoculated in the slant medium of fresh configuration, places in the biochemical incubator and cultivate, culture temperature 15-40 ℃, incubation time 2-20 days.
Two, liquid seeds is cultivated: the liquid seed culture medium prescription is: maltose 30-150 grams per liter, peptone 5.0-50 grams per liter, sal epsom 1.0-10 grams per liter, potassium primary phosphate 1.0-10 grams per liter, pH value 5-8; Culture temperature 15-40 ℃, shaking speed 50-300 rev/min, incubation time 2-20 days.The slant strains of being cultivated in the step 1 transferred shaking of level liquid seed culture medium is housed carries out the level liquid seed culture in the bottle, this nutrient solution is the level liquid seed that contains bacterial classification.The level liquid seed of being cultivated transferred to shake carry out the secondary liquid seeds in the bottle and cultivate, inoculum size counts 10% by volume, and this nutrient solution is the secondary liquid seeds that contains bacterial classification.
Three, ferfas liquid submerged fermentation and Optimum of culture medium thereof: bottle is shaken in the secondary liquid seeds switching of being cultivated in the step 2 carry out liquid submerged fermentation, inoculum size counts 10% by volume, leavening temperature 15-40 ℃, shaking speed 50-300 rev/min, fermentation time 2-20 days.The inorganic salt composition is sal epsom 0.1-10 grams per liter, potassium primary phosphate 0.1-10 grams per liter in the described liquid submerged fermentation substratum; Carbon source is maltose 50 grams per liters, lactose 30-150 grams per liter, fructose 80 grams per liters or semi-lactosi 80 grams per liters; Nitrogenous source is urea 7 grams per liters, yeast extract paste 34 grams per liters, beef extract 34 grams per liters, ammonium sulfate 15.6 grams per liters, ammonium chloride 12.6 grams per liters, SODIUMNITRATE 20 grams per liters, saltpetre 24 grams per liters, ammonium nitrate 9 grams per liters; Initial pH value is 2-9.According to the optimum result of carbon source, nitrogenous source, pH value, obtain a kind of liquid submerged fermentation culture medium prescription: lactose 30-150 grams per liter, peptone 5-50 grams per liter, sal epsom 1.0-10 grams per liter, potassium primary phosphate 1.0-10 grams per liter, pH value are 2-9.With this nutrient media components water dissolution, transfer to specified pH value then, place the interior 121 ℃ of sterilizations of high-pressure sterilizing pot promptly to finish the preparation of this liquid submerged fermentation substratum after 20 minutes.
Four, the mensuration of truffles exopolysaccharide: contained mycelial fermented liquid centrifugal (5000 rev/mins, 30 minutes) with what obtain in the step 3, get the mensuration that its supernatant liquor is used for truffles exopolysaccharide.Adopt ethanol precipitation to obtain the truffles exopolysaccharide that liquid submerged fermentation obtains, fermented supernatant fluid adds the long-pending dehydrated alcohol of tetraploid, standing over night behind the mixing, get precipitation after centrifugal 5 minutes for 13000 rev/mins, 80% ethanol cleaning back with equivalent just can obtain the higher exocellular polysaccharide of purity in centrifugal 5 minutes down at 13000 rev/mins, and its concentration is measured with the vitriol oil-phynol method.The detailed step document that sees reference, M.Dubois, K.A.Gilles, J.K.Hamilton, et al.Colorimetric method for determination of sugars andrelated substances, Anal.Chem.1956, (28): 350-356.
Beneficial effect of the present invention: because ferfas costs an arm and a leg, the suitability for industrialized production of truffle polysaccharides does not have on market at present, the present invention has avoided extracting unfavorable factors such as the raw material finite sum costs an arm and a leg in the polysaccharide process from kames, reduce its production cost greatly, be expected to fill up the blank of truffle polysaccharides suitability for industrialized production.By the optimization to carbon source, nitrogenous source, initial pH, the optimal liquid deep layer substratum that the present invention obtained can improve the output of truffle polysaccharides greatly, is more suitable for the suitability for industrialized production of truffle polysaccharides simultaneously.
Embodiment
Embodiment 1
The bacterial classification that adopts: Chinese ferfas (Tuber sinense Taoet Liu).
A, slant strains are cultivated: the slant strains culture medium prescription that present embodiment adopted is: maltose 50 grams per liters, sal epsom 5 grams per liters, potassium primary phosphate 5 grams per liters, agar 15 grams per liters, 1.0 liters of potato extracting solutions, pH value 5 or 6 or 7 or 8.Sterilising conditions is: 121 ℃, 20 minutes.Culture temperature is 15 or 17 or 19 or 24 or 27 or 29 or 34 or 36 or 40 ℃, and incubation time is 2 or 5 or 7 or 9 or 14 or 16 or 17 or 20 days.
B, liquid seeds are cultivated: the prescription of level liquid seed, secondary liquid seed culture medium is in the present embodiment: maltose 70 grams per liters, peptone 25 grams per liters, sal epsom 5 grams per liters, potassium primary phosphate 6 grams per liters, pH value 5 or 6 or 7 or 8.Slant strains transferred shaking in the bottle of level liquid seed culture medium is housed carries out the level liquid seed culture.Culture temperature is 15 or 17 or 19 or 24 or 27 or 29 or 34 or 36 or 40 ℃, and shaking speed is 200 rev/mins, and incubation time is 2 or 5 or 7 or 9 or 14 or 16 or 17 or 20 days, and liquid amount is that per 250 ml shake flasks are adorned 50 milliliters of liquid; Bottle is shaken in cultured level liquid seed switching carry out the cultivation of secondary liquid seeds, the culture medium prescription of secondary liquid seeds is identical with the prescription of above level liquid seed culture medium, culture temperature is identical with the A step, shaking speed is 200 rev/mins, incubation time is 2 or 4 or 6 or 8 or 10 days, inoculum size is 5 milliliters, and liquid amount is that per 250 ml shake flasks are adorned 50 milliliters of liquid.
C, ferfas liquid submerged fermentation and medium optimization thereof: the liquid submerged fermentation culture medium prescription that is adopted is: maltose 80 grams per liters, and peptone 30 grams per liters, sal epsom 5 grams per liters, potassium primary phosphate 6 grams per liters, pH are 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9.Fermentation condition is: temperature, shake identical with A step, 200 rev/mins of shaking speed, incubation time goes on foot identical with A.Inoculum size is 5 milliliters, and liquid amount is that per 250 ml shake flasks are adorned 50 milliliters of liquid.
The mensuration main reference document M.Dubois of exocellular polysaccharide, K.A.Gilles, J.K.Hamilton, et al.Colorimetric method for determination of sugars and related substances, Anal.Chem.1956, (28): 350-356.Concrete implementation step is as follows: will contain centrifugal (5000 rev/mins of mycelial fermented liquid after fermentation finishes, 30 minutes), the mensuration that the supernatant liquor of fermented liquid centrifugal back acquisition is used for truffles exopolysaccharide, adopt ethanol precipitation to measure the truffles exopolysaccharide that liquid submerged fermentation obtained, get the dehydrated alcohol that the supernatant liquor of 200 microlitres after centrifugal adds 800 microlitres, standing over night behind the mixing, get precipitation after centrifugal 4-6 minute for 13000 rev/mins, clean the back with 1 milliliter of 80% ethanol and just can obtain the higher exocellular polysaccharide of purity down in centrifugal 4-6 minute at 13000 rev/mins.Concentration is measured with the vitriol oil-phynol method, adds 1 milliliter in the sodium hydroxide of 1 mol in the exocellular polysaccharide that obtains, and 500 microlitre moisturizings to 1 milliliter are got in 60 ℃ of insulations 1 hour after the cooling; Add 1 milliliter of 5% phenol, add 5 milliliters of the vitriol oils behind the mixing, 25 ℃ of insulations after 25 minutes under 488nm colorimetric.With same method production standard curve, the content that goes out polysaccharide by regression equation calculation is 1.60 grams per liters at last with analytically pure glucose.
Embodiment 2-4
Carbon source is respectively 80 grams per liter lactose, 80 grams per liter fructose, 80 grams per liter semi-lactosis in the liquid submerged fermentation substratum, and other condition is identical with embodiment 1.Analytical test result, the output of truffles exopolysaccharide are respectively 1.83 grams per liters, 1.13 grams per liters, 1.17 grams per liters.
Embodiment 5-12
Carbon source is a lactose in the liquid submerged fermentation substratum, and concentration is respectively 30 grams per liters, 50 grams per liters, 60 grams per liters, 70 grams per liters, 90 grams per liters, 110 grams per liters, 130 grams per liters, 150 grams per liters, and other condition is identical with embodiment 1.Analytical test result, the output of truffles exopolysaccharide are respectively 0.88 grams per liter, 1.15 grams per liters, 1.35 grams per liters, 2.18 grams per liters, 1.60 grams per liters, 1.35 grams per liters, 1.19 grams per liters, 0.91 grams per liter.
Embodiment 13-20
Nitrogenous source is respectively urea 7 grams per liters, yeast extract paste 34 grams per liters, beef extract 34 grams per liters, ammonium sulfate 15.6 grams per liters, ammonium chloride 12.6 grams per liters, SODIUMNITRATE 20 grams per liters, saltpetre 24 grams per liters, ammonium nitrate 9 grams per liters in the liquid submerged fermentation substratum, and other condition is identical with embodiment 1.Analytical test result, the output of truffles exopolysaccharide are respectively 1.53 grams per liters, 0.28 grams per liter, 1.07 grams per liters, 0.50 grams per liter, 0.25 grams per liter, 0.45 grams per liter, 0.62 grams per liter, 0.64 grams per liter.
Embodiment 21-27
Nitrogenous source is a urea in the liquid submerged fermentation substratum, and concentration is respectively 1 grams per liter, 3 grams per liters, 5 grams per liters, 8 grams per liters, 11 grams per liters, 15 grams per liters, 20 grams per liters, and other condition is identical with embodiment 1.Analytical test result, the output of truffles exopolysaccharide are respectively 0.65 grams per liter, 1.10 grams per liters, 1.42 grams per liters, 1.85 grams per liters, 1.87 grams per liters, 1.44 grams per liters, 1.13 grams per liters.
Embodiment 28-35
Peptone concentration is 5 grams per liters, 10 grams per liters, 15 grams per liters, 20 grams per liters, 25 grams per liters, 30 grams per liters, 35 grams per liters, 50 grams per liters in the liquid submerged fermentation substratum, and other condition is identical with embodiment 1.Analytical test result, the output of truffles exopolysaccharide are respectively 1.41 grams per liters, 1.60 grams per liters, 1.85 grams per liters, 2.04 grams per liters, 2.23 grams per liters, 1.60 grams per liters, 1.46 grams per liters, 1.15 grams per liters.
Embodiment 36-42
Initial pH value is respectively 2,3,4,5,7,8,9 in the liquid submerged fermentation substratum, and other condition is identical with embodiment 1.Analytical test result, the output of truffles exopolysaccharide are respectively 0.35 grams per liter, 1.39 grams per liters, 0.93 grams per liter, 1.28 grams per liters, 1.55 grams per liters, 1.68 grams per liters, 1.01 grams per liters.
Embodiment 43-50
Magnesium sulfate concentration is 0.1 grams per liter, 0.5 grams per liter, 1 grams per liter, 2 grams per liters, 5 grams per liters, 7 grams per liters, 9 grams per liters, 10 grams per liters in the liquid submerged fermentation substratum, and other condition is identical with embodiment 1.Analytical test result, the output of truffles exopolysaccharide are respectively 0.22 grams per liter, 0.44 grams per liter, 1.10 grams per liters, 1.37 grams per liters, 1.60 grams per liters, 1.42 grams per liters, 1.13 grams per liters, 1.02 grams per liters.
Embodiment 51-58
The biphosphate potassium concn is 0.1 grams per liter, 0.5 grams per liter, 1 grams per liter, 2 grams per liters, 5 grams per liters, 6 grams per liters, 7 grams per liters, 9 grams per liters, 10 grams per liters in the liquid submerged fermentation substratum, and other condition is identical with embodiment 1.Analytical test result, the output of truffles exopolysaccharide are respectively 1.32 grams per liters, 1.58 grams per liters, 1.76 grams per liters, 2.1 5 grams per liters, 2.30 grams per liters, 1.60 grams per liters, 1.54 grams per liters, 1.38 grams per liters, 1.01 grams per liters.
Claims (2)
1, a kind of preparation method of liquid deep fermentation for producing truffle polysaccharide, it comprises the following steps:
A, slant strains cultivate: with Chinese ferfas bacterial classification inoculation on slant medium, inoculated to be placed in the biochemical incubator and cultivated, culture temperature 15-40 ℃, incubation time 2-20 days, described slant strains culture medium prescription was: maltose 30-150 grams per liter, sal epsom 1.0-10 grams per liter, potassium primary phosphate 1.0-10 grams per liter, agar 10-30 grams per liter, 1.0 liters of potato extracting solutions, the pH value is 5-8;
The cultivation of B, liquid seeds: the slant strains of cultivating in the steps A transferred shaking of level liquid seed culture medium is housed carries out the level liquid seed culture in the bottle, culture condition is: temperature 15-40 ℃, shaking speed 50-300 rev/min, time 2-20 days; This nutrient solution is the level liquid seed that contains bacterial classification, described level liquid seed culture based formulas is: maltose 30-150 grams per liter, peptone 5-50 grams per liter, sal epsom 1.0-10 grams per liter, potassium primary phosphate 1.0-10 grams per liter, pH value 5-8, and culture condition is: temperature 15-40 ℃, shaking speed 50-300 rev/min, incubation time 2-10 days; Bottle is shaken in cultured level liquid seed switching carry out the cultivation of secondary liquid seeds, the secondary liquid seed culture medium is identical with the level liquid seed culture medium, inoculum size 10%/volume ratio, and resulting nutrient solution is the secondary liquid seeds that contains bacterial classification;
C, ferfas liquid submerged fermentation: bottle is shaken in the secondary liquid seeds switching of being cultivated among the step B carry out liquid submerged fermentation, fermentation condition: inoculum size 10%/volume ratio, temperature 15-40 ℃, shaking speed 50-300 rev/min, fermentation time 2-20 days, the inorganic salt composition is sal epsom 0.1-10 grams per liter, potassium primary phosphate 0.1-10 grams per liter in the described liquid submerged fermentation substratum, and carbon source is maltose, lactose, fructose or semi-lactosi; Nitrogenous source is peptone, urea, yeast extract paste, beef extract, ammonium sulfate, ammonium chloride, SODIUMNITRATE, saltpetre, ammonium nitrate, initial pH value is 2-9, selection according to carbon source, nitrogenous source, initial pH value, the liquid submerged fermentation substratum is transferred to specified pH value after with water dissolution, place in the high-pressure sterilizing pot 121 ℃ of sterilizations promptly to finish the preparation of this liquid submerged fermentation substratum after 20 minutes.
2, a kind of substratum that is used to realize the described ferfas liquid submerged fermentation of claim 1 is characterized in that: lactose 30-150 grams per liter, peptone 5-50 grams per liter, sal epsom 0.1-10 grams per liter, potassium primary phosphate 0.1-10 grams per liter.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102224873A (en) * | 2011-05-06 | 2011-10-26 | 郭景龙 | Method for preparing food from raw materials comprising coffee beans |
| CN101724564B (en) * | 2008-10-31 | 2012-07-25 | 湖北工业大学 | Method for producing truffle active mycelia and truffle polysaccharide by intermediate feed supplementing and fermentation |
| CN102676342A (en) * | 2012-06-01 | 2012-09-19 | 郭景龙 | Truffle wine and preparation method thereof |
| CN104017841A (en) * | 2014-06-13 | 2014-09-03 | 中国林业科学研究院亚热带林业研究所 | Technique for producing truffle polysaccharide by truffle liquid fermentation |
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2006
- 2006-12-27 CN CNB2006101665031A patent/CN100564510C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724564B (en) * | 2008-10-31 | 2012-07-25 | 湖北工业大学 | Method for producing truffle active mycelia and truffle polysaccharide by intermediate feed supplementing and fermentation |
| CN102224873A (en) * | 2011-05-06 | 2011-10-26 | 郭景龙 | Method for preparing food from raw materials comprising coffee beans |
| CN102224873B (en) * | 2011-05-06 | 2012-12-05 | 郭景龙 | Method for preparing food from raw materials comprising coffee beans |
| CN102676342A (en) * | 2012-06-01 | 2012-09-19 | 郭景龙 | Truffle wine and preparation method thereof |
| CN104017841A (en) * | 2014-06-13 | 2014-09-03 | 中国林业科学研究院亚热带林业研究所 | Technique for producing truffle polysaccharide by truffle liquid fermentation |
| CN104017841B (en) * | 2014-06-13 | 2016-12-07 | 中国林业科学研究院亚热带林业研究所 | The technique that a kind of truffles liquid fermentation produces truffle polysaccharides |
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Assignee: WUHAN JIACHENG BIOTECHNOLOGY CO., LTD. Assignor: Hubei Industry University Contract record no.: 2011420000146 Denomination of invention: Culture medium for liquid deep fermentation for producing truffle polysaccharide Granted publication date: 20091202 License type: Exclusive License Open date: 20070711 Record date: 20110704 |
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| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20091202 Termination date: 20121227 |