A method for increasing the content of fruiting body polysaccharide of Volvariella volvacea and a method for screening compounds for increasing the content of fruiting body polysaccharide of Volvariella volvacea

Abstract

The application provides a method for increasing the polysaccharide content of Volvariella volvacea fruiting bodies, wherein a glucose hexaphosphate disodium solution with a concentration of 200 muM is sprayed during the sowing period of the Volvariella volvacea; the application also provides a method for screening a compound for increasing the polysaccharide content of Volvariella volvacea fruiting bodies, wherein the compound to be screened is used for Volvariella volvacea spraying and mixing for fruiting body production, then the nutritional indicators and agronomic characters of the Volvariella volvacea fruiting bodies are determined to obtain the compound for increasing the polysaccharide content of Volvariella volvacea fruiting bodies; and the application also provides the use of glucose hexaphosphate disodium in the preparation of a product for increasing the polysaccharide content of Volvariella volvacea fruiting bodies. It is found that the glucose hexaphosphate disodium solution with a concentration of 200 muM can significantly increase the polysaccharide content of Volvariella volvacea fruiting bodies.

Description

Technical Field

[0001] This invention belongs to the food field and relates to a method for increasing the polysaccharide content of mushroom fruiting bodies and a method for screening compounds that increase the polysaccharide content of mushroom fruiting bodies. Background Technology

[0002] Straw mushrooms, scientifically known as *Volvariella volvacea*, belong to the genus *Volvariella* in the family Stylophycetes of the class Agaricomycetes. Polysaccharides are one of the main nutritional components of straw mushrooms. Related studies have shown that straw mushroom polysaccharides can enhance the body's immune function and improve antioxidant capacity. Based on this, in the food industry, straw mushroom polysaccharides can be used as immune enhancers in functional foods. In the pharmaceutical field, given the immunomodulatory and antioxidant bioactivities of straw mushroom polysaccharides, they hold promise for development into drugs for treating immune-related diseases and alleviating oxidative damage. Furthermore, in the cosmetics industry, straw mushroom polysaccharides can be used in topical skincare products to provide skincare benefits, offering the cosmetics industry a new natural raw material option. In conclusion, developing technologies to increase the polysaccharide content of straw mushroom fruiting bodies is an important way to promote the quality and efficiency of the edible fungi industry.

[0003] Increasing the polysaccharide content of straw mushroom fruiting bodies can enhance their efficiency in utilizing substrate nutrients, thereby reducing resource waste. Implementing reasonable cultivation measures during straw mushroom cultivation can not only increase the polysaccharide content of the fruiting bodies but also reduce production costs, achieve efficient resource utilization, protect the ecological environment, and promote the green and sustainable development of the straw mushroom industry. Therefore, it is necessary to optimize cultivation techniques to increase the polysaccharide content of straw mushroom fruiting bodies and improve their agronomic traits. Previous studies have confirmed that spraying DGlucono-1,5-lactone (DG) compounds during the small button stage of straw mushrooms can effectively improve the frost resistance and agronomic traits of the fruiting bodies (https: / / doi.org / 10.1016 / j.postharvbio.2023.112465).

[0004] For example, this study showed that spraying straw mushrooms with DG increased their polysaccharide content by 19.61%. This means that adding compounds during the developmental stage of straw mushrooms to improve their agronomic traits is a feasible path to optimize cultivation techniques. Given that improvements in the frost resistance of straw mushrooms are closely related to increases in polysaccharide content, conducting large-scale screening of frost-resistant compounds for straw mushrooms will help identify compounds that can increase polysaccharide content, thereby solving the current technical challenge of insufficient polysaccharide enhancement effects in straw mushrooms.

[0005] This study first screened antifreeze compounds to improve the survival rate of *Volvariella fuciformis* mycelia through low-temperature stress experiments, and then conducted experiments on the effects of adding antifreeze compounds on the frost resistance of fruiting bodies. Based on this, the screened antifreeze compounds were sprayed during the sowing and watering stages of *Volvariella fuciformis*, and fruiting bodies were collected for testing agronomic traits such as polysaccharides. The aim was to provide new technical support for the development of functional *Volvariella fuciformis* with increased polysaccharide content in fruiting bodies. Summary of the Invention

[0006] To address the aforementioned technical problems in the prior art, this invention provides a method for increasing the polysaccharide content of *Flammulina velutipes* fruiting bodies and a method for screening compounds that increase the polysaccharide content of *Flammulina velutipes* fruiting bodies. This method for increasing the polysaccharide content of *Flammulina velutipes* fruiting bodies and the method for screening compounds that increase the polysaccharide content of *Flammulina velutipes* fruiting bodies aim to solve the technical problem that existing cultivation methods are not effective in increasing the polysaccharide content of *Flammulina velutipes* fruiting bodies.

[0007] This invention provides a method for increasing the polysaccharide content of straw mushroom fruiting bodies by spraying a 200 μM solution of glucose disodium hexaphosphate during the straw mushroom sowing period.

[0008] Furthermore, spray 10-20 ml of a 200 μM solution of disodium glucose hexaphosphate per square meter of cultivation material.

[0009] Furthermore, each bed (1.3 m × 1.0 m) was sprayed with 20 ml of glucose disodium hexaphosphate solution.

[0010] This invention also provides a method for screening compounds that increase the polysaccharide content of mushroom fruiting bodies, comprising the following steps:

[0011] 1) The compounds to be screened were added to PDA medium and cultured on plates of *Pleurotus ostreatus* V23. Then, the cells were subjected to low temperature stress at 4°C, and the survival rate was recorded.

[0012] 2) Based on the survival rate, select antifreeze compounds, and then conduct antifreeze experiments on fruiting bodies;

[0013] 3) Collect antifreeze phenotypes, conduct transcriptome analysis, and elucidate the antifreeze mechanism;

[0014] 4) Spraying experiments were conducted using different concentrations of the obtained antifreeze compound during the sowing and watering stages of straw mushrooms, with water treatment as the control; then, egg-shaped fruiting bodies were collected.

[0015] 5) Spray antifreeze compounds during the straw mushroom sowing and watering stages;

[0016] 6) Conduct agronomical tests on the harvested fruiting bodies for cohesion, damaged area, brittleness, resilience, hardness, gelatinousness, elasticity, chewiness, and stickiness;

[0017] 7) The polysaccharide content of straw mushroom fruiting bodies was detected, and compounds that could increase the polysaccharide content of straw mushroom fruiting bodies were screened.

[0018] Furthermore, based on low-temperature stress plate screening, antifreeze compounds that could improve the survival rate of *Pleurotus ostreatus* were identified; during the *Pleurotus ostreatus* sowing and watering periods, a 200 μM solution of disodium glucose hexaphosphate was sprayed at 5-day intervals, per bed of 1.3 m². 2 (1.3 m × 1.0 m), spray 20 ml, and then harvest the fruiting bodies at the egg-shaped stage.

[0019] Furthermore, control and experimental groups were set up. In the experimental group, different compounds were added to the PDA medium at final concentrations of 100 μM, 200 μM, and 300 μM, respectively. In the control group, no exogenous compounds were added to the PDA medium. Next, 20 mL of PDA medium was added to a 90 mm plastic plate, and 1 cm diameter straw mushroom V23 mycelial blocks were inoculated using an inoculation spatula. After inoculation, the medium was first placed in an environment of 32 ℃ for 24 h, then removed and placed in a low temperature environment of 4 ℃ for 24 h, and then placed in an environment of 32 ℃ for 72 h for further incubation.

[0020] Specifically, the V23 straw mushroom product is a commercially available product, and will not be described in detail here.

[0021] This invention also provides the application of disodium glucose hexaphosphate in the preparation of products with increased polysaccharide content in mushroom fruiting bodies.

[0022] This invention focuses on the research and development of polysaccharide enhancement technology for straw mushrooms, and comprehensively utilizes a variety of methods, including plate mycelial culture, 4℃ low temperature stress treatment, and combined with low temperature stress survival rate screening, antifreeze experiments, transcriptome sequencing, fruiting cultivation, agronomic trait determination, and polysaccharide detection.

[0023] Compared with existing technologies, the technical effects of this invention are positive and obvious, and it has the following advantages:

[0024] (1) Survival rate was determined to be a screening indicator for improving the polysaccharide content of mushrooms.

[0025] (2) It was found that disodium glucose hexaphosphate can be used as a compound to significantly increase the polysaccharide content of mushroom fruiting bodies.

[0026] (3) A method for screening compounds that enhances the polysaccharide content of mushrooms was obtained. Spraying disodium glucose hexaphosphate (DGP) at a concentration of 200 μM can significantly increase the polysaccharide content of mushroom fruiting bodies to 39.45%. Attached Figure Description

[0027] Figure 1 shows the structural formula of the small molecule compound glucose disodium hexaphosphate (DGP).

[0028] Figure 2 shows the survival rate of straw mushroom mycelium with DGP added.

[0029] Figure 3 shows the low-temperature preservation experiment of straw mushroom fruiting bodies treated with DGP at 4℃. DGP represents disodium glucose hexaphosphate, and "H" represents h.

[0030] Figure 4 shows the principal component analysis results of the expressed genes after being soaked in DGP and subjected to a low temperature of 4°C, where "H" represents h.

[0031] Figure 5 shows the enrichment analysis results of expressed genes after DGP immersion treatment and exposure to 4°C. Again, "H" represents h. The number of upregulated and downregulated genes is indicated after the KEGG pathway information.

[0032] Figure 6 shows the straw mushroom sowing stage.

[0033] Figure 7 shows the water-sprinkling stage of straw mushrooms.

[0034] Figure 8 shows a photograph of the fruiting bodies of straw mushrooms in the egg-shaped stage after DGP spraying treatment.

[0035] Figure 9 shows the changes in polysaccharide content in mushroom fruiting bodies under DGP spraying treatment. Detailed Implementation

[0036] Example 1

[0037] Survival rate statistics

[0038] Different compounds (see Table 1 for specific compound information) were selected and added to PDA medium at final concentrations of 100 μM, 200 μM, and 300 μM, respectively. 20 mL of the corresponding compound-added PDA medium was added to each 90 mm diameter plate. The *V. truncata* strain V23 (provided by the National Edible Fungus Germplasm Bank (Shanghai)) was activated by plate inoculation. Then, 1 cm diameter *V. truncata* mycelial blocks were excavated and inoculated onto individual plates.

[0039] After inoculation, the plates were incubated at 32 °C for 24 h. Then, the plates were removed and transferred to a 4 °C environment for another 24 h. After this 24 h cooling period, the plates were placed at 32 °C for 72 h.

[0040] After removing the plates from the incubator, the survival rate of the *Volvariella fuciformis* V23 mycelium blocks was calculated. The survival rate was calculated using the formula: Survival rate (%) = 100% × Survival count / Inoculation quantity. Twenty plates were prepared for each treatment, divided into three groups: 7, 7, and 6 plates.

[0041] The results showed that compounds such as α-ketoglutarate, L-serine, tetrahydrofolate, L-tyrosine, and disodium glucose hexaphosphate (DGP) could significantly improve the freeze resistance of straw mushroom V23 (Table 1).

[0042] Table 1. Survival rate (%) of straw mushrooms with different compound additions

[0043]

[0044] The control survival rate was 5 ± 8.3%. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001.

[0045] Among them, riboflavin, pyridoxine, cholecalciferol, xylan, D-mannose, and disodium ribose-5-phosphate significantly improved the survival rate at two different concentrations. α-ketoglutarate, L-serine, threonine, L-methionine, vitamin A, folic acid, tetrahydrofolate, trehalose, maltose, DGP, and DL-glyceraldehyde-3-phosphate significantly improved the survival rate of *Pleurotus ostreatus* mycelium at three different concentrations.

[0046] Compared to glucose hexaphosphate and glucose hexaphosphate sodium, disodium glucose hexaphosphate (DGP) Figure 1 The *Pleurotus ostreatus* V23 strain exhibited strong cold resistance. Specifically, a final concentration of 200 μM DGP significantly improved its resistance to cold stress (Figure 2). Therefore, DGP was further used in subsequent experiments for verification.

[0047] Antifreeze test verification

[0048] Different concentrations of DGP were used to soak the fruiting bodies of *Pleurotus ostreatus* for 2 minutes. Afterward, the treated fruiting bodies were placed in a 4℃ environment for 24 hours to test the effect of DGP soaking on improving the freeze resistance of *Pleurotus ostreatus* fruiting bodies. Compared with the control group, DGP concentrations of 100 μM, 200 μM, and 300 μM all improved the freeze resistance of *Pleurotus ostreatus* to some extent. Among them, the final concentration of 200 μM DGP showed the best effect in improving the freeze resistance of *Pleurotus ostreatus* fruiting bodies (as shown in Figure 3).

[0049] Transcriptome sequencing

[0050] Strawberry fruiting bodies were soaked in DGP at a final concentration of 200 μM, with water soaking serving as a control. The treated fruiting bodies were then subjected to 24 h of low-temperature stress at 4°C. Transcriptome sequencing was performed on the fruiting bodies to obtain gene expression profiles. Principal component analysis of the obtained gene expression profiles showed that DGP treatment significantly altered the gene expression profiles of the fruiting bodies under low-temperature stress (Figure 4). Further enrichment analysis confirmed that DGP treatment upregulated the activity of the N-Glycan biosynthesis pathway, with 20 genes upregulated and 2 genes downregulated (Figure 5).

[0051] Agronomic trait testing

[0052] During the straw mushroom sowing period (Figure 6) and watering period (Figure 7), a solution of disodium glucose hexaphosphate was sprayed at 5-day intervals, covering 1.3 m² of each bed. 2 (1.3 m × 1.0 m), spray 20 ml, and then harvest the fruiting bodies at the egg-shaped stage (Fig. 8).

[0053] Compared with the control and other treatments, the experimental group sprayed with 200 μM DGP had the highest hardness, cohesion, resilience and viscosity (Table 2).

[0054] Table 2. Agronomical characteristics of straw mushrooms under DGP spraying treatment.

[0055]

[0056] Polysaccharide index detection

[0057] Compared with the control group and other treatment groups, the experimental group sprayed with 200 μM DGP had the highest initial polysaccharide content in the fruiting bodies of *Lentinula edodes*, reaching 42.28 mg·g⁻¹. -1 (Figure 9). Compared with the control group, the polysaccharide content of the fruiting bodies in this experimental group increased by 39.45%.

[0058] Experimental results showed that the survival rate of *Volvariella volvacea* reached 65% at 4℃ after adding a final concentration of 200 μM DGP; soaking *Volvariella volvacea* in a final concentration of 200 μM DGP improved its preservation effect at 4℃ for 24 hours; transcriptome analysis confirmed that the addition of DGP stimulated the polysaccharide synthesis pathway of *Volvariella volvacea*, promoting polysaccharide synthesis. Furthermore, adding a final concentration of 200 μM DGP increased the polysaccharide content of *Volvariella volvacea* fruiting bodies by 39.45%.

[0059] In conclusion, adding DGP to a final concentration of 200 μM is an effective method to increase the polysaccharide content of mushroom shiitake.

[0060] Comparative Example 1

[0061] (1) The survival rate of the starting strain, straw mushroom V23, at 4℃ was only 5%.

[0062] (2) The low temperature stress test confirmed that the fruiting bodies of V23 after water treatment showed shrinkage, collapse and water seepage during 24h low temperature preservation (Figure 3).

[0063] (3) The polysaccharide content of the straw mushroom fruiting bodies treated with water spraying was 30.31 mg·g. -1 The concentration was lower than 42.273 mg·g⁻¹ in the fruiting bodies of *Lentinula edodes* treated with 200 μM DGP. -1 (Figure 9).

[0064] Comparative Example 2

[0065] (1) The survival rate of straw mushrooms with a final concentration of 100 μM DGP at 4℃ was only 30%.

[0066] (2) The low temperature stress test showed that the fruiting bodies of V23 treated with a final concentration of 100 μM DGP could maintain their shape during 24 h of low temperature preservation, but there was a slight water seepage at the bottom (Figure 3).

[0067] (3) The fruiting bodies of *Lentinula edodes* sprayed with a final concentration of 100 μM DGP had a polysaccharide content of 31.90 mg·g⁻¹. -1 The concentration was slightly higher than that of fruiting bodies treated with control water (30.31 mg·g⁻¹). -1 The concentration of DGP was significantly lower than that of the fruiting body of *Lentinula edodes* at 200 μM DGP (42.273 mg·g⁻¹). -1 (Figure 9).

[0068] Comparative Example 3

[0069] (1) The survival rate of straw mushrooms with a final concentration of 300 μM DGP at 4℃ was only 20%.

[0070] (2) The low temperature stress test showed that the fruiting bodies of V23 treated with a final concentration of 300 μM DGP maintained their shape and surface dryness during 24 h of low temperature preservation (Figure 3).

[0071] (3) The fruiting bodies of *Lactarius deliciosus* treated with 300 μM DGP had a polysaccharide content of 31.34 mg·g⁻¹. -1The concentration of fruiting bodies in the water treatment was slightly higher than that in the control water treatment (30.31 mg·g⁻¹). -1 The concentration of *Lactarius deliciosus* fruiting bodies (42.273 mg·g⁻¹) was significantly lower than that of *Lactarius deliciosus* fruiting bodies at a final concentration of 200 μM DGP. -1 (Figure 9).

[0072] Those skilled in the art can conceive of and make various other corresponding changes and modifications based on the technical solutions described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.

Claims

1. The application of disodium glucose hexaphosphate in increasing the polysaccharide content of mushroom fruiting bodies, characterized in that, During the straw mushroom sowing period, spray 20 ml of a 200 μM solution of glucose disodium hexaphosphate per bed of 1.3 m × 1.0 m.

2. A method for screening compounds that increase the polysaccharide content of mushroom fruiting bodies, characterized in that... Includes the following steps: 1) The compounds to be screened were added to PDA medium and cultured on plates of *Pleurotus ostreatus* V23. Then, the cells were subjected to low temperature stress at 4°C, and the survival rate was recorded. 2) Based on the survival rate, select antifreeze compounds, and then conduct antifreeze experiments on fruiting bodies; 3) Collect antifreeze phenotypes, conduct transcriptome analysis, and elucidate the antifreeze mechanism; 4) Spraying experiments were conducted using different concentrations of the obtained antifreeze compound during the straw mushroom sowing and watering stages, with water treatment as the control; then, egg-shaped fruiting bodies were collected. 5) Spray antifreeze compounds during the straw mushroom sowing and watering stages; 6) Conduct agronomical tests on the harvested fruiting bodies for cohesion, damaged area, brittleness, resilience, hardness, gelatinousness, elasticity, chewiness, and stickiness; 7) The polysaccharide content of straw mushroom fruiting bodies was detected, and compounds that could increase the polysaccharide content of straw mushroom fruiting bodies were screened.

3. The method for screening compounds that increase the polysaccharide content of mushroom fruiting bodies according to claim 2, characterized in that: In step 1), a control group and an experimental group were set up. In the experimental group, different compounds with final concentrations of 100 μM, 200 μM, and 300 μM were added to the PDA medium, respectively. In the control group, no exogenous compounds were added to the PDA medium. Next, 20 mL of PDA medium was added to a 90 mm plastic plate, and 1 cm diameter straw mushroom V23 mycelial blocks were inoculated using an inoculation spatula. After inoculation, the medium was first placed in an environment of 32 ℃ for 24 h, then removed and placed in a low temperature environment of 4 ℃ for 24 h, and then placed in an environment of 32 ℃ for 72 h for further incubation.

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