CN101724564A - Method for producing truffle active mycelia and truffle polysaccharide by intermediate feed supplementing and fermentation - Google Patents
Method for producing truffle active mycelia and truffle polysaccharide by intermediate feed supplementing and fermentation Download PDFInfo
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Abstract
The invention discloses a method for producing truffle active mycelia and truffle polysaccharide by intermediate feed supplementing and fermentation. In the invention, in a submerged fermentation process of tuber liquid, a certain amount of carbon source and/or nitrogen source is supplemented in a culture system at a proper time, thereby effectively promoting the growth of the mycelia and the biosynthesis of the truffle polysaccharide, and improving the biomass and the yield of the truffle polysaccharide. The method of the invention improves the equipment capacity factor, further saves the production cost, and lays a foundation for the industrial production of the active mycelia and the truffle polysaccharide by the submerged fermentation of the tuber liquid.
Description
Technical field
The present invention relates to the cell culture processes of ferfas, relate in particular to the fermentation method for producing of a kind of ferfas active mycelium body and truffle polysaccharides, belong to the microbial fermentation field.
Background technology
Common ferfas includes black truffle (Tuber melanosporum), summer truffle (Tuberaestivum), white ferfas (Tuber magnatum), India truffle (Tuber indicum) and Chinese ferfas (Tuber sinense).Wherein, ferfas (Truffles) belongs to Ascomycotina (Ascomycotina), Tuberale (Tuberales), Tuberaceae (Tuberaceae), Tuber (Tuber).Ferfas is to be grown in the subterranean medicine-food two-purpose fungi of forest, and its main active ingredient has alpha-androstanol, ceramide, truffle polysaccharides etc. on the taxology.Truffle polysaccharides has antitumor and participates in function such as immunomodulatory.Ferfas on the market is mainly derived from wild and half manual simulation cultivation, and the price of fresh black truffle up to more than 1000 dollars/kilograms, is called " underground gold " and " black diamond " on American-European market.Ferfas requires harsh to growth conditions, the ferfas of occurring in nature is and the root system symbiosis that grows in seeds (as: Oak Tree, robur) in the alkaline soil environment such as calcium carbonate that output is limited.Adopt half manual simulation cultivation, ferfas is vulnerable to the influence of other ectomycorrhizas, from being inoculated into results, generally needs the time of 7-9.
Ferfas has the property of cultivation as a kind of medicine-food two-purpose fungi.Effective constituents such as fermentative Production ferfas biological activity mycelium and truffle polysaccharides, exist with short production cycle, need the labor force few and be subjected to advantages such as external environment influence is little, than manual simulation's cultivation, be a kind of more efficiently method.
At present, adopting effective constituents such as fermentative Production ferfas biological activity mycelium and truffle polysaccharides mainly is to adopt batch culture method (batch culture: be meant microorganism is placed certain volumetrical substratum, through incubation growth, the training method of last results.In batch culture, substratum once adds, and will not replenish, no longer change.)。Its biomass and meta-bolites output (ferfas intracellular polyse, exocellular polysaccharide) are lower, and usage ratio of equipment is low.
Publication number is that (open day: Chinese patent application on June 27th, 2007) disclosed a kind of preparation method of truffle polysaccharides to CN1986827A, and the output of the resulting truffles exopolysaccharide of this method is up to 2.30 grams per liters.Publication number is that (open day: Chinese patent application on July 11st, 2007) disclosed a kind of substratum of liquid deep fermentation for producing truffle polysaccharide to CN1995322A, adopt this substratum to carry out liquid submerged fermentation than existing substratum, the output of truffle polysaccharides has had raising to a certain degree.
(Tang YJ such as Tang Yajie, Zhu LL, Li DS, Mi ZY, Li HM.Significance ofinoculation density and carbon source on the mycelial growth and Tuberpolysaccharides production by submerged fermentation of Chinese truffle Tubersinense.Process Biochemistry 2008,43:576-586.) reported a kind of optimization cultural method of ferfas fermentation culture, this method is to inoculum size (160,320,487,653 milligrams of dry cell weights/liter) screen and optimize, under the condition of the highest inoculum size (653 milligrams dry cell weight/liter), can make the output of exocellular polysaccharide (EPS) reach 1.97 ± 0.08 grams per liters (exocellular polysaccharide adopts ethanol precipitation to extract).Simultaneously, this method has also disclosed carbon source kind (glucose, maltose, sucrose, lactose) to the influence of ferfas liquid fermenting process, test-results shows: sucrose helps the raising (14.97 ± 0.08 grams per liter) of cell concentration most.Further investigated the influence of sucrose initial concentration (20-125 grams per liter) pair cell amount, experimental result shows: in 20-80 grams per liter scope, the maximum cell amount increases with the rising of sucrose initial concentration, when 80 grams per liters, reach peak value (22.81 ± 1.03 grams per liter), the intracellular polyse of this moment also reaches maximum value (2.92 ± 0.20 grams per liter), and exocellular polysaccharide also adopts ethanol precipitation to extract in this method.
(Liu RS such as Liu, Li DS, Li HM, Tang YJ.Response surface modeling thesignificance of nitrogen source on the submerged cultivation of Chinese truffleTuber sinense.Process Biochemistry 2008,43:868-876.) investigated the carbon nitrogen source the most adaptive ratio of ferfas fermentation, utilize statistics experimental design method (Box-Behnken design) to study glucose, yeast extract paste, the peptone initial concentration is to the influence of ferfas liquid fermenting process.The mathematical model that draws according to experiment is as can be known: glucose, and yeast extract paste, the suitableeest initial concentration proportioning of peptone is respectively 60 grams per liters, 30 grams per liters, 10 grams per liters.Under this condition, the highest biomass and EPS output and IPS output all are to obtain under the optimum condition of prediction, be respectively 23.94 ± 0.49 grams per liters, 1.59 ± 0.06 grams per liters and 1.48 ± 0.26 grams per liters (exocellular polysaccharide adopts ethanol precipitation to extract).
Tang Yajie etc. are (Tang YJ in another piece document, Zhu LL, Liu RS, Li YY, Li DS, Mi ZY, Li HM.Quantitative response of cell growth and Tuber polysaccharidesbiosynthesis by medicinal mushroom Chinese truffle Tuber sinense to metal ion inculture medium.Bioresource Technology 2008 99:7606-7615.) has disclosed the influence to ferfas fermentation of micro-kind and optimal concentration: by the single-factor experimental technique to metal ion (Ca
2+, Na
+, K
+And Mg
2+) screen and optimize, proved 50 mmole Mg
2+With 10 mmole k
+It is the generation that helps exocellular polysaccharide (EPS) most.Then utilize total divisor experimental design (Full factorialdesign), investigated the synergy of magnesium ion and potassium ion.Experimental result shows, 30 mmole Mg
2+With 5 mmole K
+Be the suitableeest combination, can make exocellular polysaccharide (EPS) output reach maximum value 5.86 ± 0.21 grams per liters (exocellular polysaccharide adopts ethanol precipitation to extract).
In sum, in the fermentation method for producing of existing ferfas active mycelium body and truffle polysaccharides, the output of its active mycelium body and truffle polysaccharides all has the space of further raising, how to improve ferfas active mycelium body and truffle polysaccharides output, improve its plant factor and further save production cost, be that the fermentation method for producing of existing ferfas active mycelium body and truffle polysaccharides is demanded the technical issues that need to address urgently.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, and the fermentation method for producing of a kind of ferfas active mycelium body and truffle polysaccharides is provided, and this method can effectively improve the output of ferfas active mycelium body and truffle polysaccharides.
In order to solve the problems of the technologies described above, the present invention has adopted following technical scheme:
The fermentation method for producing of a kind of Tuber (Tuber) active mycelium body and truffle polysaccharides comprises slant culture, level liquid seed culture, the cultivation of secondary liquid seeds and liquid submerged fermentation; Wherein, in the liquid submerged fermentation process, in culture system, add carbon source and/or nitrogenous source.
As one of optimized technical scheme of the present invention: in the liquid submerged fermentation process, in culture system, added the peptone of 1-3 1-20 grams per liter and the yeast extract powder of 1-30 grams per liter every 1-6 days, make in whole fermentation process that total input concentration of peptone and yeast extract powder reaches 5-20 grams per liter and 20-50 grams per liter respectively in the culture system.
As two of optimized technical scheme of the present invention: when the remaining sugar concentration in the culture system is the 0.1-15 grams per liter, adds carbon source and make remaining sugar concentration in the culture system maintain the level of 0.1-80 grams per liter.
Wherein, described carbon source can for example, can be sucrose, maltose, glucose or fructose etc. for various various monose or the polysaccharide that are used for microorganism culturing, and its concentration is preferably the 10-50 grams per liter.
The inventor finds through further test, adds to be aided with in these means of carbon source in liquid submerged fermentation and adds a certain amount of nitrogenous source, can more effectively improve the output of ferfas active mycelium body and truffle polysaccharides; As a kind of optimized technical scheme: when remaining sugar concentration during at the 0.1-15 grams per liter, adding carbon source makes carbon source concentration in the culture system maintain the level of 0.1-80 grams per liter, can in 1-3 days time period of liquid submerged fermentation, add simultaneously 1 peptone and yeast extract powder, make the concentration of peptone and yeast extract powder in the culture system reach the level of 5-20 grams per liter and 20-50 grams per liter respectively; Also can be in 1-3 days time period of liquid submerged fermentation, in culture system, to add 1 peptone and yeast extract powder, then in 4-7 days time period of liquid submerged fermentation, in culture system, add 1 peptone and yeast extract powder again, in whole fermentation process, make the total concn of peptone in the culture system and yeast extract powder reach the level of 5-20 grams per liter and 20-50 grams per liter respectively equally; Can also be in culture system, to add 1 peptone and yeast extract powder respectively at the 3rd day, the 6th day of liquid submerged fermentation and the 9th day, make in the whole fermentation process total concn of the peptone in the culture system and yeast extract powder reach the level of 5-20 grams per liter and 20-50 grams per liter respectively; Wherein, the peptone concentration that is added is preferably the 1-20 grams per liter, and yeast extract powder concentration is preferably the 1-30 grams per liter.
Described Tuber (Tuber) is preferably Chinese ferfas (Tuber sinense), India truffle (Tuberindium), black truffle (Tuber melanosporum), white ferfas (Tuber magnatum) or summer truffle (Tuber aestivum); These Tuber bacterial strains all can be bought from commercial channels and obtain.For example can be available from Mianyang, Sichuan edible mushrooms institute, Chinese common micro-organisms culture presevation administrative center etc.
Described slant culture, the level liquid seed culture, cultivation of secondary liquid seeds and the used substratum of liquid submerged fermentation and culture condition are conventional substratum and the conventional culture condition in the ferfas cell cultures, these all are known in those skilled in the art, as a reference, described substratum can carry out (Tang according to the disclosed content of relevant document with relevant culture condition, Y.J., Zhu, L.L., Li.D.S., Mi, Z.Y., Li, H.M..Significance of inoculation density and carbon source on themycelial growth and Tuber polysaccharides production by submerged fermentationof Chinese truffle Tuber sinense.Process Biochemistry 2008,43:576-586.).
As a reference:
Described slant medium is the PDA nutrient agar: the magnesium sulfate heptahydrate of 0.15 grams per liter, the potassium primary phosphate of 0.30 grams per liter, the glucose of 20 grams per liters, the vitamins B of 0.05 grams per liter
1Potato with 200 grams per liters; The slant culture condition is: 25 ℃ of culture temperature, incubation time are 5-7 days.
Described level liquid seed culture medium is: glucose 35 grams per liters, peptone 5 grams per liters, yeast extract powder 2.5 grams per liters, magnesium sulfate heptahydrate 0.5 grams per liter, potassium primary phosphate 1 grams per liter, vitamins B
10.05 grams per liter; Level liquid seed culture condition is: 25 ℃ of temperature, rotary shaking table, rotating speed are 80-180 rev/min.
Described secondary liquid seed culture medium is: glucose 35 grams per liters, peptone 5 grams per liters, yeast extract powder 2.5 grams per liters, magnesium sulfate heptahydrate 0.5 grams per liter, potassium primary phosphate 1 grams per liter, vitamins B
10.05 grams per liter; Secondary liquid seeds culture condition is: 25 ℃ of temperature, rotary shaking table, rotating speed are 80-180 rev/min.
Described liquid submerged fermentation substratum is: sucrose 35-40 grams per liter, peptone 5 grams per liters, yeast extract powder 5 grams per liters, 30 mmole Mg
2+With 10 mmole k
+, vitamins B
10.05 grams per liter; Liquid submerged fermentation condition: temperature 20-30 ℃, rotary shaking table, rotating speed are 80-180 rev/min.
With the content of the resulting product of cell culture processes of the present invention with the vitriol oil-phynol method determination block granulose, measurement result shows, method of the present invention is than existing batch culture, the black truffle mycelium has improved 55.8% than cultivate in batches in the cultured products, especially truffles exopolysaccharide rate ratio batch culture method has improved 222.2%, and the ferfas intracellular polyse has also increased by 103.2% than culture method in batches in the ferfas cell cultures.This is the maximum of the truffle polysaccharides that obtained in the ferfas cell cultures of being reported at present.This shows adds carbon source and/or nitrogenous source in the liquid submerged fermentation process, for the biosynthesizing of truffle polysaccharides promoter action is significantly arranged, for the activeconstituents in the suitability for industrialized production ferfas is laid a good foundation.
The inventive method is added a certain amount of carbon source and/or nitrogenous source suitable opportunity in the ferfas fermenting process, can effectively promote the cell growth, improves biomass, significantly improve outside the ferfas born of the same parents and the output of intracellular polyse, improve plant factor, reduced production cost, had industrial prospect.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
One, test materials: black truffle (Tuber melanosporum), Chinese ferfas (Tubersinense), India truffle (Tuber indium), white ferfas (Tuber magnatum), summer truffle (Tuberaestivum), all buy from Sichuan Province Mianyang City edible mushrooms institute.
Two, the truffle polysaccharides content detecting method: cell is weighed after 60 ℃ of oven dry, the cell of oven dry is according to document (Tang, Y.J., Zhu, L.L., Li.D.S., Mi, Z.Y., Li, H.M..Significance ofinoculation density and carbon source on the mycelial growth and Tuberpolysaccharides production by submerged fermentation of Chinese truffle Tubersinense.Process Biochemistry 2008 is after method break process 43:576-586.), with the content of the vitriol oil-phynol method determination block granulose.
Comparative experimental example 1 adopts batch culture method fermentative production truffle polysaccharides
(1) bacterial classification of Cai Yonging is: black truffle (Tuber melanosporum);
(2) slant medium: PDA, culture condition: 25 ℃, cultivated 5-7 days;
(3) level liquid seed culture medium (grams per liter): glucose 35, peptone 5, yeast extract powder 2.5, magnesium sulfate heptahydrate 0.5, potassium primary phosphate 1, vitamins B
10.05, in 5.5,50 milliliters of substratum of pH/250 ml shake flasks; Culture condition: 25 ℃ of temperature, rotary shaking table, rotating speed are 120 rev/mins.
(4) secondary liquid seed culture medium (grams per liter): glucose 35, peptone 5, yeast extract powder 2.5, magnesium sulfate heptahydrate 0.5, potassium primary phosphate 1, vitamins B
10.05,5.5,200 milliliters of substratum of pH/500 ml shake flasks; Culture condition: 25 ℃ of temperature, rotary shaking table, rotating speed are 120 rev/mins.
(5) the initial substratum (grams per liter) that ferments: sucrose 35, peptone 5, yeast extract powder 5,30 mmole Mg
2+With 10 mmole K
+, vitamins B
10.05 grams per liter, 5.5,50 milliliters of substratum of pH/250 ml shake flasks; Culture condition: 25 ℃ of temperature, rotary shaking table, rotating speed are 120 rev/mins; Cultivate after 30 days, carry out following assay determination:
The mensuration of A, exopolysaccharides: under 30 micron pore size membrane filtrations, get 34.49 grams per liter dry cell weights; Add the long-pending dehydrated alcohol of tetraploid in the fermented liquid, mixing is spent the night, and obtains truffle polysaccharides outside the born of the same parents in 13000rpm centrifugation.Through 1 mol NaOH dissolving, adopting the vitriol oil-phynol method to measure exopolysaccharides is 2.2 grams per liters;
The Determination on content of B, intracellular polyse: 100 milligrams of mycelium, add 1 mol NaOH dissolving, it is 7.28 milligrams/100 milligrams dry weights that the vitriol oil-phynol method is measured intracellular polyse content, and intracellular polyse output is 2.18 grams per liters (intracellular polyse content multiply by dry cell weight).
Embodiment 1
One, test materials and culture condition
Bacterial classification, slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and the culture condition thereof, the initial substratum of fermentation and the culture condition thereof that adopt are with comparative experimental example 1.
Two, test method
Be divided into test group and control group:
1, control group: the ferfas bacterial classification inoculation is cultivated to slant medium, carried out level liquid seed culture, the cultivation of secondary liquid seeds and liquid submerged fermentation more successively;
2, test group is divided into three groups, different are with control group: 1 group of (1) test: the 3rd day of the liquid submerged fermentation process adding peptone and yeast extract powder (peptone concentration is that 5 grams per liters, yeast extract powder concentration are 25 grams per liters) to culture system makes peptone in the culture system concentration that always feeds intake maintain 10 grams per liters, and yeast extract powder concentration maintains the level (volume of whole fermentation process culture system preferably is controlled at 50 milliliters constant level) of 30 grams per liters; (2) test is 2 groups: respectively in the 3rd of liquid submerged fermentation process, added peptone and yeast extract powder (peptone concentration is that 2.5 grams per liters, yeast extract powder concentration are 12.5 grams per liters) to culture system in 6 days and make the peptone of the culture system concentration that always feeds intake maintain 15 grams per liters, yeast extract powder concentration maintains the level (volume of whole fermentation process culture system preferably is controlled at 50 milliliters constant level) of 35 grams per liters; (3) test is 3 groups: respectively in the 3rd of liquid submerged fermentation process, 6, added peptone and yeast extract powder (peptone concentration is that 1.67 grams per liters, yeast extract powder concentration are 8.33 grams per liters) to culture system in 9 days and make peptone in the culture system concentration that always feeds intake maintain 20 grams per liters, yeast extract powder concentration maintains the level (volume of whole fermentation process culture system preferably is controlled at 50 milliliters constant level) of 45 grams per liters;
In addition, used substratum of test group and control group and culture condition are all identical.
Cultivate after 30 days, measure the output of dry cell weight and polysaccharide respectively, measuring method is with comparative experimental example 1, and measurement result sees Table 1.
Three kinds of additional ways of table 1 nitrogenous source are to the influence of black truffle liquid submerged fermentation
aThe mean value of three samples and standard deviation
bDry cell weight reaches maximum incubation time
As known from Table 1, compare with control group (not adding nitrogenous source), after test group was added nitrogenous source, active mycelium body output obtained raising to a certain degree, has improved 90.71% than control group.This shows an amount of nitrogenous source that adds in ferfas liquid submerged fermentation process, can promote the production of ferfas active mycelium body.
Embodiment 2
One, test materials and culture condition
Bacterial classification, slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and the culture condition thereof, the initial substratum of fermentation and the culture condition thereof that adopt are with comparative experimental example 1.
Two, test method
Be divided into test group and control group:
1, control group: the ferfas bacterial classification inoculation is cultivated to slant medium, carried out level liquid seed culture, the cultivation of secondary liquid seeds and liquid submerged fermentation more successively;
2, test group is divided into 3 groups, different are with control group: 1 group of (1) test: made sucrose concentration in the fermented liquid reach the level (volume of whole fermentation process culture system preferably is controlled at 50 milliliters constant level) of 30-80 grams per liter to culture system interpolation sucrose when remaining sugar concentration is in the level of 0.1-15 grams per liter in the 3rd day of the liquid submerged fermentation process; (2) test is 2 groups: add sucrose respectively at the 3rd, 7 day of the liquid submerged fermentation process (remaining sugar concentration is in the level of 0.1-15 grams per liter in culture system this moment after testing) to culture system and make sucrose concentration in the fermented liquid reach the level (volume of whole fermentation process culture system preferably is controlled at 50 milliliters constant level) of 0.1-80 grams per liter; (3) test is 3 groups: add sucrose respectively at the 3rd, 7,10 day of the liquid submerged fermentation process (remaining sugar concentration is in the level of 0.1-15 grams per liter in culture system this moment after testing) to culture system and make sucrose concentration in the culture system reach the level (volume of whole fermentation process culture system preferably is controlled at 50 milliliters constant level) of 0.1-80 grams per liter;
In addition, used substratum of test group and control group and culture condition are all identical.
Cultivate after 30 days, measure the output of dry cell weight and polysaccharide respectively, measuring method is with comparative experimental example 1, and measurement result sees Table 2.
Table 2 carbon source is added the influence to black truffle liquid submerged fermentation process
aThe mean value of three samples and standard deviation
bDry cell weight reaches maximum incubation time
As known from Table 2, compare with control group (not adding carbon source), test group ferfas active mycelium body has increased by 111.4% than control group, and the yield increased group of truffles exopolysaccharide has increased by 98.8%, and the yield increased group of ferfas intracellular polyse has improved 160.3%.
Embodiment 3
One, test materials and culture condition
Bacterial classification, slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and the culture condition thereof, the initial substratum of fermentation and the culture condition thereof that adopt are with comparative experimental example 1.
Two, test method
Be divided into test group and control group:
1, control group: the ferfas bacterial classification inoculation is cultivated to slant medium, carried out level liquid seed culture, the cultivation of secondary liquid seeds and liquid submerged fermentation more successively;
2, different are for test group and control group: 1 group of (1) test: the 3rd day of the liquid submerged fermentation process adding peptone and yeast extract powder (peptone concentration is that 1.67 grams per liters, yeast extract powder concentration are 8.33 grams per liters) to culture system makes peptone in the culture system concentration that always feeds intake maintain 20 grams per liters, and yeast extract powder concentration maintains the level of 50 grams per liters; When finding that remaining sugar concentration is in the level of 0.1-15 grams per liter, the sucrose that adds 30 grams per liters to culture system makes sucrose concentration in the fermented liquid reach the level (volume of whole fermentation process culture system preferably is controlled at 50 milliliters constant level) of 35-80 grams per liter in the liquid submerged fermentation process; (2) test is 2 groups: respectively added peptone and yeast extract powder (peptone concentration is that 1.67 grams per liters, yeast extract powder concentration are 8.33 grams per liters) to culture system respectively in the 3rd day, the 6th day of the liquid submerged fermentation process and make peptone in the culture system concentration that always feeds intake maintain 15 grams per liters, yeast extract powder concentration maintains the level of 40 grams per liters; When finding that remaining sugar concentration is in the level of 0.1-15 grams per liter, the sucrose that adds 30 grams per liters to culture system makes remaining sugar concentration in the culture system maintain the level (volume of whole fermentation process culture system preferably is controlled at 50 milliliters constant level) of 0.1-80 grams per liter in the liquid submerged fermentation process; (3) test is 3 groups: add peptone and yeast extract powder (peptone concentration is that 1.67 grams per liters, yeast extract powder concentration are 8.33 grams per liters) the 3rd day, the 6th day of the liquid submerged fermentation process and the 9th day to culture system respectively and make peptone in the culture system concentration that always feeds intake maintain 10 grams per liters, yeast extract powder concentration maintains the level of 20 grams per liters; When finding that remaining sugar concentration in the culture system is in the level of 0.1-15 grams per liter, the sucrose that adds 30 grams per liters to culture system makes sucrose concentration in the culture system reach the level (volume of whole fermentation process culture system preferably is controlled at 50 milliliters constant level) of 0.1-80 grams per liter in the liquid submerged fermentation process;
In addition, used substratum of test group and control group and culture condition are all identical.
Cultivate after 30 days, measure the output of dry cell weight and polysaccharide respectively, measuring method is with comparative experimental example 1.Measurement result sees Table 3.
Table 3 carbon, nitrogenous source mix the influence of adding black truffle liquid submerged fermentation process
aThe mean value of three samples and standard deviation
bDry cell weight reaches maximum incubation time
As known from Table 3, compare with control group (not adding carbon nitrogen source), test group is increasing substantially of all obtaining of the output of cell concentration or truffles exopolysaccharide output, ferfas intracellular polyse, output than cell concentration, truffles exopolysaccharide output and the ferfas intracellular polyse of control group has improved 230.4% respectively, 213.7% and 246.1%, this shows that nitrogenous source and the effective bonded carbon nitrogen source of intermittent injecting sucrose mixing feed supplement cultivation, the biosynthesizing that can more effectively improve truffle polysaccharides are added in pulse in ferfas liquid submerged fermentation process.
Embodiment 4
One, test materials and culture condition
The bacterial classification that adopts is: Chinese ferfas (Tuber sinense); Slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and culture condition thereof, the initial substratum of fermentation and culture condition thereof are with comparative experimental example 1.
Two, test method
In the liquid submerged fermentation process the 3rd, 6, in culture system, added peptone and yeast extract powder (peptone concentration is that 3 grams per liters, yeast extract concentration are powder 6 grams per liters) in 9 days respectively, make peptone in the culture system concentration that always feeds intake maintain 10 grams per liters, yeast extract powder concentration maintains the level of 30 grams per liters; When remaining sugar concentration is in the level of 10 grams per liters, add 40 grams per liter maltose to culture system, make remaining sugar concentration in the culture system reach the level (volume of whole fermentation process culture system preferably is controlled at 50 milliliters constant level) of 0.1-80 grams per liter.Cultivate results mycelium and fermented liquid after 30 days, carry out analysis of effective component (measuring method is with comparative experimental example 1), analytical results is: the output of cell concentration, exocellular polysaccharide, intracellular polyse reaches 55 grams per liters, 7.3 grams per liters, 4.6 grams per liters respectively.
Embodiment 5
One, test materials and culture condition
The bacterial classification that adopts is: summer truffle (Tuber aestivum); Slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and culture condition thereof, the initial substratum of fermentation and culture condition thereof are with comparative experimental example 1.
Two, test method
In the liquid submerged fermentation process the 3rd, 6, added peptone and yeast extract powder (peptone concentration is that 5 grams per liters, yeast extract powder concentration are 10 grams per liters) to culture system respectively in 9 days, make peptone in the culture system concentration that always feeds intake maintain 20 grams per liters, yeast extract powder concentration maintains the level of 40 grams per liters; When remaining sugar concentration is in the level of 12 grams per liters, add 50 grams per liter glucose to culture system, make remaining sugar concentration in the culture system maintain the level of 0.1-80 grams per liter.Cultivate results mycelium and fermented liquid after 30 days, carry out analysis of effective component (measuring method is with comparative experimental example 1), analytical results is: the output of cell concentration, exocellular polysaccharide, intracellular polyse reaches 60 grams per liters, 7.8 grams per liters, 4.86 grams per liters respectively.
Embodiment 6
One, test materials and culture condition
The bacterial classification that adopts is: India truffle (Tuber indium); Slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and culture condition thereof, the initial substratum of fermentation and culture condition thereof are with comparative experimental example 1.
Two, test method
In the liquid submerged fermentation process the 3rd, 6, in culture system, added peptone and yeast extract powder (peptone concentration is that 15 grams per liters, yeast extract powder concentration are 20 grams per liters) in 9 days respectively, make peptone in the culture system concentration that always feeds intake maintain 20 grams per liters, yeast extract powder concentration maintains the level of 40 grams per liters; When remaining sugar concentration is in the level of 7 grams per liters, add 50 grams per liter fructose, make remaining sugar concentration in the culture system maintain the level of 0.1-80 grams per liter.Cultivate results mycelium and fermented liquid after 30 days, carry out analysis of effective component (measuring method is with comparative experimental example 1), analytical results is: the output of cell concentration, exocellular polysaccharide, intracellular polyse reaches 63 grams per liters, 6.8 grams per liters, 4.45 grams per liters respectively.
Embodiment 7
One, test materials and culture condition
The bacterial classification that adopts is: white ferfas (Tuber magnatum); Slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and culture condition thereof, the initial substratum of fermentation and culture condition thereof are with comparative experimental example 1.
Two, test method
In the liquid submerged fermentation process the 4th, 7, in culture system, added peptone and yeast extract powder (peptone concentration is that 12 grams per liters, yeast extract powder concentration are 10 grams per liters) in 10 days respectively, make peptone in the culture system concentration that always feeds intake maintain 20 grams per liters, yeast extract powder concentration maintains the level of 40 grams per liters; When remaining sugar concentration is in the level of 4 grams per liters, add 40 grams per liter maltose to culture system, make remaining sugar concentration in the culture system maintain the level of 0.1-50 grams per liter.Cultivate results mycelium and fermented liquid after 30 days, carry out analysis of effective component (measuring method is with comparative experimental example 1), analytical results is: the output of cell concentration, exocellular polysaccharide, intracellular polyse reaches 55 grams per liters, 5.8 grams per liters, 4.83 grams per liters respectively.
Embodiment 8
One, test materials and culture condition
The bacterial classification that adopts is: summer truffle (Tuber aestivum); Slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and culture condition thereof, the initial substratum of fermentation and culture condition thereof are with comparative experimental example 1.
Two, test method
In the liquid submerged fermentation process the 2nd, 5, in culture system, added peptone and yeast extract powder (peptone concentration is that 12 grams per liters, yeast extract powder concentration are 10 grams per liters) in 8 days respectively, make peptone in the culture system concentration that always feeds intake maintain 20 grams per liters, yeast extract powder concentration maintains the level of 40 grams per liters; When remaining sugar concentration is in the level of 7 grams per liters, add 30 grams per liter maltose to culture system, make remaining sugar concentration in the culture system maintain the level of 0.1-40 grams per liter.Cultivate results mycelium and fermented liquid after 30 days, carry out analysis of effective component (measuring method is with comparative experimental example 1), analytical results is: the output of cell concentration, exocellular polysaccharide, intracellular polyse reaches 52 grams per liters, 6.8 grams per liters, 4.34 grams per liters respectively.
Embodiment 9
One, test materials and culture condition
The bacterial classification that adopts is: Chinese ferfas (Tuber sinense); Slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and culture condition thereof, the initial substratum of fermentation and culture condition thereof are with comparative experimental example 1.
Two, test method
In the liquid submerged fermentation process the 1st, 4, in culture system, added peptone and yeast extract powder (peptone concentration is that 12 grams per liters, yeast extract powder concentration are 10 grams per liters) in 7 days respectively, make peptone in the culture system concentration that always feeds intake maintain 20 grams per liters, yeast extract powder concentration maintains the level of 40 grams per liters; When remaining sugar concentration is in the level of 9 grams per liters, add 50 grams per liter fructose to culture system, make remaining sugar concentration in the culture system maintain the level of 0.1-40 grams per liter.Cultivate results mycelium and fermented liquid after 30 days, carry out analysis of effective component (measuring method is with comparative experimental example 1), analytical results is: the output of cell concentration, exocellular polysaccharide, intracellular polyse reaches 56 grams per liters, 6 grams per liters, 5 grams per liters respectively.
Embodiment 10
One, test materials and culture condition
The bacterial classification that adopts is: black truffle (Tuber melanosporum); Slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and culture condition thereof, the initial substratum of fermentation and culture condition thereof are with comparative experimental example 1.
Two, test method
In the liquid submerged fermentation process the 3rd, 6, in culture system, added peptone and yeast extract powder (peptone concentration is that 12 grams per liters, yeast extract powder concentration are 10 grams per liters) in 9 days respectively, make peptone in the culture system concentration that always feeds intake maintain 20 grams per liters, yeast extract powder concentration maintains the level of 40 grams per liters; When remaining sugar concentration is in the level of 5.7 grams per liters, add 40 grams per liter maltose to culture system, make remaining sugar concentration in the culture system maintain the level of 0.1-50 grams per liter.Cultivate results mycelium and fermented liquid after 30 days, carry out analysis of effective component (measuring method is with comparative experimental example 1), analytical results is: the output of cell concentration, exocellular polysaccharide, intracellular polyse reaches 55 grams per liters, 5.8 grams per liters, 4.83 grams per liters respectively.
Embodiment 11
One, test materials and culture condition
The bacterial classification that adopts is: India truffle (Tuber indium); Slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and culture condition thereof, the initial substratum of fermentation and culture condition thereof are with comparative experimental example 1.
Two, test method
In the liquid submerged fermentation process the 4th, 6, in culture system, added peptone and yeast extract powder (peptone concentration is that 7.8 grams per liters, yeast extract powder concentration are 10 grams per liters) in 8 days respectively, make peptone in the culture system concentration that always feeds intake maintain 20 grams per liters, yeast extract powder concentration maintains the level of 40 grams per liters; When remaining sugar concentration is in the level of 7.9 grams per liters, add 49 grams per liter glucose to culture system, make in the culture system in remaining sugar concentration maintain the level of 0.1-60 grams per liter.Cultivate results mycelium and fermented liquid after 30 days, carry out analysis of effective component (measuring method is with comparative experimental example 1), analytical results is: the output of cell concentration, exocellular polysaccharide, intracellular polyse reaches 53 grams per liters, 6.84 grams per liters, 4.23 grams per liters respectively.
Embodiment 12
One, test materials and culture condition
The bacterial classification that adopts is: white ferfas (Tuber magnatum); Slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and culture condition thereof, the initial substratum of fermentation and culture condition thereof are with comparative experimental example 1.
Two, test method
In the liquid submerged fermentation process the 3rd, 6, in culture system, added peptone and yeast extract powder (peptone concentration is that 12 grams per liters, yeast extract powder concentration are 10 grams per liters) in 9 days respectively, make peptone in the culture system concentration that always feeds intake maintain 20 grams per liters, yeast extract powder concentration maintains the level of 40 grams per liters; When remaining sugar concentration is in the level of 3 grams per liters, add 40 grams per liter sucrose to culture system, make remaining sugar concentration in the culture system maintain the level of 0.1-45 grams per liter.Cultivate results mycelium and fermented liquid after 30 days, carry out analysis of effective component (measuring method is with comparative experimental example 1), analytical results is: the output of cell concentration, exocellular polysaccharide, intracellular polyse reaches 55 grams per liters, 5.8 grams per liters, 4.83 grams per liters respectively.
Embodiment 13
One, test materials and culture condition
The bacterial classification that adopts is: India truffle (Tuber indium); Slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and culture condition thereof, the initial substratum of fermentation and culture condition thereof are with comparative experimental example 1.
Two, test method
In the liquid submerged fermentation process the 1st, 5, added peptone and yeast extract powder (peptone concentration is that 10 grams per liters, yeast extract powder concentration are 8 grams per liters) to culture system respectively in 7 days, make peptone in the culture system concentration that always feeds intake maintain 20 grams per liters, yeast extract powder concentration maintains the level of 40 grams per liters; When remaining sugar concentration is in the level of 2.5 grams per liters, add 35 grams per liter sucrose, make remaining sugar concentration in the culture system maintain the level of 0.1-40 grams per liter.Cultivate results mycelium and fermented liquid after 30 days, carry out analysis of effective component (measuring method is with comparative experimental example 1), analytical results is: the output of cell concentration, exocellular polysaccharide, intracellular polyse reaches 58 grams per liters, 6.1 grams per liters, 4.93 grams per liters respectively.
Embodiment 14
One, test materials and culture condition
The bacterial classification that adopts is: Chinese ferfas (Tuber sinense); Slant medium and culture condition thereof, level liquid seed culture medium and culture condition thereof, secondary liquid seed culture medium and culture condition thereof, the initial substratum of fermentation and culture condition thereof are with comparative experimental example 1.
Two, test method
In the liquid submerged fermentation process the 2nd, 5, added peptone and yeast extract powder (peptone concentration is that 12 grams per liters, yeast extract powder concentration are 10 grams per liters) to culture system respectively in 7 days, make peptone in the culture system concentration that always feeds intake maintain 20 grams per liters, yeast extract powder concentration maintains the level of 40 grams per liters; When remaining sugar concentration is in the level of 5.4 grams per liters, add 36 grams per liter maltose to culture system, make remaining sugar concentration in the culture system maintain the level of 0.1-40 grams per liter.Cultivate results mycelium and fermented liquid after 30 days, carry out analysis of effective component (measuring method is with comparative experimental example 1), analytical results is: the output of cell concentration, exocellular polysaccharide, intracellular polyse reaches 57 grams per liters, 6.31 grams per liters, 4.75 grams per liters respectively.
Claims (7)
1. the method that middle fed-batch fermentation is produced ferfas active mycelium body and truffle polysaccharides comprises slant culture, and level liquid seed culture, secondary liquid seeds are cultivated and liquid submerged fermentation; It is characterized in that: in the liquid submerged fermentation process, in culture system, add carbon source and/or nitrogenous source.
2. in accordance with the method for claim 1, it is characterized in that: when the remaining sugar concentration in the culture system is the 0.1-15 grams per liter, adds carbon source and make remaining sugar concentration in the fermented liquid maintain the level of 0.1-80 grams per liter.
3. according to claim 1 or 2 described methods, it is characterized in that: described carbon source comprises sucrose, glucose, maltose or fructose.
4. according to claim 1 or 2 described methods, it is characterized in that: in the liquid submerged fermentation process, at least in culture system, added the peptone of 1-3 1-20 grams per liter and the yeast extract powder of 1-30 grams per liter in every interval 1-6 days, make in whole fermentation process that the total concn of peptone and yeast extract powder reaches 5-20 grams per liter and 20-50 grams per liter respectively in the culture system.
5. according to the described method of claim 1-4, it is characterized in that: described ferfas is selected from black truffle (Tuber melanosporum), Chinese ferfas (Tuber sinense), India truffle (Tuber indium), white ferfas (Tuber magnatum) or summer truffle (Tuberaestivum).
6. according to the described method of claim 1-4, it is characterized in that: contain an amount of inorganic salt and VITAMIN in the described liquid submerged fermentation substratum.
7. according to the described method of claim 1-4, it is characterized in that: the culture condition of described liquid submerged fermentation is: culture temperature is 20-30 ℃, and the rotating speed that circles round is 80-180 rev/min.
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Cited By (3)
| 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 |
| CN107488624A (en) * | 2017-10-19 | 2017-12-19 | 青岛农业大学 | A kind of India truffle mycelia quick separating and cultural method |
| CN115443851A (en) * | 2022-08-18 | 2022-12-09 | 云南农业大学 | Application of V8 vegetable juice in culture of truffle mycelium, culture of truffle mycelium and synthesis method of truffle mycorrhiza |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1141392C (en) * | 2001-03-20 | 2004-03-10 | 华东理工大学 | Process for preparing ganoderic polyose and ganoderic acid by fermentation during which raw materials are supplemented |
| CN1986827B (en) * | 2006-12-27 | 2010-05-19 | 湖北工业大学 | Truffle polyose preparing process |
| CN100564510C (en) * | 2006-12-27 | 2009-12-02 | 湖北工业大学 | A kind of substratum of liquid deep fermentation for producing truffle polysaccharide |
| CN101153299B (en) * | 2007-08-31 | 2010-06-02 | 清华大学 | Method for high production of microorganism grease by stuffing batch culture |
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Cited By (5)
| 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 |
| CN102224873B (en) * | 2011-05-06 | 2012-12-05 | 郭景龙 | Method for preparing food from raw materials comprising coffee beans |
| CN107488624A (en) * | 2017-10-19 | 2017-12-19 | 青岛农业大学 | A kind of India truffle mycelia quick separating and cultural method |
| CN115443851A (en) * | 2022-08-18 | 2022-12-09 | 云南农业大学 | Application of V8 vegetable juice in culture of truffle mycelium, culture of truffle mycelium and synthesis method of truffle mycorrhiza |
| CN115443851B (en) * | 2022-08-18 | 2024-01-26 | 云南农业大学 | Application of V8 vegetable juice in culturing truffle mycelium, method for culturing truffle mycelium and synthesizing truffle mycorrhiza |
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