AU2013248826A1 - Liquid fermentation method for increasing yield of Cordyceps polysaccharide by expansin - Google Patents

Abstract

Provided is a liquid fermentation method for increasing the yield of

Description

1 Description Liquid fermentation method for increasing yield of Cordyceps polysaccharide by expansin 5 Technical Field The present invention relates to a liquid fermentation method for increasing the yield of Cordycepic polysaccharide by an expansin, which belongs to biotechnology fermentation engineering field. Background 10 Cordyceps militaris (Cordyceps militaris) is a pattern stain belonging to Ascomycota subphylum (Ascomycota), Hypocreales order (Hypocreales), Clavicipitaceae family (Clavicipitaceae), Cordyceps genus (Cordyceps). Its scientific name is Cordyceps militaris, and it is also named North Cordyceps militaris, North Cordyceps sinensis, Cordyceps, etc. It comprises of stroma (the part of grass) and sclerotium (the part of worm's body). Cordyceps militaris is worldwide distribution but the natural resources are few in number. The 15 traditional Chinese doctor thought, Cordyceps militaris can infiltrate into the lung and kidney channels, supply the Yin of lung and the Yang of kidney, mainly treat the kidney deficiency, waist and knee paining, vacuity following illness, bloody phlegm caused by chronic cough, perspire during sleep and so on, and is the only one traditional Chinese medicine that can balance and regulate Yin and Yang. Abundant modern pharmacological researches indicated that Cordyceps militaris not only has special nutritional value, but also 20 possess notable medicinal value. The cordyceps polysaccharide thereof is one of the most abundant and most important biological active substances in Cordyceps militaris. It has been widely noticed because it functions in a wide range of pharmacological effects such as antitumor, antioxidant, antiradiation, improving the body's immune function, promoting adrenal function, increasing the insulin secretion in the human body, and reducing blood sugar levels in diabetics. The cordyceps polysaccharide is a highly branched galactomannan, 25 and it can activate the macrophages to stimulate the antibody production, promote lymphocyte transformation, increase the content of serum IgG antibody, improve the body's immune function and enhance the body's capacity on antitumor. Moreover, it has been used in treating malignant tumor clinically. In addition, the cordyceps polysaccharide also can improve the respiratory system, anti-arrhythmia, anti-myocardial ischemia, expand peripheral vascular, lower the blood pressure and blood fat, inhibit platelet aggregation and so on. 30 Currently the demands of Cordyceps militaris and cordycepic polysaccharide are rapidly growing. Cordycepic polysaccharide of the wild Cordyceps militaris is low. Furthermore, crazy picking of the wild Cordyceps makes it increasingly scarce and expensive. So the resource of material constrains severely the application scope of Cordyceps militaris polysaccharide. The technics of chemosynthesis method is too complex, and the yield is very poor. The method using liquid fermentation has many advantages such as short producing period, 35 economic work force, etc. And studies have shown that cordycepic polysaccharide obtained from Cordyceps militaris fruiting body, mycelium and fermentation broth have basically the same biochemical structure. So it is considered that the fermentation method is an effective substitute method for extracting polysaccharide 2 from wild Cordyceps militaris. However, the fermentation level of Cordyceps militaris polysaccharide is low and the yield is poor, which constrains the development of industrial production. Compared with many studies in the epiphyte polysaccharide field, the research reports of Cordyceps militaris polysaccharide at home and abroad is few. These reports are mostly developing in the recent several years, which focus basically on the 5 technical aspects such as fermentation, product extract and separation, etc. Chinese patent CN101067005 (application No. 200710052357) discloses a process of producing cordycepic polysaccharide with rice, which includes the steps of compounding material, inoculating, culturing, harvesting cordycepic polysaccharide, radiation to sterilize and packing. The material includes rice 45.0-49.0 wt%, mixed corn and bean cake powder 0.9-2 wt %, and nutrient solution 49.0-54.1 wt%; and the 10 nutrient solution is compounded with potato 180~220g, sugar 18~22g, peptone 13~18g, potassium dihydrogen phosphate 1.5~2.2g, magnesium sulfate 0.8~1.2g, and water 900~ 1100g. However, there is no research analyzing based on the angle of the biological metabolism of Cordyceps militaris polysaccharides at present, therefore it is limited in improving the fermentation level of cordycepic militaris polysaccharide. The fermentation period is long, the price is high, and the overall producing efficiency is low, which make it far to 15 meet the need of modern industrial fermentation production. Expansin is a new type of protein species found in plant cell walls in the recent years. Expansin is first obtained by separating and purifying the elongation field of cucumber hypocotyls. It has been proved that expansin also exists in the cell walls of oat coleoptile, Trichosanthes kirilowii root tip, tomato, strawberry, arabidopsis, paddy, cotton fibrin, corn, soybean, etc, and is considered existing widely in the cell walls of 20 various dicotyledonous and monocotyledonous plant. It is considered that expansin is related to promoting the cell physiological growth, affecting the physiological growth processes such as vegetative growth, morphogenesis, pollination and fertilization, and fruit softening, etc. Experiments about cell wall recombination have shown that expansin has the function of recovering the thermal inactivated cell wall in vitro to extend, which is different with other enzyme proteins for cell wall found formerly. It is supposed that 25 expansin can regulate physiological activities such as acid-dependent cell wall extension and stress relaxation by breaking the hydrogen bonds between the cell wall polymers, and may be an important regulatory factor for physiological regulation and cell wall extension process in the period of plant growth. However, the function mechanism of expansin is mostly speculated and supposed at precent. There is yet no unambiguous study conclusion and mechanism illumination at home and abroad. Currently it is not reported at home and 30 abroad that the expansin is applied in liquid fermentation of Cordyceps militaris for increasing the yield of secondary metabolites such as cordycepic polysaccharide. Summary of The Invention In view of the defects of the prior art, the present invention aim to provide a liquid fermentation method 35 for increasing the yield of Cordyceps polysaccharide by an expansin. The technical scheme of the present invention is as follows. A liquid fermentation method for increasing the yield of Cordyceps polysaccharide by an expansin, comprises the following steps of: 3 (1) inoculating Cordyceps militaris strains into the PD liquid fermentation medium to perform activation culture, and transferring to a seed fermentation medium to a volume ratio of 10-15% to perform seed culture in the swing bed at 20-25 C for 2-4 days so as to obtain a seed liquid; (2) inoculating the seed liquid obtained in step (1) into a liquid fermentation medium to a volume ratio of 5 5-10% to perform liquid fermentation culture at 20-25 C for 1-6 days, then adding an expansin solution to a concentration of 0.01-0.85 mg/mL to perform further culture for 2-9 days, and separating to obtain the mycelium of Cordyceps militaris and the fermentation broth; and (3) extracting the exopolysaccharide of Cordyceps militaris from the fermentation broth obtained in the step (2), extracting the intracellular polysaccharide from the Cordyceps militaris mycelium obtained in the 10 step (2), and mixing the exopolysaccharide and the intracellular polysaccharide so as to obtain Cordyceps polysaccharide. Preferably, according to the invention, each litre of the PD liquid fermentation medium in the step (1) comprises the following components: potato 200g, glucose 20g, diluted to 1000 mL by distilled water. 15 According to the invention, the preferred activation culture in the step (1) is processed in the darkroom with a shaking speed of 100 - 160 r/min at 20 - 25C for 3-5 days. Preferably, according to the invention, each litre of the seed fermentation medium in the step (1) comprises the following components: glucose 3g, yeast extract supernatant 1.5g, MgSO 4 0.1g, CaCl 2 0.03g, KH 2

PO

4 0.1g, K 2

HPO

4 0.1g, 2-5 20 pieces of glass beads with 3-6 mm diameter. After cultured in the seed fermentation medium, the amount of mycelium pellet may increase more than 60%, and the diameter of mycelium pellet may reduce more than 40%. The mycelium is loose and uniform. Preferably, according to the invention, each litre of the liquid fermentation medium in the step (2) comprises the following components: 25 lactose 3g, molasses 2g, soybean powder 2g, peptone 1.5g, MgSO 4 0.1g, CaCl 2 0.03g, KH 2

PO

4 0.1g,

K

2

HPO

4 0.1g. Preferably, according to the invention, the expansin concentration in the step (2) is 0.15-0.7 mg/mL; further preferably, 0.25-0.5 mg/mL; most preferably, the expansin concentration in the step (2) is 0.5 mg/mL. The preparation of the expansin solution in the step (2) may refer to the prior technique, such as the 30 method described in "Two endogenous proteins that induce cell wall extension in plants. McQueen-Mason et al. Plant Cell , 1992, 4: 1425-1433. McQueen-Mason S J, Durachko D M, Cosgrove D J". The expansin solution in step (2) may also be prepared by the method which comprises the following steps of: sterilizing a broad bean or cucumber seed for 4-6 minutes with 0.05-0.15wt.% mercury chloride (HgCl 2 ), washing with running water for 5-7 hours, culturing in the darkroom for 4-6 days at 15-28"C, 35 taking 3-4 cm of seedling hypocotyl apices, precooling for 0.5 hours at -20 C, adding a homogenate buffer solution that is pre-cooled to 4"C, filtering with a nylon net having an aperture of 70gm after homogenate, washing the filter residue with a homogenate buffer solution, adding the filter residue in the homogenate 4 buffer solution, settling for 1-3 hours to obtain a settled solution, adding an extracting solution in the settled solution, extracting for 44-50 hours at 4 C, slowly adding 0.3-0.5g/mL ammonium sulfate ((NH4) 2

SO

4 ) in the filtrate while stirring to prevent a partial supersaturation of the (NH4) 2

SO

4 , settling for 45-50 hours, centrifuging for 5-10 minutes at 4 C, dissolving the sediment with an acid buffer solution, dialyzing in a 5 dialysis bag with a molecular weight of 3000 Da at 4"C, centrifuging the dialyzate at 20000g for 10 minutes, and taking the supernatant so as to obtain the expansin solution. In the above preparation method of the expansin solution, the homogenate buffer solution has a pH of 7.0, and comprises 25 mmol/L HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 1.5 mmol/L Na 2

S

2 0 5 , 2 mmol/L EDTA and 0.1 wt.% Triton X-100. The extracting solution has a pH of 6.0, and comprises 10 15 mmol/L 4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid, 1.0 mmol/L EDTA, 1.5 mmol/L Na 2

S

2 0 5 and 0.5 mmol/L NaCl. The acid buffer solution is prepared by dissolving 2.05g of sodium acetate in water, adjusting pH to 4.0 with glacial acetic acid, and adding water to IL. Preferably, according to the invention, the separation in the step (2) is carried out by centrifuging with a rotate speed of 15000 r/min at 4 0 C for 5~ 10 minutes. 15 Preferably, according to the invention, the extraction method of exopolysaccharide of Cordyceps militaris in the step (3) comprises the following steps of: adding 4 times volume of 95% ethanol to the fermentation broth obtained in the step (2), mixing to uniform and static settling for 5 hours at 4 C, 12000 rpm centrifuging for 10 minutes, discarding the supernatant, dissolving the precipitate into 6iL of IM NaOH solution at 60'C for 1 hour so as to obtain 20 Cordyceps militaris extracellular polysaccharide. Preferably, according to the invention, the extraction method of intracellular polysaccharide of Cordyceps militaris in the step (3) comprises the following steps of: drying Cordyceps militaris mycelium obtained in the step (2) at 65 C and grinding to power, adding 6mL of IM NaOH solution per gram of Cordyceps militaris mycelium powder, extracting at 60'C for 2 hours, 25 then centrifuging at 12000 rpm for 5 minutes and separating supernatant so as to obtain Cordyceps militaris intracellular polysaccharide. Compared with the prior art, the present invention has the following advantages: 1. The expansin is applied in the method of the invention for producing Cordecepic polysaccharide of Cordyceps militaris by liquid fermentation, therefore the yield of the intracellular polysaccharide may achieve 30 1.73 g/L and the yield of the exopolysaccharide 7.83 g/L, which is respectively raised for more than 1.5 times and 6.9 times compared with the prior art. And the yield of Cordyceps polysaccharide of the whole Cordyceps militaris can be increased significantly. So the method in the invention has excellent prospects for industrial application. 2. The method of the invention adopts liquid aerobic fermentation method, which has generally a 35 fermentation time of 8-10 days. Compared to the prior art, it has advantages of high-yield, short-cycle, high production efficiency, and dispensing with static culture and inducing synthetic products processes. The generative polysaccharide of Cordyceps militaris can be directly used in the preparation of drugs for 5 regulating immunity, antitumor and reducing blood sugar, etc. 3. The expansin mentioned in the invention can be extracted from most dicotyledonous and monocotyledonous plants, which is widely-available and low-cost. The preferred method of the invention is relatively simple, so it can be applied for scale extraction production and also has a good effect on promoting 5 and upgrading the active substance production of Cordyceps militaris such as Cordycepic polysaccharide. 4. The liquid fermentation method of Cordyceps militaris in the invention is simple, environment friendly, non-toxic and low raw material costs. The whole fermentation process is easily controlled and not restricted by external environmental conditions, so it is suitable for production in industrial-scale and popularization. 10 5. The method in the invention optimizes the formulation of the seed fermentation medium and liquid fermentation medium, and increases significantly the yeild of Cordycepic polysaccharide of Cordyceps militaris. Figure Description 15 Figure 1 is the curve of the different concentrations of expansin solution effect on the output of the exopolysaccharide and the intracellular polysaccharide of Cordyceps militaris. Embodiment The following is the detail description of the present invention with reference to examples, but the scope 20 of the present invention is not limited thereto. Raw Materials and Medium Cordyceps chrysalides (Cordyceps militaris) fermentation strains described in Examples, with Culture Collection Number of CGMCC No.5.699 and CGMCC No. 3.4655, were purchased from China General Microbiological Culture Collection Center. 25 The expansin solution of examples can be prepared by the method which comprises the following steps of: sterilizing the soybean (Glycine max L. Merr. CV. M40; purchased from Jinan Weili Seed Industry Co.,Ltd) or cucumber seed (Cucumis sativus L. CV Jinnian No 6; purchased from Jinan Weili Seed Industry Co., Ltd.) with 0.1 wt% HgCl 2 for 5 minutes, washing with running water for 6 h, planting in wet vermiculite, 30 dark culturing for 4 days at 27C, taking 3-4 cm of seedling hypocotyl apices, e.g. growing area that about 100g, setting at -20'C for 0.5 hour to pre-cool, adding homogenate buffer solution that pre-cooled to 4 C, filtering with a nylon net having an aperture of 70 gm after high speed dividing, washing the filter residue with homogenate buffer solution, adding the filter residue in the homogenate buffer solution, settling for 2 hours to obtain a settled solution, adding an extracting solution in the settled solution, extracting for 48 hours 35 at 4 C, slowly adding 0.4 g/mL ammonium sulfate ((NH4)2sO 4 ) in the filtrate while stirring to prevent a partial supersaturation of the (NH4) 2

SO

4 , settling for 28 hours, centrifuging at 25000g at 4 C for 10 minutes, dissolving the sediment with an acid buffer solution, dialyzing in a polyvinylidene fluoride (PVDF) dialysis 6 bag (purchased from Beijing Lubrizol Wright Science and Technology Co., Ltd.) with a molecular weight of 3000 Da at 4"C, centrifuging at 20000g the dialyzate for 10 minutes, taking the supernatant so as to obtain the expansin solution and reserving at 4 C. Other steps that are not described can consult the descriptions in "Two endogenous proteins that induce cell wall extension in plants. McQueen-Mason et al. Plant Cell, 1992, 5 4: 1425-1433. McQueen-Mason S J, Durachko D M, Cosgrove D J". The homogenate buffer solution mentioned as above has a pH of 7.0, and comprises 25 mmol/L HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 1.5 mmol/L Na 2

S

2 0 5 , 2 mmol/L EDTA and 0.1 wt.% Triton X-100. The extracting solution has a pH of 6.0, and comprises 15 mmol/L 4-(2-hydroxyethyl)-1 10 piperazineethanesulfonic acid, 1.0 mmol/L EDTA, 1.5 mmol/L Na 2

S

2 0 5 and 0.5 mmol/L NaCl. The acid buffer solution is prepared by dissolving 2.05g of sodium acetate in water, adjusting pH to 4.0 with glacial acetic acid, and adding water to 1 L. The concentration determination of the expansin solution can employ Coomassie brilliant blue method, which may specifically refer to the operations of Coomassie blue method described in "Quintessence of 15 Protein Science Laboratory Manual" (ISBN: 703018086, publication date: 1900-1-1), by using bovine serum albumin as the standard curve. The expansin concentration of the expansin solution detected by the above method and the result is 0.27 g/mL. Each litre of the PD liquid fermentation medium described in examples comprises the following components: potato 200g, glucose 20g, dilute to 1000 ml with distilled water. 20 Each litre of the seed fermentation medium described in examples comprises the following components: glucose 3g, yeast extract supernatant 1.5g, MgSO 4 0.1g, CaCl 2 0.03g, KH 2

PO

4 0.1g, K 2

HPO

4 0.1g, 2-5 pellets of 3-6 mm diameter glass beads. Each litre of the liquid fermentation medium described in Examples comprise the following components: lactose 3g, molasses 2g, soybean powder 2g, peptone 1.5g, MgSO 4 0.1g, CaCl 2 0.03g, KH 2

PO

4 0.1g, K 2

HPO

4 25 0.1g/L. Example 1 A liquid fermentation method for increasing the yield of Cordycepic polysaccharide of Cordyceps militaris by an expansin, comprises the following steps of: (1) inoculating Cordyceps chrysalis (Cordyceps militaris) strains which has a strain number of CGMCC 30 No.5.699 into the PD liquid fermentation medium to perform activation culture in the darkroom with a shaking speed of 150 r/min at 25 C for 96 hours, and transferring to a seed fermentation medium to a volume ratio of 15% to perform seed culture in the swing bed at 25C for 3 days so as to obtain a seed liquid; (2) inoculating the seed liquid obtained in step (1) into a liquid fermentation medium to a volume ratio of 10% to perform liquid fermentation culture at 20-25C for 1 days, then adding an expansin solution to a 35 concentration of 0.15 mg/mL to perform further culture for 9 days, centrifuging at 15000 r/min for 10 minutes, separating to obtain the mycelium of Cordyceps militaris and the fermentation broth; and (3) extracting the exopolysaccharide of Cordyceps militaris from the fermentation broth obtained in the 7 step (2), extracting the intracellular polysaccharide from the Cordyceps militaris mycelium obtained in the step (2), and mixing the exopolysaccharide and the intracellular polysaccharide so as to obtain Cordyceps polysaccharide. The extraction method of exopolysaccharide of Cordyceps militaris mentioned as above comprises the 5 following steps of: adding 200mL 95% ethanol to 50mL of the fermentation broth obtained in the step (2), mixing to uniform and static settling for 5 hours at 4 C, 12000 rpm centrifuging for 10 minutes, discarding the supernatant, dissolving the precipitate into 6iL of IM NaOH solution at 60C for 1 hour so as to obtain Cordyceps militaris extracellular polysaccharide. 10 The extraction method of intracellular polysaccharide of Cordyceps militaris mentioned as above comprises the following steps of: drying Cordyceps militaris mycelium obtained in the step (2) at 65C and grinding to power, adding 0.1g Cordyceps militaris mycelium powder into 6mL of IM NaOH solution, extracting at 60C for 2 hours, then 12000 rpm centrifuging for 5 minutes and separating supernatant so as to obtain Cordyceps militaris 15 intracellular polysaccharide. Determination of the Cordycepic polysaccharide Conventional phenol-dense sulphuric acid method in the art may be used, which can be referred to "Biochemistry experiment method and technology. ISBN: 9787030106858, 2009-07-01, published by Science Press". 20 (1) Establishment of Standard Curve The standard curve basing on glucose can be built by the method which comprises the following steps of: weighing up the glucose powder which has been dried at 10i for 2 hours and cooled to room temperature, then dissolving to 0.01 wt% standard solution, accurately sucking up OmL, 0.lmL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, 1.OmL standard solution of mannitol into 11 tubes of 25mL with 25 stopper, and separately added distilled water to 2.OmL, then adding 1.OmL 5% phenol solution and 5.OmL dense sulphuric acid, placing for 10 minutes and shaking to uniform, placing at 20 2 EMwater bath to keep temperature for 15 minutes, mensurating absorbency value of reaction solution by spectrophotometer at 490nm, and setting the glucose content (mg/mL) as X-coordinate and the absorbency value A 490 as Y coordinate so as to obtain the standard curve. 30 (2) Determination of the yield of Cordyceps militaris extracellular polysaccharide The yield of Cordyceps militaris extracellular polysaccharide is determined by the method which comprises the following steps of: taking 5 gL Cordyceps militaris extracellular polysaccharide sample into tubes, diluting to 1 L with distilled water and shaking to uniform, using 1.OmL water as the control group, then separately adding 1.OmL 5% phenol solution and adding 5.OmL dense sulphuric acid by drop rapidly, shaking 35 to uniform and cooling to room temperature, mensurating OD value at 490nm. According to the standard curve, we can calculate the concentration of polysaccharide and the result is 241.26gg. The yield of extracellular polysaccharide per litre fermentation broth is: 241.26 ggx (6 mL/5 8 gL) x20=5.79 g. (3) Determination of the yield of Cordyceps militaris intracellular polysaccharide The yield of Cordyceps militaris intracellular polysaccharide is determined by the method which comprises the following steps of: taking 5 gL Cordyceps militaris intracellular polysaccharide sample into 5 tubes, diluting to 1 L with distilled water and shaking to uniform, using 1.0mL water as the control group, then separately adding 1.0mL 5% phenol solution and adding 5.0mL dense sulphuric acid by drop rapidly, shaking to uniform and cooling to room temperature, mensurating OD value at 490nm. According to the standard curve, we can calculate the concentration of polysaccharide and the result is 19.16gg. The yield of intracellular polysaccharide from mycelium per litre fermentation broth is: 19.16 ggx (6 10 mL/5 gL) x10x5.87 (dry weight of the mycelium per fermentation broth) =1.35g. The result is shown in Figure 1. Therefore, the total yield of Cordyceps polysaccharide per liter fermentation broth is 5.79+1.35=7.14g. Example 2 It is the same as the liquid fermentation method described in Example 1, except that in the step (2) the 15 liquid fermentation culture is performed ?C lbr 3 days, then adding an expansin solution to a concentration of 0.5 mg/mL to perform further culture for 7 days. After been detected and calculated, each milliliter fermentation broth has 7.83mg of Cordyceps militaris extracellular polysaccharide, i.e. each liter fermentation broth has 7.83g of Cordyceps militaris extracellular polysaccharide; each gram (dry weight) of mycelium has 260.15mg of Cordyceps militaris intracellular 20 polysaccharide, i.e. the mycelium produced by each litre fermentation broth has 1.73g of Cordyceps militaris intracellular polysaccharide. The result is shown in Figure 1. Therefore, the total yield of Cordyceps polysaccharide per liter fermentation broth is 7.83+1.73=9.56g. Example 3 It is the same as the liquid fermentation method described in Example 1, except that in the step (2) the 25 liquid fermentation culture is performed ?C lbr 5 days, then adding an expansin solution to a concentration of 0.85 mg/mL to perform further culture for 5 days. After been detected and calculated, each milliliter fermentation broth has 4.91mg of Cordyceps militaris extracellular polysaccharide, i.e. each liter fermentation broth has 4.91g of Cordyceps militaris extracellular polysaccharide; each gram (dry weight) of mycelium has 230.58mg of Cordyceps militaris intracellular 30 polysaccharide, i.e. the mycelium produced by each litre fermentation broth has 0.92g of Cordyceps militaris intracellular polysaccharide. The result is shown in Figure 1. Therefore, the total yield of Cordyceps polysaccharide per liter fermentation broth is 4.91 +0.92=5.83g. Example 4 It is the same as the liquid fermentation method described in Example 1, except that Cordyceps 35 chrysalides (Cordyceps militaris) fermentation strains used in the example is purchased from China General Microbiological Culture Collection Center and having Culture Collection No. of CGMCC No. 3.4655. In the step (2) the liquid fermentation culture is performed at 2SC for 3 days, then adding an expansin solution to a concentration of 0.5 mg/mL to perform further culture for 7 days.

9 After been detected and calculated, each milliliter fermentation broth has 7.11mg of Cordyceps militaris extracellular polysaccharide, i.e. each liter fermentation broth has 7.11g of Cordyceps militaris extracellular polysaccharide; each gram (dry weight) of mycelium has 243.99mg of Cordyceps militaris intracellular polysaccharide, i.e. the mycelium produced by each litre fermentation broth has 1.58g of Cordyceps militaris 5 intracellular polysaccharide. The result is shown in Figure 1. Therefore, the total yield of Cordyceps polysaccharide per liter fermentation broth is 7.11 + 1.58=8.69g. Comparative Example 1 It is the same as the liquid fermentation method described in Example 1, except that in the step (2) the expansin-free acidic buffer is instead of the expansin protein solution added in the examples, then performs 10 further culture at 25 0 C for 10 days. After been detected and calculated, each milliliter fermentation broth has 2.3mg of Cordyceps militaris extracellular polysaccharide, i.e. each liter fermentation broth has 2.3g of Cordyceps militaris extracellular polysaccharide; each gram (dry weight) of mycelium has 193.21mg of Cordyceps militaris intracellular polysaccharide, i.e. the mycelium produced by each litre fermentation broth has 0.74g of Cordyceps militaris 15 intracellular polysaccharide. The result is shown in Figure 1. Therefore, the total yield of Cordyceps polysaccharide per liter fermentation broth is 2.3 + 0.74 = 3.04g. Comparative Example 2 It is the same as the determination steps of Cordyceps polysaccharide described in Example 1, except that the fermentation broth and Cordyceps militaris mycelium is produced by the method described in the 20 examples in the patent literature CN101067005 (application number: 200710052357) . After been detected and calculated, each milliliter fermentation broth has 0.99mg of Cordyceps militaris extracellular polysaccharide, i.e. each liter fermentation broth has 0.99g of Cordyceps militaris extracellular polysaccharide; each gram (dry weight) of mycelium has 183.91mg of Cordyceps militaris intracellular polysaccharide, i.e. the mycelium produced by each litre fermentation broth has 0.69g of Cordyceps militaris 25 intracellular polysaccharide. The result is shown in Figure 1. Therefore, the total yield of Cordyceps polysaccharide per liter fermentation broth is 0.99 + 0.69 = 1.68g.

Claims (7)

1. A liquid fermentation method for increasing the yield of Cordyceps polysaccharide by an expansin, comprises the following steps of: 5 (1) inoculating Cordyceps militaris strains into the PD liquid fermentation medium to perform activation culture, and transferring to a seed fermentation medium to a volume ratio of 10-15% to perform seed culture in the swing bed at 20-25C for 2~ 4 days so as to obtain a seed li qui d; (2) inoculating the seed liquid obtained in step (1) into a liquid fermentation medium to a volume ratio of

5-10% to perform liquid fermentation culture at 20~-2% for 1~6 days, then adding an expansin solution to a 10 concentration of 0.01-0.85 mg/mL to perform further culture for 2-9 days, and separating to obtain the mycelium of Cordyceps militaris and the fermentation broth; and (3) extracting the exopolysaccharide of Cordyceps militaris from the fermentation broth obtained in the step (2), extracting the intracellular polysaccharide from the Cordyceps militaris mycelium obtained in the step (2), and mixing the exopolysaccharide and the intracellular polysaccharide so as to obtain Cordyceps 15 polysaccharide. The expansin solution in step (2) may be prepared by the method which comprises the following steps of: sterilizing a broad bean or cucumber seed for 4-6 minutes with 0.05-0.15wt.% mercury chloride (HgCl 2 ), washing with running water for 5-7 hours, culturing in the darkroom for 4-6 days at 15-2C, taking 3-4 cm of seedling hypocotyl apices, precooling for 0.5 hours at -20 'C, adding a homogenate buffer solution 20 that is pre-cooled to 4 0 C, filtering with a nylon net having an aperture of 70 1tn after homogenate, washing the filter residue with a homogenate buffer solution, adding the filter residue in the homogenate buffer solution, settling for 1-3 hours to obtain a settled solution, adding an extracting solution in the settled solution, extracting for 44-50 hours at C, slowly adding 0.3~0.5grnL ammonium sulfate ((NH 4 ) 2 SO 4 ) in the filtrate while stirring to prevent a partial supersaturation of the (NH 4 ) 2 SO 4 , settling for 45-50 hours, centrifuging for 25 5-10 minutes at 4C, dissolving the sediment with an acid buffer solution, dialyzing in a dialysis bag with a molecular weight of 3000 Da afGl, centrifuging the dialyzate at 20000g for 10 minutes, and taking the supernatant so as to obtain the expansin solution. 2. The method according to claim 1, wherein activation culture in the step (1) is processed in the 30 darkroom with a shaking speed of 100-160 r/min at 20-25C for 3~ 5 days. 3. The method according to claim 1, wherein each litre of the seed fermentation medium in the step (1) comprises the following components: glucose 3g, yeast extract supernatant 1.5g, MgSO 4 0.1g, CaCl 2 0.03g, KH 2 PO 4 0.1g, K 2 HPO 4 0.1g, 2-5 35 pieces of glass beads with 3-6 mm diameter. 4. The method according to claim 1, wherein each litre of the liquid fermentation medium in the step (2) 11 comprises the following components: lactose 3g, molasses 2g, soybean powder 2g, peptone 1.5g, MgSO 4 0.1g, CaCl 2 0.03g, KH 2 PO 4 0.1g, K 2 HPO 4 0.1g. 5 5. The method according to claim 1, wherein the expansin concentration in the step (2) is 0.15~0.7mg/mL.

6. The method according to claim 5, wherein the expansin concentration in the step (2) is 0.25-0.5 mg/mL; most preferably, 0.5 mg/mL. 10

7. The method according to claim 6, wherein the homogenate buffer solution mentioned as above has a pH of 7.0, and comprises 25 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1.5 mmol/L Na 2 S 2 0 5 , 2 mmol/L EDTA and 0.1 wt.% Triton X-100. The extracting solution has a pH of 6.0, and comprises 15 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1.0 mmol/L ethylene diamine tetraacetic acid 15 (EDTA), 1.5 mmol/L Na 2 S 2 0 5 and 0.5 mmol/L NaCl. The acid buffer solution is prepared by dissolving 2.05g of sodium acetate in water, adjusting pH to 4.0 with glacial acetic acid, and adding water to IL.

8. The method according to claim 1, wherein the separation in the step (2) is carried out by 15000 rpm centrifuging for 5-10 minutes. 20

9. The method according to claim 1, wherein the extraction method of exopolysaccharide of Cordyceps militaris in the step (3) comprises the following steps of: adding 4 times volume of 95% ethanol to the fermentation broth obtained in the step (2), mixing to uniform and static settling for 5 hours at 4 0 C, 12000 rpm centrifuging for 10 minutes, discarding the 25 supernatant, dissolving the precipitate into 6iL of IM NaOH solution at 60 0 C for 1 hour so as to obtain Cordyceps militaris extracellular polysaccharide.

10. The method according to claim 1, wherein the extraction method of intracellular polysaccharide of Cordyceps militaris in the step (3) comprises the following steps of: 30 drying Cordyceps militaris mycelium obtained in the step (2) at 60 and grinding to power, adding 6mL of IM NaOH solution per gram of Cordyceps militaris mycelium powder, extracting at 0 6Dfor 2 hours, then 12000 rpm centrifuging for 5 minutes and separating supernatant so as to obtain Cordyceps militaris intracellular polysaccharide. 35