Method for inducing generation of water-soluble pachymaran

Abstract

The invention belongs to the technical field of fermentation, and particularly relates to a method for inducing generation of water-soluble pachymaran. According to the method, Wolfiporia cocos and Trichoderma longibrachiatum are subjected to staged co-culture, and low-temperature stress at the temperature of 15 DEG C is applied to the key stage of co-culture. The specific strain combination and low-temperature induction have a synergistic effect, so that a water-soluble polysaccharide synthesis and secretion pathway of the poria cocos bacteria is remarkably activated, and the technical bottlenecks of low water-soluble polysaccharide proportion and low yield in the existing fermentation process are solved. The yield of the prepared water-soluble pachymaran can reach 2.35 g / L, the immunoregulatory activity of the water-soluble pachymaran is excellent, macrophages can be effectively promoted to secrete key cell factors such as IL-6 and IL-1beta, and a new technical scheme is provided for developing efficient and high-value pachymaran products.

Description

Technical Field

[0001] This invention belongs to the field of fermentation technology, specifically relating to a method for inducing the generation of water-soluble Poria cocos polysaccharides. Background Technology

[0002] Poria cocos polysaccharides are the core active ingredient of Poria cocos, possessing biological functions such as immunomodulation, anti-tumor activity, anti-inflammation, and cell proliferation promotion. Poria cocos polysaccharides enhance the body's non-specific immune response by activating macrophages, T lymphocytes, and NK cells. Studies have shown that Poria cocos polysaccharides have an inhibition rate of 30%–45% against solid tumors such as nasopharyngeal carcinoma and gastric cancer, with lower toxicity and side effects than traditional chemotherapy drugs. Furthermore, as a low-concentration interferon inducer, Poria cocos polysaccharides show potential in the antiviral field. With the increasing clinical demand for natural immunomodulators, the market size of Poria cocos polysaccharides continues to expand; however, the depletion of wild resources and the long cultivation cycle result in high costs.

[0003] Currently, the extraction of Poria cocos polysaccharides mainly relies on the fruiting body. However, wild Poria cocos resources are endangered, and artificial cultivation is limited by climate and soil conditions, resulting in limited yield. In addition, the traditional water extraction method yields less than 10% of polysaccharides, requiring repeated extraction, which is time-consuming. Although the alkali extraction method can increase the yield to 15%~20%, the strong alkaline environment destroys the tertiary structure of polysaccharides, leading to a decrease in biological activity.

[0004] Liquid fermentation, which uses Poria cocos mycelium to replace fruiting bodies, offers advantages such as short cycle time, low cost, and high product consistency. However, existing fermentation processes still have some problems, such as a low proportion of water-soluble polysaccharides and a high proportion of basic polysaccharides. The poor bioactivity of basic polysaccharides limits the high-value utilization of Poria cocos polysaccharides. There is an urgent need to develop methods to improve the production of water-soluble Poria cocos polysaccharides to expand their applications. Existing studies have shown that moderate low temperatures (15-20℃) can induce the synthesis of fungal secondary metabolites, but there are no reports on low-temperature induction to increase the production of Poria cocos polysaccharides, nor are there any reports on the relationship between low-temperature induction and the production of water-soluble Poria cocos polysaccharides. Therefore, investigating whether low-temperature stimulation can induce the production of water-soluble Poria cocos polysaccharides is of significant research importance. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention screened and obtained *Trichoderma longibrachiatum*, a fungus that can be co-cultured with *Wolfiporia cocos*. Through extensive experimentation, the co-culture process was optimized, and a staged co-culture process was finally selected. This process, employing low-temperature stress stimulation in the second stage of fermentation, synergistically induces the production and secretion of water-soluble *Poria cocos* polysaccharides. Furthermore, the water-soluble *Poria cocos* polysaccharides prepared using this process exhibit slightly superior immunomodulatory activity compared to carboxymethyl *Poria cocos* polysaccharides. This invention provides a new technical solution for developing efficient and high-value *Poria cocos* polysaccharide products.

[0006] On one hand, the present invention provides a method for inducing the generation of water-soluble Poria cocos polysaccharides, the method comprising the following steps: S1. First stage fermentation: Wolfiporia cocos was inoculated into the fermentation medium and cultured with shaking at 25-26℃ and 150-155 r / min for 3-4 days to obtain the first stage fermentation broth. The preservation number of Wolfiporia cocos is CGMCC 5.78. S2. Second stage fermentation: Trichoderma longibrachiatum was inoculated into the first stage fermentation broth and co-cultured with shaking at 20-22℃ and 150-160 r / min for 2-3 days. Then, the culture temperature was lowered to 15℃ and co-cultured with shaking at 140-160 r / min for another 2-3 days to obtain the co-culture fermentation broth. The preservation number of Trichoderma longibrachiatum is CGMCC 3.1029. S3. Polysaccharide extraction: Extract water-soluble Poria cocos polysaccharides from the co-culture fermentation broth.

[0007] Furthermore, in the method, in step S1, the inoculation amount of Poria cocos is 4% to 6% of the total mass of the fermentation culture medium.

[0008] Furthermore, in the method, in step S2, the inoculation amount of Trichoderma longifolia is 8% to 12% of the fermentation liquid volume in the first stage.

[0009] Further, in the method, in step S1, the fermentation medium contains the following components: glucose 20~30 g / L, yeast extract 3.5~4.5 g / L, peptone 3.5~4.5 g / L, K2HPO4 1 g / L, MgSO4·7H2O 0.5 g / L, the solvent is distilled water, and the initial pH is 5.4~5.6.

[0010] Further, in the method, step S3, the extraction of water-soluble Poria cocos polysaccharides includes: a) Centrifuge the co-culture fermentation broth to separate the supernatant 1 and bacterial cells; b) The bacterial cells were ultrasonically disrupted and then centrifuged to collect the supernatant 2; c) Combine the supernatant 1 and supernatant 2, concentrate them, and then add ethanol to precipitate them; d) Collect the precipitate, and after deproteinization, dialysis and drying, obtain the water-soluble Poria cocos polysaccharide.

[0011] Furthermore, in the method, in step c), the concentration is achieved by rotary evaporation to 20% of the original volume; the final concentration of ethanol is 30%, and the precipitation condition is standing at 4°C.

[0012] Furthermore, in the method, in step d), the deproteinization is performed using the Sevag method, and the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 Da.

[0013] Secondly, a water-soluble Poria cocos polysaccharide prepared by the method described in this invention is also provided.

[0014] Finally, the application of the water-soluble Poria cocos polysaccharide described in this invention in the preparation of immunomodulators is also provided.

[0015] Furthermore, in the application, the immunomodulator is used to promote the secretion of IL-6 and / or IL-1β cytokines by macrophages.

[0016] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (1) By co-fermenting *Wolfiporia cocos* and *Trichoderma longibrachiatum*, combined with a low-temperature stress treatment of 15°C during the second stage of co-fermentation, the yield of water-soluble *Poria cocos* polysaccharides was significantly increased. The yield of water-soluble *Poria cocos* polysaccharides prepared by the method of this invention reached 2.06~2.35 g / L, which is significantly higher than the yield of 0.01~0.35 g / L of water-soluble *Poria cocos* polysaccharides from single-strain fermentation and the yield of 1.04 g / L of water-soluble *Poria cocos* polysaccharides from co-culture without low-temperature induction. This method overcomes the problem of low yield of water-soluble polysaccharides in traditional fermentation methods.

[0017] (2) The water-soluble Poria cocos polysaccharide prepared by the method of the present invention also exhibits slightly better immunomodulatory activity than conventional products. At different concentrations, its ability to stimulate mouse macrophages RAW264.7 to secrete inflammatory cytokines IL-6 and IL-1β is superior to that of carboxymethyl Poria cocos polysaccharide, which is used as a positive control, indicating that the water-soluble Poria cocos polysaccharide prepared by the method of the present invention has better immune activation potential. Detailed Implementation

[0018] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0019] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention.

[0020] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0021] Trichoderma longibrachiatum, accession number: CGMCC 3.1029, purchased from China General Microbiological Culture Collection Center, hereinafter referred to as Trichoderma longibrachiatum CGMCC 3.1029.

[0022] Wolfiporia cocos, accession number CGMCC 5.78, was purchased from the China General Microbiological Culture Collection Center and will be referred to as Wolfiporia CGMCC 5.78 below.

[0023] Example 1 This example describes the preparation of a seed culture medium for Poria cocos CGMCC 5.78.

[0024] Remove Poria cocos CGMCC 5.78 from the -80℃ freezer and streak it onto the surface of a PDA slant culture medium. Incubate in the dark at 28℃ for 7-10 days until the mycelium covers the slant. Use an inoculation loop to pick up one loopful of activated Poria cocos CGMCC 5.78 and inoculate it into 200mL of PDA liquid medium. Incubate in a shake flask at 28℃ for 2 days to obtain the seed culture of Poria cocos CGMCC 5.78.

[0025] Example 2 This example describes the preparation of seed culture of Trichoderma longifolia CGMCC 3.1029.

[0026] Trichoderma longifolia CGMCC 3.1029 was taken out of the -80℃ freezer and streaked onto the surface of PDA slant medium. It was then cultured in a constant temperature incubator at 28℃ for 5 days. One loopful of activated Trichoderma longifolia CGMCC 3.1029 was picked up with an inoculation loop and inoculated into 250 mL of PDA liquid medium. The mixture was then cultured at 28℃ with shaking at 150 r / min for 3 days to obtain the Trichoderma longifolia CGMCC 3.1029 seed culture.

[0027] Example 3 In this embodiment, a staged co-culture of Poria cocos CGMCC 5.78 and Trichoderma longifolia CGMCC 3.1029 was used to induce the production of water-soluble Poria cocos polysaccharides at low temperature.

[0028] Fermentation medium: 20g glucose, 3.5g yeast extract, 3.5g peptone, 1g K2HPO4, 0.5g MgSO4·7H2O, 1L distilled water, initial pH 5.4.

[0029] The seed culture of Poria cocos CGMCC 5.78 prepared in Example 1 was inoculated into the fermentation medium at 5% of the total mass of the fermentation medium, and cultured at 25°C and 150 r / min for 3 days to obtain the first stage fermentation broth.

[0030] Second stage of fermentation The *Trichoderma longicornis* CGMCC3.1029 seed culture prepared in Example 2 was inoculated at 10% (by volume) of the first-stage fermentation broth. The mixture was co-cultured at 20°C with shaking at 160 rpm for 2 days, then cooled to 15°C and co-cultured with shaking at 160 rpm for another 2 days to obtain the co-culture fermentation broth. This broth was labeled as #1 co-culture fermentation broth.

[0031] Example 4 In this embodiment, a staged co-culture of Poria cocos CGMCC 5.78 and Trichoderma longifolia CGMCC 3.1029 was used to induce the production of water-soluble Poria cocos polysaccharides at low temperature.

[0032] Fermentation medium: 30g glucose, 4.5g yeast extract, 4.5g peptone, 1g K2HPO4, 0.5g MgSO4·7H2O, 1L distilled water, initial pH 5.5.

[0033] The seed culture of Poria cocos CGMCC 5.78 prepared in Example 1 was inoculated into the fermentation medium at 4% of the total mass of the fermentation medium, and cultured at 26°C and 155 r / min for 4 days to obtain the first stage fermentation broth.

[0034] Second stage of fermentation The *Trichoderma longicornis* CGMCC3.1029 seed culture prepared in Example 2 was inoculated at 12% (by volume) of the first-stage fermentation broth. The mixture was co-cultured at 22°C with shaking at 150 rpm for 3 days, then cooled to 15°C and co-cultured with shaking at 160 rpm for another 3 days to obtain the co-culture fermentation broth. This broth was labeled as #2 co-culture fermentation broth.

[0035] Example 5 In this embodiment, a staged co-culture of Poria cocos CGMCC 5.78 and Trichoderma longifolia CGMCC 3.1029 was used to induce the production of water-soluble Poria cocos polysaccharides at low temperature.

[0036] Fermentation medium: 25g glucose, 4.0g yeast extract, 4.0g peptone, 1g K2HPO4, 0.5g MgSO4·7H2O, 1L distilled water, initial pH 5.6.

[0037] The seed culture of Poria cocos CGMCC 5.78 prepared in Example 1 was inoculated into the fermentation medium at 6% of the total mass of the fermentation medium, and cultured at 25°C and 150 r / min for 4 days to obtain the first stage fermentation broth.

[0038] Second stage of fermentation The seed culture of Trichoderma longifolia CGMCC 3.1029 prepared in Example 2 was inoculated at 8% of the volume percentage of the first stage fermentation broth. The culture was co-cultured at 21°C and 160 r / min for 2 days with shaking. Then the temperature was lowered to 15°C and the culture was co-cultured at 140 r / min for 36 hours to obtain the co-culture fermentation broth, which was labeled as No. 3 co-culture fermentation broth.

[0039] Comparative Example 1 The only difference between this comparative example and Example 3 is that the co-fermentation at 15°C was not performed in the second stage of fermentation.

[0040] The second stage of this comparative fermentation: 10% of the volume of the fermentation broth from the first stage was inoculated with the *Trichoderma longicornis* CGMCC 3.1029 seed culture prepared in Example 2, and co-cultured at 20°C and 160 r / min for 2 days with shaking. Then, the co-culture was continued at 20°C and 160 r / min for another 2 days with shaking to obtain the co-culture fermentation broth, which was labeled as #1 control fermentation broth.

[0041] Comparative Example 2 The only difference between this comparative example and Example 3 is the fermentation of a single strain of *Poria cocos* CGMCC 5.78. In the second stage of fermentation, the *Trichoderma longifolia* CGMCC 3.1029 seed culture from Example 3 was replaced with the *Poria cocos* CGMCC 5.78 seed culture. All other conditions were the same as in Example 3. The resulting fermentation broth was labeled as #2 control fermentation broth.

[0042] Comparative Example 3 The difference between this comparative example and Example 3 is that it uses a single strain of Poria cocos CGMCC 5.78 for fermentation, and the second stage of fermentation was not carried out by cooling to 15°C.

[0043] The second stage of this comparative fermentation was as follows: 10% of the volume of the fermentation broth from the first stage was inoculated with *Poria cocos* CGMCC 5.78 seed culture, and co-cultured at 20°C with shaking at 160 rpm for 2 days. Subsequently, co-cultured at 20°C with shaking at 160 rpm for another 2 days to obtain the fermentation broth. This broth was labeled as #3, the control fermentation broth.

[0044] Comparative Example 4 The only difference between this comparative example and Example 4 is the fermentation of a single strain of *Trichoderma longicornis* CGMCC 3.1029. In the first stage of fermentation, the CGMCC 5.78 seed culture from Example 3 was replaced with the *Trichoderma longicornis* CGMCC 3.1029 seed culture; all other conditions were the same as in Example 3. The resulting fermentation broth was labeled as #4 control fermentation broth.

[0045] Example 6 This embodiment extracts water-soluble Poria cocos polysaccharides from different fermentation broths.

[0046] Using the co-culture fermentation broths (1#~3#) prepared in Examples 3~5 and the comparative fermentation broths (1#~4#) prepared in Comparative Examples 1~4 as samples, water-soluble Poria cocos polysaccharides were extracted from each test group.

[0047] Take 1 L each of the co-culture fermentation broth (1#~3#) and the control fermentation broth (1#~4#), centrifuge at 8000 r / min, 4℃ for 15 min, and collect the supernatant. Add water to the residual bacterial cells in each fermentation broth at 10 times the bacterial cell volume, then sonicate on ice for 5 min (ultrasonic disruption power 200W), followed by centrifugation at 8000 r / min, 4℃ for 15 min, and collect the supernatant from each group.

[0048] The supernatants of the fermentation broths and the supernatants of the cell disruption from each group were combined to obtain combined supernatants. Each combined supernatant was rotary evaporated at 60℃ to 20% of its original volume to obtain a concentrated solution. Five times the volume of 30% ethanol was added to the concentrated solution, and the mixture was allowed to stand at 4℃ for 24 hours. The precipitate was then collected by filtration. The precipitate was reconstituted with ultrapure water to prepare a 20 mg / mL solution. Crude water-soluble Poria cocos polysaccharide was obtained. Sevag reagent (chloroform: n-butanol = 4:1) was added to the crude water-soluble Poria cocos polysaccharide to remove proteins. This process was repeated three times. The supernatant polysaccharide solution was carefully aspirated and dialyzed with distilled water for 48 hours (6 water changes) using a 3500 Da molecular weight cutoff dialysis bag to remove small molecule impurities. The solution was pre-frozen at -80℃ for 6 hours and then vacuum dried to obtain powdered water-soluble Poria cocos polysaccharide. The mass of each group of water-soluble Poria cocos polysaccharide was weighed, and the yield of the water-soluble polysaccharide was calculated. The results are shown in Table 1.

[0049] Table 1. Results of yield determination of water-soluble Poria cocos polysaccharides under different fermentation methods Experimental grouping Yield of water-soluble Poria cocos polysaccharides (g / L) #1 co-culture fermentation broth 2.11 #2 co-culture fermentation broth 2.35 #3 co-culture fermentation broth 2.06 1# Comparative Fermentation Broth 1.04 2# Comparative Fermentation Broth 0.35 3# Comparative Fermentation Broth 0.15 4# Comparative Fermentation Broth 0.01 As shown in Table 1, fermentation using a single strain of *Trichoderma longifolia* CGMCC 3.1029 yielded almost no water-soluble *Poria cocos* polysaccharides in the fermentation broth. This may be related to the fact that 30% ethanol precipitation can only precipitate large-molecule polysaccharides, while the polysaccharides produced by *Trichoderma longifolia* CGMCC 3.1029 are small-molecule polysaccharides. In contrast, the water-soluble *Poria cocos* polysaccharide yield in the control fermentation broth (No. 3) using a single strain of *Poria cocos* CGMCC 5.78 without 15℃ cooling was 0.15 g / L, while the water-soluble *Poria cocos* polysaccharide yield in the control fermentation broth (No. 2) using a single strain of *Poria cocos* CGMCC 5.78 with 15℃ cooling was 0.35 g / L. The water-soluble polysaccharide yield was 2.33 times that of the control fermentation broth (No. 3). This suggests that 15℃ low-temperature stress may activate the stress metabolic pathway of *Poria cocos* mycelium, inducing increased activity of key enzymes in polysaccharide synthesis, and simultaneously altering cell membrane permeability to promote polysaccharide secretion. Comparative Example 1 also reflects the same trend.

[0050] The yield of water-soluble Poria cocos polysaccharides in the fermentation broths prepared in Examples 3-5 of this invention (Cultivation Broth #1-Cultivation Broth #3) was 2.06-2.35 g / L, significantly higher than that in the control fermentation broths (Cultivation Broth #2-Cultivation Broth #4). This suggests that the synergistic effect of *Trichoderma longifolia* CGMCC 3.1029, *Poria cocos* CGMCC 5.78, and cold stimulation significantly increases the yield of water-soluble Poria cocos polysaccharides. This may be related to the synergistic effect of specific metabolites of *Trichoderma longifolia* CGMCC 3.1029 and cold stimulation on the fermentation products of *Poria cocos*, promoting the conversion of water-soluble polysaccharides in *Poria cocos*.

[0051] Example 7 This embodiment measures the activity of water-soluble Poria cocos polysaccharides prepared from the co-culture fermentation broths of Examples 3 to 5.

[0052] RAW264.7 mouse monocytes / macrophages were cultured using standard methods. First, the RAW264.7 cells, cryopreserved in liquid nitrogen, were rapidly thawed in a 37°C water bath with shaking. They were then transferred to 15mL centrifuge tubes, and DMEM complete medium containing 10% fetal bovine serum and 1% penicillin 100 U / mL + streptomycin 100 μg / mL was slowly added. After centrifugation at 1000 rpm for 5 min to remove the cryopreservation solution, the cells were resuspended in fresh medium and seeded into culture flasks. The cells were then cultured at 37°C in a 5% CO2 incubator. When the cells reached 80%–90% confluence, they were passaged. The cell layer was washed 1–2 times with PBS, and after adding complete medium, adherent cells were gently scraped using a cell scraper and pipetted to form a single-cell suspension. This suspension was then seeded into new culture flasks at an appropriate ratio, and fresh DMEM high-glucose complete medium was added for further culture. Typically, after 2–3 passages, the cells were stable and ready for subsequent experimental studies.

[0053] The water-soluble Poria cocos polysaccharides prepared by combining the co-culture fermentation broths of #1 to #3 were prepared into different concentrations and labeled as co-fermented water-soluble Poria cocos polysaccharides of different concentrations for later use. The positive control carboxymethyl Poria cocos polysaccharide (CMP) was prepared into different concentrations for later use.

[0054] Mouse RAW264.7 mononuclear macrophages were treated with different concentrations of co-fermented water-soluble Poria cocos polysaccharide and a positive control group. A negative control group (CK) of mouse RAW264.7 mononuclear macrophages without any treatment was also set up. After culturing at 37℃ and 5% CO2 for 24 h, the concentrations of cytokines IL-6 and IL-1β in the cell supernatant were detected according to the instructions of the ELISA kit. The results are shown in Tables 2 and 3.

[0055] Table 2. Results of IL-6 concentration determination under different treatments Experimental grouping IL-6 (pg / mL) CK 801.35 0.05 mg / mL CMP 998.13 0.25 mg / mL CMP 1115.97 0.5 mg / mL CMP 1236.12 1.0 mg / mC M P 1139.06 0.05 mg / mL co-fermented water-soluble Poria cocos polysaccharide 1198.05 0.25 mg / mL co-fermented water-soluble Poria cocos polysaccharide 1284.31 0.5 mg / mL co-fermented water-soluble Poria cocos polysaccharide 1305.82 1.0 mg / mL co-fermented water-soluble Poria cocos polysaccharide 1187.39 Table 3. Results of concentration determination of IL-1β cytokine under different treatments Experimental grouping IL-1β (pg / mL) CK 2400.15 0.05 mg / mL CMP 2700.81 0.25 mg / mL CMP 2801.56 0.5 mg / mL CMP 2831.35 1.0 mg / mC M P 2794.08 0.05 mg / mL co-fermented water-soluble Poria cocos polysaccharide 3105.94 0.25 mg / mL co-fermented water-soluble Poria cocos polysaccharide 3213.24 0.5 mg / mL co-fermented water-soluble Poria cocos polysaccharide 3105.16 1.0 mg / mL co-fermented water-soluble Poria cocos polysaccharide 3078.95 As shown in Table 2, the co-fermented water-soluble Poria cocos polysaccharide can stimulate IL-6 secretion, and its ability to stimulate IL-6 secretion is slightly better than that of the positive control carboxymethyl Poria cocos polysaccharide (CMP).

[0056] As shown in Table 3, the co-fermented water-soluble Poria cocos polysaccharide was significantly better than the positive control carboxymethyl Poria cocos polysaccharide (CMP) in stimulating the secretion of IL-1β by cells.

[0057] In summary, the test results of this embodiment show that the water-soluble Poria cocos polysaccharide prepared by the method of the present invention has good biological activity.

[0058] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A method for inducing the formation of water-soluble Poria cocos polysaccharides, characterized in that, Includes the following steps: S1. First stage fermentation: Wolfiporia cocos was inoculated into the fermentation medium and cultured with shaking at 25-26℃ and 150-155 r / min for 3-4 days to obtain the first stage fermentation broth. The preservation number of Wolfiporia cocos was CGMCC 5.

78. S2. Second stage fermentation: Trichoderma longibrachiatum was inoculated into the first stage fermentation broth and co-cultured with shaking at 20-22℃ and 150-160 r / min for 2-3 days. Then, the culture temperature was lowered to 15℃ and co-cultured with shaking at 140-160 r / min for another 2-3 days to obtain the co-culture fermentation broth. The preservation number of Trichoderma longibrachiatum is CGMCC 3.1029. S3. Polysaccharide extraction: Extract water-soluble Poria cocos polysaccharides from the co-culture fermentation broth.

2. The method according to claim 1, characterized in that, In step S1, the inoculation amount of Poria cocos is 4% to 6% of the total mass of the fermentation medium.

3. The method according to claim 1, characterized in that, In step S2, the inoculation amount of Trichoderma longifolia is 8% to 12% of the fermentation liquid volume in the first stage.

4. The method according to claim 1, characterized in that, In step S1, the fermentation medium contains the following components: glucose 20-30 g / L, yeast extract 3.5-4.5 g / L, peptone 3.5-4.5 g / L, K2HPO4 1 g / L, MgSO4·7H2O 0.5 g / L, distilled water as solvent, and an initial pH of 5.4-5.

6.

5. The method according to claim 1, characterized in that, In step S3, the extraction of water-soluble Poria cocos polysaccharides includes: a) Centrifuge the co-culture fermentation broth to separate the supernatant 1 and bacterial cells; b) The bacterial cells were ultrasonically disrupted and then centrifuged to collect the supernatant 2; c) Combine the two supernatants 2 and 2, concentrate them, and then add ethanol to precipitate them; d) Collect the precipitate, and after deproteinization, dialysis and drying, obtain the water-soluble Poria cocos polysaccharide.

6. The method according to claim 5, characterized in that, In step c), the concentration is achieved by rotary evaporation to 20% of the original volume; the final concentration of ethanol is 30%, and the precipitation condition is standing at 4°C.

7. The method according to claim 5, characterized in that, In step d), the deproteinization is performed using the Sevag method, and the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 Da.

8. A water-soluble Poria cocos polysaccharide, characterized in that, It is prepared by the method according to any one of claims 1 to 7.

9. The application of the water-soluble Poria cocos polysaccharide according to claim 8 in the preparation of immunomodulators.

10. The application according to claim 9, characterized in that, The immunomodulator is used to promote the secretion of IL-6 and / or IL-1β cytokines by macrophages.