CN105695530A - Liquid fermentation medium for high yielding of Qaidam agaricus bitorguis polysaccharide and application - Google Patents
Liquid fermentation medium for high yielding of Qaidam agaricus bitorguis polysaccharide and application Download PDFInfo
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Abstract
The invention discloses a liquid fermentation medium for high yielding of Qaidam agaricus bitorguis polysaccharides and an application. The liquid fermentation medium is prepared from raw materials in percentage by mass as follows: 2.0%-4.5% of maltose, 0.4%-0.8% of peptone, 0.05%-0.25% of vitamin B complex, 0.005%-0.025% of calcium chloride, 0.4%-0.6% of a yeast extract, 3%-4% of fructose and the balance of water. With the adoption of the liquid fermentation medium, the production period of the Qaidam agaricus bitorguis polysaccharide can be shortened, the components and the content of the polysaccharides are relatively stable, main components are basically consistent, and the yield of the Qaidam agaricus bitorguis polysaccharides is high.
Description
Technical field
The present invention relates to a kind of liquid fermentation medium and application, be specifically related to liquid fermentation medium and the application of a kind of high yield Qaidam Agaricus bitorqui polysaccharide。
Background technology
Agaricus bitorqui [(SpringAgaricusAgaricusOrUrbanAgaric) Agaricusbitorguis]. belong to Agaricales Agaricus edibilis edible fungi, have another name called double-deck ring umbrella。Agaricus bitorqui bacteria cover diameter 6～20cm, first hemispherical, rear flat hemispherical, top horizontal or slightly recessed, color is white, and rear dimmed yellow, without scale；White in bacterium, abundant closely hinder slightly thin out redness, and time young, variable color is slower；Lamella white, after become pink to pitchy, dense, narrow, from life, Length discrepancy: stem is short, middle reality, closely cylindrical；Collarium is double-deck, white, and film quality is born in the middle part of stem。Spore dark brown, oval is to subsphaeroidal, smooth。Qaidam Agaricus bitorqui is a kind of raw bacterium of low form grass being born on meadow, and the polysaccharide in the Agaricus bitorqui of Qaidam has the biological activitys such as long aging, antitumor, antiviral, antiinflammatory and raising immunity, especially has the effect of anti-hypoxia。The areas such as Qinghai, Hebei, Xinjiang it are distributed in China, wherein, Qaidam Agaricus bitorqui is distributed mainly on Gobi desert, the Caidamu Basin, saline and alkaline sandy beach in Qinghai, special owing to being grown in, under ecological environmental condition, it has the features such as bacterial context close structure, water content be low, high resilience, resistance to accumulating, wide adaptability, strong stress resistance, is that a kind of edibility is significantly high, highly important Wild Edible Macrofungi of classifying。Therefore locals is commonly called as " sandy beach mushroom ", " royal agaric ", there are the features such as sporophore is very large, the plump delicacy of bacterial context, delicious flavour, low, the resistance to accumulating of water content。
Fungus polysaccharide is isolated metabolite from fungus sporophore, mycelium and fermentation liquid thereof, is the natural polymer polymer that is formed by connecting with glycosidic bond by the monosaccharide of more than 10 of a class。It has the triple helices conformation of β-(1 → 3)-D-glucosan mostly, thus has special biological activity, as controlled cell division differentiation, regulating Growth of Cells aging, antitumor, antiviral, antiinflammatory and raising immunity etc.。At present, polysaccharide and complex thereof to a large amount of originated from fungus, such as Agaricus Bisporus polysaccharide, lentinan, ganoderan, larynx granulose, grifolan, its traditional cultural method cycle is long, the polysaccharide therefrom extracted is far from meeting the market demand, and the polysaccharide component of every batch of product and content differ bigger。And the aboundresources of Qaidam Agaricus bitorqui, but owing to the place of production is remote, it is impossible to pluck on time, and the harvesting custom of peasants and herdsmen and fermentation level low etc., cause yielding poorly of Qaidam Agaricus bitorqui polysaccharide, limit the development of Qaidam Agaricus bitorqui polysaccharide industrialized production。Therefore; explore one can make that Qaidam Agaricus bitorqui polysaccharide is with short production cycle, polysaccharide component relative with content stable, Main Components is basically identical; and the liquid fermentation medium of high yield, for exploitation Qaidam Agaricus bitorqui polysaccharide scale and industrialized production, there is important using value。
Summary of the invention
The technical problem to be solved, it is provided that the liquid fermentation medium of a kind of high yield Qaidam Agaricus bitorqui polysaccharide and application。
This invention address that its technical problem employed technical scheme comprise that, a kind of liquid fermentation medium of high yield Qaidam Agaricus bitorqui polysaccharide, it is made up of the raw material of following mass fraction, 2.0～4.5% maltose (preferably 3.2%), 0.4～0.8% peptone (preferably 0.56%), 0.05～0.25% compound vitamin B (preferably 0.1%), 0.005～0.025% calcium chloride (preferably 0.011%), 0.4～0.6% yeast leaches cream (preferably 0.5%), 3～4% fructose (preferably 3.5%), and surplus is water。
The present invention solves its technical problem further and employed technical scheme comprise that, a kind of application of the liquid fermentation medium of high yield Qaidam Agaricus bitorqui polysaccharide, by Qaidam Agaricus bitorqui strain through slant activation, after liquid culture seed, then 6～16% liquid seeds are seeded on the liquid fermentation medium of described high yield Qaidam Agaricus bitorqui polysaccharide, condition of culture is fermentation temperature 11～21 DEG C (preferred fermentation temperature is 19 DEG C), initial ph value 5～9 (preferable ph is 6), shaking speed 110～170r/min (preferred shaking speed 130r/min), shaken cultivation 1～12 day, obtain Qaidam Agaricus bitorqui mycelium and the extracellular polysaccharide of Qaidam Agaricus bitorqui, finally separate according to a conventional method, extract the Qaidam extracellular polysaccharide of Agaricus bitorqui。
The liquid fermentation medium of the present invention can make that Qaidam Agaricus bitorqui polysaccharide is with short production cycle, polysaccharide component relative with content stable, Main Components is basically identical, gained Qaidam Agaricus bitorqui polysaccharide yield is high。It is demonstrated experimentally that adopt the liquid fermentation medium of the present invention that Qaidam Agaricus bitorqui polysaccharide can be made up to 2.67g/L。
Accompanying drawing explanation
Fig. 1 is glucose standard curve。
Fig. 2 is the different carbon source impact on hypha biomass and polyoses content。
Fig. 3 is the different nitrogen sources impact on mycelial biomass and polyoses content。
Fig. 4 is the impact on Biomass and polyoses content of the somatomedin kind。
Fig. 5 is the impact on Biomass and polyoses content of the trace element kind。
Fig. 6 is Qaidam Agaricus bitorqui growth curve。
Fig. 7 is the impact on mycelial biomass and polyoses content of different temperature。
Fig. 8 is the different pH impact on mycelial biomass and polyoses content。
Fig. 9 is the impact on mycelial biomass and polyoses content of different inoculum concentration。
Figure 10 is the impact on mycelial biomass and polyoses content of different shaking speed。
Figure 11 is each factor contour map to the extracellular polysaccharide reciprocal effect of Qaidam Agaricus bitorqui。Wherein, (a) is maltose and peptone reciprocal effect contour map, and (b) is the contour map of peptone and the mutual shadow contour map of calcium chloride, (c) maltose and peptone reciprocal effect Biomass。
Figure 12 is each factor contour map to Qaidam Agaricus bitorqui mycelial biomass reciprocal effect。Wherein, the contour map of (a) maltose and calcium chloride reciprocal effect Biomass, the contour map of (b) peptone and calcium chloride reciprocal effect Biomass, the contour map of (c) maltose and peptone reciprocal effect Biomass。
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the invention will be further described。
Embodiment 1: the preparation of the liquid fermentation medium of high yield Qaidam Agaricus bitorqui polysaccharide
By 32g maltose, 5.6g peptone, 1g compound vitamin B, 1.1g calcium chloride, 5g yeast leaches cream, 35g fructose, adds water to 1000ml。
Embodiment 2: the liquid fermentation method of Qaidam Agaricus bitorqui polysaccharide
1, seed slant activation: take a fritter thalline to PDA slant medium (in advance 15 days preparation) from Qaidam Agaricus bitorqui original seed, it is placed in incubator lucifuge and cultivates, adopting initial 48 hours cultivation temperature is 16 DEG C, cultivation temperature is the alternating temperature control of 18 DEG C afterwards, cultivate 12-15 days, activated inclined plane can be made standby。
2, prepared by liquid seeds: the liquid fermentation medium of high yield Qaidam Agaricus bitorqui polysaccharide embodiment 1 prepared, and liquid amount 100mL/250mL triangular flask is standby after sterilizing。Sterile working inoculation shovel 0.5-1cm2Size 2-3 truffle, in inoculation aforesaid liquid culture medium, 18 DEG C, 130r/min shaking table shaken cultivation 10～14 days, to growing more uniform mycelium pellet, obtain Qaidam Agaricus bitorqui liquid culture seed。
3, liquid fermentation and culture: inoculation 6～16% liquid seeds in the liquid fermentation medium of the high yield Qaidam Agaricus bitorqui polysaccharide of embodiment 1 preparation, condition of culture is fermentation temperature 11～21 DEG C, initial ph value 5～9, shaking speed 110～170r/min, shaken cultivation 1～12 day, obtain Qaidam Agaricus bitorqui mycelium and the extracellular polysaccharide of Qaidam Agaricus bitorqui, finally separate according to a conventional method, extract the Qaidam extracellular polysaccharide of Agaricus bitorqui。
4, assay method
(1) the mensuration ethanol precipitation of extracellular polysaccharide content
Take the ferment filtrate in 250mL conical flask, carry out precipitation process with 75% ethanol and (namely first measure ferment filtrate volume V1, then with formula: C*V2=75%* (V1+V2) calculate required ethanol volume, wherein C is concentration of alcohol；V2It is the ethanol volume needing to measure), seal followed by preservative film, put into 4 DEG C of refrigerators and stand 48h, then adopt dry weight method, first filter paper is put into and drying baker is dried to constant weight, duration used is 24h, and temperature is 60 DEG C。Take out every other day, claim dry weight successively and make marks, recording the data obtained W1, then the ferment filtrate after precipitate with ethanol is filtered, solid content (i.e. crude polysaccharides) is placed 24-48h in 60 DEG C of baking ovens together with filter paper and is dried to constant weight, and weigh after taking-up (W2), W2-W1Namely extracellular polysaccharide content is obtained。
(2) the mensuration Phenol sulfuric acid procedure of extracellular polysaccharide
1. the making of glucose standard curve
Accurately weigh standard glucose 10mg in 100mL volumetric flask, add distilled water to scale, draw 0.2,0.4,0.6,0.8,1.0,1.2,1.4 and 1.6mL liquid respectively, respectively mend to 2.0mL with distilled water, it is subsequently adding 6% phenol 0.5mL and 98% concentrated sulphuric acid 5.0mL, shaking up cooling, room temperature places 20min later in 490nm densitometric, using 2.0mL distilled water by same operation as blank, abscissa is polysaccharide concentration, vertical coordinate is optical density value, drawing standard curve, in Table 1 and Fig. 1。Gained equation of linear regression y=13.976x-0.0058, R2=0.9962。
Table 1 glucose standard curve makes
2. the mensuration of fermentation liquid extracellular polysaccharide content
Accurately measure fermentation liquid 1mL (extension rate of fermentation liquid carries out taking the circumstances into consideration to adjust for benchmark) in 100mL volumetric flask with the optical density value finally measured, add distilled water to scale, draw 2mL liquid in color comparison tube, it is subsequently adding 6% phenol 0.5mL and concentrated sulphuric acid 5.0mL, shakes up cooling room temperature and place 20min later in 490nm densitometric。Measure every time and take double; two sample comparison, calculate polyoses content with standard curve。Noting: determine the amount of sampling during mensuration according to optical density value, optical density value is preferably between 0.1-0.3。Such as: when can consider to take sample under less than 0.1, take 2mL, the sample liquid taking 0.5mL as reduced by half more than 0.3 measures。
Computing formula:
In formula: C-polysaccharide fermentation liquid cumulative volume (mL)；
N-diluted sample multiple；
A-calculates polyoses content (mg/mL) in survey timed samples according to standard curve；
The polysaccharide fermentation liquid volume (mL) drawn when V-measures。
Embodiment 3: the optimum experimental of the liquid fermentation of Qaidam Agaricus bitorqui polysaccharide
One, Orthogonal Experiment and Design
With inoculum concentration, incubation time, initial tri-condition of culture of pH, carry out L9(33) orthogonal test。Experimental design is shown in table 2 below。
Table 2-orthogonal test factor level table
Two, response surface experiments
According to single factor test result, selecting maltose, peptone, calcium chloride is investigation factor, adopts SAS central combination design, and the designed response surface tests, and the experiment factor and level are in Table 3。
Table 3-empirical factor level and coding
Three, experimental result and analysis
1, single factor experiment
(1) experiment of single factor of medium optimization
1. the different carbon source impact on mycelial biomass and extracellular polysaccharide content
Investigating its impact on the extracellular polysaccharide of Qaidam Agaricus bitorqui and mycelial biomass with 3.5% fructose, sucrose, maltose, lactose, starch, cellulose, Semen Tritici aestivi, wheat bran, glucose respectively, experimental result is shown in Fig. 2。
As shown in Figure 2, Qaidam Agaricus bitorqui may utilize the several kinds of carbon source such as monosaccharide, disaccharide and polysaccharide, during using maltose as carbon source, Qaidam Agaricus bitorqui mycelial biomass relative maximum is for 11.06g/L, extracellular polysaccharide content is 0.86g/L, during using fructose as carbon source, grassland one mycelial biomass is for 9.83g/L, and extracellular polysaccharide content is 0.82g/L。
Owing to the price of fructose is higher than maltose, it is unfavorable for industrialized great production。Considering Biomass and polyoses content and cost these three factor, selection maltose is optimum carbon source。
2. the different nitrogen sources impact on mycelial biomass and extracellular polysaccharide content
Investigating its impact on the extracellular polysaccharide of Qaidam Agaricus bitorqui and mycelial biomass with different nitrogen sources such as 0.5% ammonium oxalate, yeast extract, ammonium sulfate, ammonium nitrate, carbamide, peptones respectively, experimental result is shown in Fig. 3。
From the figure 3, it may be seen that during using peptone as nitrogenous source, Qaidam Agaricus bitorqui mycelial biomass 10.28g/L and polyoses content 0.90g/L, during using ammonium oxalate as nitrogenous source, though the highest 0.97g/L of polyoses content, but the relatively low only 3.31g/L of Biomass, therefore select peptone as optimum nitrogen source。
3. the different vitamin impacts on mycelial biomass and extracellular polysaccharide content
With 0.25% vitamin B1, vitamin B2, vitamin B12, vitamin C, the different vitamin such as compound vitamin B investigate it to the extracellular polysaccharide of Qaidam Agaricus bitorqui and the impact on mycelial biomass, and experimental result is shown in Fig. 4。
As shown in Figure 4, consider Biomass and polyoses content, when with compound vitamin B for somatomedin, Biomass 13.81g/L and polyoses content 0.96g/L, all reach a relatively high value, therefore, selecting compound vitamin B is the optimum growh factor。
4. the different trace element impacts on mycelial biomass and extracellular polysaccharide content
With 0.005% magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, aluminum sulfate, ferrous sulfate, zinc sulfate, manganese sulfate for trace element, investigate it to the extracellular polysaccharide in grassland one and the impact on mycelial biomass。Experimental result is shown in Fig. 5。
As shown in Figure 5, considering Biomass and polyoses content, when with calcium chloride for trace element, Biomass and polyoses content all reach a relatively high value, and therefore, selective chlorination calcium is best trace element。
5. the amount that carbon source the is different impact on mycelial biomass and extracellular polysaccharide content
The experiment F value=6.387 > F of the not commensurability screening of carbon source0.01(4.46), illustrating that the difference between the effects of polyoses content is extremely significant by the amount that carbon source is different, LSD method carries out multiple comparisons, and experimental result is in Table 4。
The significance of difference that affects of mycelial biomass and polyoses content is compared (LSD method) by the amount that table 4-carbon source is different
| Maltose (%) | 4.5 | 4.0 | 3.5 | 3.0 | 2.5 | 2.0 |
| Biomass (g/L) | 8.35cdC | 11.66aA | 9.32bB | 8.60cBc | 8.15cdC | 8.13dC |
| Polyoses content (g/L) | 0.89bB | 0.98cC | 0.91bB | 1.12aA | 0.72bB | 0.62bB |
Note: lower case, capitalization represent the significant difference in 0.05,0.01 level respectively, table 4-is same to table 8。
As shown in Table 4, maltose is conducive to the optimum dosage 4% of mycelial growth, is conducive to the optimum dosage 3% of extracellular polysaccharide, considers and determines that the suitableeest dosage of maltose is 3.5%。
6. the amount that nitrogenous source the is different impact on mycelial biomass and polyoses content
The experiment F value=119.037 > F of the not commensurability screening of nitrogenous source0.01(7.023), illustrate that the difference between the effects of mycelial biomass is extremely significant by the different amount of nitrogenous source；F value=6.784 > F0.01(1.215), illustrate that the difference between the effects of polyoses content is extremely significant by the different amount of nitrogenous source。LSD method carries out multiple comparisons, and experimental result is in Table 5。
The significant difference that affects of mycelial biomass and polyoses content is compared (LSD method) by the different amount of table 5-nitrogenous source
| Peptone (%) | 0.8 | 0.7 | 0.6 | 0.5 | 0.4 |
| Biomass (g/L) | 10.82bAB | 11.21abA | 9.56aA | 7.89abA | 6.18cB |
| Polyoses content (g/L) | 0.84bB | 0.94bB | 0.98aA | 0.86bB | 0.70bB |
As shown in Table 5, peptone is conducive to mycelial growth optimum dosage to be 0.7%, and being conducive to the optimum dosage producing polysaccharide is 0.6%, it is thus determined that the optimum add amount of peptone is 0.6%。
7. the amount that somatomedin the is different impact on mycelial biomass and extracellular polysaccharide content
The experiment F value=5.436 > F of the not commensurability screening of somatomedin0.01(0.523), illustrate that the difference between the effects of mycelial biomass is extremely significant by the different amount of somatomedin；F value=5.436 > F0.01(0.523), illustrating that the difference between the effects of polyoses content is extremely significant by the different amount of somatomedin, LSD method carries out multiple comparisons, and experimental result is in Table 6。
Mycelial biomass is affected significant difference and compares (LSD method) by the different amount of table 6-somatomedin
| Compound vitamin B (%) | 0.25 | 0.2 | 0.15 | 0.1 | 0.05 |
| Biomass (g/L) | 12.8bAb | 13.00abA | 14.51aA | 13.40abA | 12.35cB |
| Polyoses content (g/L) | 0.73bAB | 0.82abA | 0.98abA | 1.24aA | 0.72cB |
As shown in Table 6, compound vitamin B is conducive to mycelial growth optimum dosage to be 0.15%, and being conducive to the optimum dosage producing polysaccharide is 0.1%。
8. the amount that trace element the is different impact on mycelial biomass and extracellular polysaccharide content
The experiment F value=67.923 > F of the not commensurability screening of trace element0.01(4.25), illustrate that the difference between the effects to mycelial biomass of measuring that carbon source is different is extremely significant；F value=5.436 > F0.01(0.523), illustrate that the difference between the effects to polyoses content of measuring that carbon source is different is extremely significant。LSD method carries out multiple comparisons, and experimental result is in Table 7。
Mycelial biomass is affected significant difference and compares (LSD method) by the different amount of table 7-trace element
| Calcium chloride (%) | 0.025 | 0.02 | 0.015 | 0.01 | 0.005 |
| Biomass (g/lL) | 10.56bB | 9.79bB | 14.94aA | 13.66bB | 11.29bB |
| Polyoses content (g/L) | 0.935bB | 0.99bB | 0.97bB | 1.46 | 0.03bB |
As shown in Table 7, calcium chloride is conducive to mycelial growth optimum dosage to be 0.015%, and being conducive to the optimum dosage producing polysaccharide is 0.01%, it is contemplated that to obtain the main target of polysaccharide, it is determined that the suitableeest addition of calcium chloride is 0.01%。
(2) experiment of single factor of fermentation condition optimization
1. the impact on mycelial biomass and extracellular polysaccharide content of the different fermentation times
Experimental result is shown in Fig. 6, it will be appreciated from fig. 6 that along with longer fermentation times, hypha biomass and polyoses content all increase, within the 5th day, polyoses content reaches maximum (1.54g/L)；Within 6th day, mycelial biomass reaches maximum (12.01g/L)。Owing to the present invention mainly attaches most importance to inspection target with polyoses content, it is thus determined that best fermentation time is 5 days。
2. the impact on mycelial biomass and extracellular polysaccharide content of the different temperature
Qaidam Agaricus bitorqui is different to the Utilization ability of nutrient substance in the environment that temperature is different, thus causing that mycelium upgrowth situation in the different amounts of culture medium of trace element is different, thus affecting its Biomass and yield of extracellular polysaccharide further, experimental result is shown in Fig. 7。
As shown in Figure 7, when temperature is 15 DEG C, mycelial biomass (12.59g/L) and extracellular polysaccharide content (1.24g/L) all reach peak, it is thus determined that 15 DEG C is optimum fermentation temperature。
3. the different pH impact on mycelial biomass and extracellular polysaccharide content
Qaidam Agaricus bitorqui is different to the Utilization ability of nutrient substance in the environment that pH is different, thus causing that mycelium upgrowth situation in the different amounts of culture medium of trace element is different, thus affecting its Biomass and yield of extracellular polysaccharide further, experimental result is shown in Fig. 8。
As shown in Figure 8, when initial pH is 6, extracellular polysaccharide content 1.76g/L reaches the highest, and mycelial biomass 13.91g/L is also higher, it is thus determined that initial pH6 is best。
4. the impact on mycelial biomass and extracellular polysaccharide content of the different inoculum concentrations
Respectively with inoculum concentration 6%, 8%, 10%, 12%, 14%, 16%, investigate its impact on the extracellular polysaccharide of Qaidam Agaricus bitorqui and mycelial biomass, result is shown in Fig. 9。
As shown in Figure 9, when inoculum concentration is 10%, polyoses content and Biomass all reach peak, therefore choose the optimal value that inoculum concentration 10% is fermentation culture conditions inoculum concentration。
5. the impact on mycelial biomass and polyoses content of the different shaking speed
Investigating the impact on the extracellular polysaccharide of Qaidam Agaricus bitorqui and mycelial biomass with 110r/min, 130r/min, 150r/min, 170r/min rotating speed respectively, experimental result is shown in Figure 10。
As shown in Figure 10, when rotating speed is 130r/min, polyoses content and Biomass all reach peak, therefore choose the optimal value that rotating speed 130r/min is fermentation culture conditions rotating speed。
2, orthogonal test
According to experiment of single factor result, with initial pH, inoculum concentration, fermentation temperature for factor, with extracellular polysaccharide content for index, carrying out the orthogonal test of Three factors-levels, experimental result is in Table 8。
The orthogonal experiments of polysaccharide training systern is produced in the Agaricus bitorqui fermentation of table 8-Qaidam
By table 8 extreme difference it can be seen that the influence degree of Qaidam Agaricus bitorqui polyoses content is R by each factorA> RC> RB, illustrate that pH factor is the most notable on the impact of experiment, what obtain optimal culture condition is combined as A2B2C3, optimal culture condition is: initial ph value 6, and inoculum concentration is 12%, and temperature is 19 DEG C；It is R to Qaidam Agaricus bitorqui Biomass influence degreeB> RC> RAIllustrate that inoculum concentration is the most notable on the impact of test, obtain best cultivation and be combined as A1B2C2, it is determined that optimal culture condition is: pH value is 5, and temperature is 17 DEG C, and inoculum concentration is 12%。
3, response surface optimization test
(1) polysaccharide response surface optimization
From single factor experiment, different fermentations culture medium composition is different on the impact of Qaidam Agaricus bitorqui polyoses content, in order to obtain the formula of optimal medium, respond interview to test, altogether devise 17 experiments, 4th, 6,11,12,15 experiments are the center experimental point repeated, and experimental result is in Table 9。
Table 9-polysaccharide response surface experiments result
| Maltose | Peptone | Calcium chloride | Polyoses content (g/L) | |
| 1 | 2.5 | 0.8 | 0.01 | 1.85 |
| 2 | 4.5 | 0.8 | 0.01 | 1.98 |
| 3 | 1.5 | 0.6 | 0.005 | 1.92 |
| 4 | 3.5 | 0.6 | 0.01 | 2.54 |
| 5 | 2.5 | 0.4 | 0.01 | 2.03 |
| 6 | 3.5 | 0.6 | 0.01 | 2.47 |
| 7 | 4.5 | 0.6 | 0.015 | 2.07 |
| 8 | 3.5 | 0.8 | 0.015 | 1.97 |
| 9 | 3.5 | 0.8 | 0.005 | 2.01 |
| 10 | 4.5 | 0.4 | 0.01 | 2.17 |
| 11 | 3.5 | 0.6 | 0.01 | 2.49 |
| 12 | 3.5 | 0.6 | 0.01 | 2.51 |
| 13 | 3.5 | 0.4 | 0.005 | 2.17 |
| 14 | 3.5 | 0.4 | 0.015 | 2.10 |
| 15 | 3.5 | 0.6 | 0.01 | 2.46 |
| 16 | 2.5 | 0.6 | 0.015 | 2.01 |
| 17 | 4.5 | 0.6 | 0.005 | 2.17 |
The variance analysis of table 10-polysaccharide regression equation model
| Source | Quadratic sum | Degree of freedom | Mean square | F value | P value | Significance |
| Model | 0.729249118 | 9 | 0.08102768 | 28.58119215 | 0.0001 | significant |
| A | 0.0435125 | 1 | 0.0435125 | 15.34832451 | 0.0058 | ** |
| B | 0.05445 | 1 | 0.05445 | 19.20634921 | 0.0032 | ** |
| C | 0.0021125 | 1 | 0.0021125 | 0.745149912 | 0.4166 | |
| AB | 0.231537895 | 1 | 0.231537895 | 81.67121507 | < 0.0001 | ** |
| AC | 0.189237895 | 1 | 0.189237895 | 66.75058015 | < 0.0001 | ** |
| BC | 0.135664211 | 1 | 0.135664211 | 47.85333705 | 0.0002 | ** |
| Correlation coefficient (R2) | 0.973508002 |
Note: * * represents that difference is extremely notable；* significant difference is represented
Carrying out significance analysis according to table 10, model equation extremely notable (P=0.001), this other factor B has appreciable impact (0.01 < P≤0.05)。It is carried out regression fit analysis, the binary regression equation of corresponding independent variable maltose (A) peptone (B) of extracellular polysaccharide predictive value (Y) and calcium chloride (C):
Y (g/L)=1.454+0.07375A-0.0825B-0.01625C-0.2345A2-0.212B2-0.1795C2-0.0025AB-0.045AC+0.0075BC
The determination coefficients R of model2=0.9735, illustrate that this equation can explain the transmutability of this model data to a certain extent (more than 97%), according to the response surface of regression equation drafting as shown in figure 11。Through SAS software analysis it can be seen that Qaidam Agaricus bitorqui fermentation polysaccharides medium optimization optimum combination is: maltose 3.2%, peptone 0.56%, calcium chloride 0.014%, with this understanding, polysaccharide predicted maximum is 2.67g/L。
(2) mycelial biomass response surface optimization
By single factor experiment it can be seen that different fermentations culture medium forms the difference of the impact on Qaidam Agaricus bitorqui polyoses content, in order to obtain the formula of optimal medium, respond interview to test, altogether devising 17 experiments, the 4th, 6,11,12,15 experiments are the center experiment repeated, and result is in Table 11。
Table 11-mycelial biomass response surface experiments result
The variance analysis of table 12-mycelial biomass regression equation model
Significance analysis according to table 12, the synthesis of mycelial biomass is had pole appreciable impact (P < 0.01) by the first order of B factor, and the binary regression equation of the corresponding independent variable maltose (A) of mycelial biomass predictive value (Y), peptone (B) and calcium chloride (C) is: Y (g/L)=7.24+0.975A+0.325B-0.825C+0.905A2+1.555B2-1.595C2+0.9AB-0.8AC+0.2BC
The determination coefficients R of model2=0.92, illustrate that this equation can explain the transmutability of this model data to a certain extent (more than 92%), according to response surface such as Figure 12 that regression equation is drawn。Through SAS software analysis it can be seen that the optimum combination of Qaidam Agaricus bitorqui mycelial biomass culture medium is: maltose 4.5%, peptone 0.66%, calcium chloride 0.011%, with this understanding, mycelial biomass predicted maximum is 17.28g/L。
(3) response surface confirmatory experiment
For checking the reliability of response phase method acquired results, doing 3 parallel tests with the optimal medium that the response surface obtains, be comparison with the initial culture medium being not optimised, inoculating 12% Qaidam Agaricus bitorqui seed liquor respectively ferments, utilizing LSD method to carry out multiple comparisons, experimental result is in Table 13-14。
The multiple comparisons (LSD method) that mycelial biomass is affected by table 13-Optimal Medium and initial medium
Note: * represents that significant difference, * * represent that difference is extremely notable
The multiple comparisons (LSD method) that polyoses content is affected by table 14-Optimal Medium and initial medium
Note: * represents that significant difference, * * represent that difference is extremely notable
By table 13 and table 14 it can be seen that hypha biomass is 17.63g/L, polyoses content is 2.56g/L, has been respectively increased 43.11% and 22.30% than comparison (hypha biomass is 11.45g/L, and polyoses content is 1.67g/L)。
Utilize LSD method that experimental data is carried out multiple comparisons, it is shown that A1And A2Between relatively reached difference pole significance degree, illustrate Qaidam Agaricus bitorqui mycelial biomass optimal medium formula relatively initial incubation based formulas on Biomass impact bigger；The extracellular polysaccharide optimal medium formula relatively initial incubation based formulas of Qaidam Agaricus bitorqui is on polysaccharide yield impact greatly。
Claims (4)
1. the liquid fermentation medium of a high yield Qaidam Agaricus bitorqui polysaccharide, it is characterized in that, it is made up of the raw material of following mass fraction, 2.0～4.5% maltose, 0.4～0.8% peptone, 0.05～0.25% compound vitamin B, 0.005～0.025% calcium chloride, 0.4～0.6% yeast leaches cream, 3～4% fructose, and surplus is water。
2. the liquid fermentation medium of high yield Qaidam according to claim 1 Agaricus bitorqui polysaccharide, it is characterized in that, it is made up of the raw material of following mass fraction, 3.2% maltose, 0.56% peptone, 0.1% compound vitamin B, 0.014% calcium chloride, 0.5% yeast leaches cream, 3.5% fructose, and surplus is water。
3. the application of the liquid fermentation medium of high yield Qaidam according to claim 1 and 2 Agaricus bitorqui polysaccharide, it is characterized in that, by Qaidam Agaricus bitorqui strain through slant activation, after liquid culture seed, then 6～16% liquid seeds are seeded on the liquid fermentation medium of described high yield Qaidam Agaricus bitorqui polysaccharide, condition of culture is fermentation temperature 11～21 DEG C, initial ph value 5～9, shaking speed 110～170r/min, shaken cultivation 1～12 day, obtain Qaidam Agaricus bitorqui mycelium and the extracellular polysaccharide of Qaidam Agaricus bitorqui, finally separate according to a conventional method, extract the Qaidam extracellular polysaccharide of Agaricus bitorqui。
4. the application of the liquid fermentation medium of high yield Qaidam according to claim 1 Agaricus bitorqui polysaccharide, it is characterised in that described condition of culture is fermentation temperature 19 DEG C, initial ph value 6, shaking speed 130r/min；Described inoculum concentration is 12%。
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| CN105961548A (en) * | 2016-06-24 | 2016-09-28 | 青海大学 | Making technology of agaricus bitorguis biscuits |
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| CN109937794A (en) * | 2019-03-26 | 2019-06-28 | 阜阳职业技术学院 | A kind of big fat mushroom cultural hypha base and its preparation method and application |
| CN112791095A (en) * | 2021-01-22 | 2021-05-14 | 青海伊纳维康生物科技有限公司 | Application of intracellular polysaccharide of pleurotus ferulae mycelium in preparation of immunoregulation medicine, health-care product or food |
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