Method for improving polysaccharide content of straw mushroom sporocarp and method for screening compound for improving polysaccharide content of straw mushroom sporocarp

Abstract

The invention provides a method for increasing the polysaccharide content of volvariella volvacea sporocarp, which comprises the following step: spraying a glucose disodium hexaphosphate solution with the concentration of 200 mu M in the sowing period of volvariella volvacea. The invention further provides a method for screening the compound capable of improving the polysaccharide content of the straw mushroom sporocarp, the compound to be screened is used for a straw mushroom spraying and mixing fruiting experiment, then the straw mushroom sporocarp is subjected to nutritional index and agronomic character determination, and the compound capable of improving the polysaccharide content of the straw mushroom sporocarp is obtained. The invention also provides an application of the glucose disodium hexaphosphate in preparation of a product for improving the polysaccharide content of the volvariella volvacea sporocarp. The invention finds that the spraying concentration of the glucose disodium hexaphosphate is 200 [mu] M, so that the content of the polysaccharide in the volvariella volvacea sporocarp can be obviously increased.

Description

Technical Field

[0001] The invention belongs to the field of food, and relates to a method for increasing the polysaccharide content of a straw mushroom fruiting body and a method for screening a compound for increasing the polysaccharide content of a straw mushroom fruiting body. Background Art

[0002] Volvariella volvacea, whose scientific name is Volvariella volvacea , belonging to the genus Agaricus of the family Agaricus, class Agaricaceae, polysaccharide is one of the main nutrients of Volvariella volvacea. Relevant studies have shown that Volvariella volvacea polysaccharide has the effect of enhancing the body's immune function and improving antioxidant capacity. Based on this, in the food field, Volvariella volvacea polysaccharide can be used as an immunopotentiator for functional foods. In the medical field, given that Volvariella volvacea polysaccharide has biological activities such as immunomodulation and antioxidant, it is expected to be developed into a drug for the treatment of immune-related diseases and the alleviation of antioxidant damage. In addition, in the field of cosmetics, Volvariella volvacea polysaccharide can be used in skin topical preparations to play a role in beauty and skin care, providing the cosmetics industry with a new natural raw material option. In summary, the research and development of technologies to increase the polysaccharide content of Volvariella volvacea fruiting bodies is an important way to promote the quality and efficiency of edible fungi-related industries.

[0003] Increasing the polysaccharide content of Volvariella volvacea fruiting bodies can enhance the efficiency of the mushroom's use of substrate nutrients, thereby reducing resource waste. Implementing appropriate cultivation practices during Volvariella volvacea cultivation not only increases polysaccharide content but also reduces production costs, achieving efficient resource utilization, and protecting the ecological environment, promoting the green and sustainable development of the Volvariella volvacea industry. Therefore, optimizing cultivation techniques is necessary to increase the polysaccharide content of Volvariella volvacea fruiting bodies and improve their agronomic traits. Previous studies have demonstrated that spraying DGlucono-1,5-lactone (DG) during the button stage of Volvariella volvacea effectively improves frost tolerance and agronomic traits (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 suggests that adding compounds during the developmental stage of straw mushrooms to improve their agronomic traits is a viable path to optimizing cultivation techniques. Given that improved frost resistance in straw mushrooms is closely related to increased polysaccharide content, large-scale screening of antifreeze compounds in straw mushrooms will help identify compounds that can increase polysaccharide content, thereby addressing the current technical challenge of insufficient polysaccharide enhancement.

[0005] This study first screened for antifreeze compounds that improve the survival rate of Volvariella volvacea mycelium through low-temperature stress experiments. Subsequently, experiments were conducted to determine the effects of adding these compounds on the freezing tolerance of the fruiting bodies. Furthermore, the selected antifreeze compounds were sprayed during the sowing and watering periods, and the fruiting bodies were collected for testing of agronomic traits such as polysaccharides. This study aims to provide new technical support for the development of functional Volvariella volvacea mushrooms that increase the polysaccharide content of their fruiting bodies. Summary of the Invention

[0006] In response to the above-mentioned technical problems in the prior art, the present invention provides a method for increasing the polysaccharide content of straw mushroom fruiting bodies and a method for screening compounds that increase the polysaccharide content of straw mushroom fruiting bodies. The method for increasing the polysaccharide content of straw mushroom fruiting bodies and the method for screening compounds that increase the polysaccharide content of straw mushroom fruiting bodies are intended to solve the technical problem that the cultivation methods in the prior art are not effective in increasing the polysaccharide content in straw mushroom fruiting bodies.

[0007] The invention provides a method for increasing the polysaccharide content of straw mushroom fruiting bodies. During the straw mushroom sowing period, a glucose hexaphosphate disodium solution with a concentration of 200 μM is sprayed.

[0008] Furthermore, 10 to 20 ml of 200 μM glucose hexaphosphate disodium solution was sprayed per square meter of the cultivation material.

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

[0010] The present invention also provides a method for screening a compound for increasing the polysaccharide content of a fruiting body of a Volvariella volvacea, comprising the following steps: The compounds to be screened were added to PDA medium and cultured on plates of Volvariella volvacea V23, and then subjected to low temperature stress at 4°C, and the survival rate was calculated; Based on the survival rate, antifreeze compounds were selected and then subjected to fruiting body antifreeze experiments; Collect antifreeze phenotypes, conduct transcriptome analysis, and analyze the antifreeze mechanism; The obtained antifreeze compound was used to spray different concentrations of Volvariella volvacea at the sowing and watering stages, with water treatment used as a control. Then, the egg-shaped fruiting bodies were collected. During the straw mushroom sowing and watering period, spray antifreeze compounds; The harvested fruiting bodies are tested for agronomic traits such as cohesion, damaged area, brittleness, resilience, hardness, gelatinity, elasticity, chewiness, and stickiness; The polysaccharide content of the fruiting body of the Volvariella volvacea is detected, and compounds that can increase the polysaccharide content of the fruiting body of the Volvariella volvacea are screened.

[0011] Furthermore, antifreeze compounds that can improve the survival rate of Volvariella volvacea were screened based on low temperature stress plates; during the sowing period and watering period of Volvariella volvacea, 200 μM glucose hexaphosphate disodium solution was sprayed every 5 days, 1.3 m per bed. 2 (1.3 m×1.0 m), spray 20 ml, and then harvest the fruiting bodies in the egg-shaped stage.

[0012] Furthermore, 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 culture medium, respectively; while the PDA culture medium of the control group did not add any exogenous compounds. Next, 20 mL of PDA culture medium was added to a 90 mm plastic plate, and then 1 cm diameter Volvariella V23 mushroom blocks were inoculated with an inoculation spatula. After inoculation, the culture medium was first cultured at 32°C for 24 h, then taken out and placed in a low temperature environment of 4°C for 24 h, and then placed in a 32°C environment for further culture for 72 h.

[0013] Specifically, Volvariella volvacea V23 is a commercially available product and will not be described in detail here.

[0014] The present invention also provides the use of disodium glucose hexaphosphate in the preparation of a product with increased polysaccharide content in straw mushroom fruiting bodies.

[0015] The present invention carries out research and development around the technology of enhancing polysaccharides of Volvariella volvacea, and comprehensively utilizes a variety of means, including flat mycelium culture, 4°C low-temperature stress treatment, and combines low-temperature stress survival rate screening, antifreeze experiments, transcriptome sequencing, mushroom cultivation, agronomic trait determination and polysaccharide detection.

[0016] Compared with the existing technology, the present invention has positive and obvious technical effects and has the following advantages: (1) It was determined that the survival rate can be used as a screening indicator for improving the polysaccharide content of Volvariella volvacea.

[0017] (2) It was clarified that disodium glucose hexaphosphate can be used as a compound to significantly increase the polysaccharide content in the fruiting body of Volvariella volvacea.

[0018] (3) A method for screening compounds that increase the polysaccharide content of Volvariella volvacea was obtained. Spraying disodium glucose hexaphosphate (DGP) at a concentration of 200 μM can significantly increase the polysaccharide content of Volvariella volvacea fruiting bodies, reaching 39.45%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG1 is the structural formula of the small molecule compound disodium glucose hexaphosphate (DGP).

[0020] Figure 2 shows the survival rate of Volvariella volvacea mycelium under the addition of DGP.

[0021] Figure 3 shows the 4°C low-temperature preservation experiment of Volvariella volvacea fruiting bodies treated with DGP. DGP stands for disodium glucose hexaphosphate, and “H” stands for h.

[0022] Figure 4 presents the principal component analysis results of the expressed genes after DGP immersion treatment and low temperature condition of 4°C, where “H” represents h.

[0023] Figure 5 shows the enrichment analysis results of genes expressed after DGP immersion and exposure to 4°C. Similarly, "H" stands for h. The KEGG pathway information is followed by the number of upregulated and downregulated genes.

[0024] Figure 6 shows the sowing stages of straw mushrooms.

[0025] Figure 7 shows the watering stage of straw mushrooms.

[0026] Figure 8 shows photos of fruiting bodies of Volvariella volvacea at the egg-shaped stage under DGP spraying treatment.

[0027] FIG9 shows the changes in polysaccharide content in fruiting bodies of Volvariella volvacea under DGP spraying treatment. DETAILED DESCRIPTION Example

[0028] ① Survival rate statistics Different compounds (see Table 1 for specific compound information) were added to PDA culture medium at final concentrations of 100 μM, 200 μM, and 300 μM. 20 mL of PDA culture medium supplemented with the corresponding compound was added to a 90 mm diameter plate. Volvariella volvacea V23 strain (provided by the National Edible Fungi Germplasm Resource Bank (Shanghai)) was activated on the plates. Subsequently, 1 cm diameter pieces of Volvariella volvacea V23 were excised and inoculated onto each plate.

[0029] After the inoculation operation is completed, the plates are placed in an environment with an environment of 32°C for 24 hours. Then, the plates are removed and transferred to a cold environment at 4°C for another 24 hours. After the 24-hour cold treatment, the plates are placed in an environment with an environment of 32°C for 72 hours.

[0030] After removing the plates from the incubator, the survival rate of Volvariella volvacea V23 clumps was calculated. The survival rate was calculated as follows: Survival rate (%) = 100% × number of surviving clumps / number of inoculated clumps. Each treatment had 20 plates, divided into three groups of 7, 7, and 6.

[0031] The results showed that compounds such as α-ketoglutaric acid, L-serine, tetrahydrofolic acid, L-tyrosine, and disodium glucose hexaphosphate (DGP) can significantly improve the frost resistance of Volvariella volvacea V23 (Table 1).

[0032] The survival rate of the control group was 5±8.3 (%). P<0.05 ;**represent P<0.01 ;***represent P<0.001 .

[0033] Among them, six compounds—riboflavin, pyridoxine, cholecalciferol, xylan, D-mannose, and disodium ribose-5-phosphate—significantly increased survival at two different concentrations. Eleven compounds—α-ketoglutarate, L-serine, threonine, L-methionine, vitamin A, folic acid, tetrahydrofolate, trehalose, maltose, DGP, and DL-glyceraldehyde-3-phosphate—significantly increased survival at three different concentrations.

[0034] Compared with glucose hexaphosphate and sodium glucose hexaphosphate, disodium glucose hexaphosphate (DGP) ( Figure 1 ) exhibited strong freezing tolerance. Specifically, DGP at a final concentration of 200 μM significantly enhanced the cold stress tolerance of Volvariella volvacea V23 (Figure 2). Therefore, DGP was further used in subsequent experimental verification.

[0035] ③ Antifreeze test verification Volvariella volvacea fruiting bodies were immersed in different concentrations of DGP for 2 minutes. The treated fruiting bodies were then placed at 4°C for 24 hours to test the effect of DGP on improving freezing tolerance. Compared with the control group, DGP at final concentrations of 100 μM, 200 μM, and 300 μM all improved freezing tolerance to some extent. A final concentration of 200 μM DGP exhibited the greatest effect (Figure 3).

[0036] ③ Transcriptome sequencing Fruiting bodies of Volvariella volvacea were soaked in DGP at a final concentration of 200 μM, while fruiting bodies soaked in water served as a control. The treated fruiting bodies were then subjected to cold stress at 4°C for 24 hours. Transcriptome sequencing was then performed to obtain gene expression profiles. Principal component analysis of the resulting gene expression profiles revealed that DGP treatment significantly altered the gene expression profiles of the fruiting bodies under cold stress (Figure 4). Further enrichment analysis confirmed that DGP treatment upregulated the activity of the polysaccharide biosynthesis pathway (N-glycan biosynthesis), with 20 genes upregulated and 2 genes downregulated (Figure 5).

[0037] ④ Agronomic trait testing During the sowing period of straw mushrooms (Figure 6) and the watering period (Figure 7), the concentration of glucose hexaphosphate disodium solution was sprayed every 5 days, 1.3 m per bed. 2 (1.3 m × 1.0 m), spray 20 ml, and then harvest the fruiting bodies at the egg-shaped stage (Figure 8).

[0038] 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).

[0039]

[0040] ⑥ Polysaccharide index detection Compared with the control group and other treatment groups, the initial polysaccharide content of the fruiting body of Volvariella volvacea in the experimental group sprayed with 200 μM DGP was the highest, reaching 42.28 mg·g -1 (Figure 9) Compared with the control group, the polysaccharide content of the fruiting body of the experimental group increased by 39.45%.

[0041] Experimental results showed that the survival rate of Volvariella volvacea reached 65% after adding DGP at a final concentration of 200 μM at 4°C. Soaking Volvariella volvacea in DGP at a final concentration of 200 μM improved their preservation at 4°C for 24 hours. Transcriptome analysis confirmed that DGP stimulated the polysaccharide biosynthesis pathway in Volvariella volvacea, promoting polysaccharide synthesis. Furthermore, adding DGP at a final concentration of 200 μM increased the polysaccharide content in Volvariella volvacea fruiting bodies by 39.45%.

[0042] In summary, adding DGP at a final concentration of 200 μM is an effective method to increase the polysaccharide content of Volvariella volvacea.

[0043] Comparative Example 1 (1) The survival rate of the starting strain Volvariella volvacea V23 at 4°C was only 5%.

[0044] (2) The low temperature stress tolerance test confirmed that the V23 fruiting bodies treated with water shrank, collapsed, and leaked water during 24 h of low temperature storage (Figure 3).

[0045] (3) The polysaccharide content of the fruiting body of the straw mushroom treated with water was 30.31 mg·g -1 , which is lower than the 42.273 mg·g of the fruiting body of Volvariella volvacea treated with 200μM DGP. -1 (Figure 9).

[0046] Comparative Example 2 (1) The survival rate of Volvariella volvacea at 4°C after addition of DGP at a final concentration of 100 μM was only 30%.

[0047] (2) The low temperature stress tolerance test showed that the V23 fruiting bodies treated with DGP at a final concentration of 100 μM could maintain their mushroom shape during 24 h of low temperature storage, but there was a slight water seepage at the bottom (Figure 3).

[0048] (3) The polysaccharide content of the fruiting body of Volvariella volvacea sprayed with a final concentration of 100 μM DGP was 31.90 mg·g -1 , slightly higher than that of the fruiting bodies treated with control water (30.31 mg·g -1 ), which was significantly lower than that of the fruiting body of Volvariella volvacea with a concentration of 200 μM DGP (42.273 mg·g -1 )(Figure 9).

[0049] Comparative Example 3 (1) When DGP was added to a final concentration of 300 μM at 4°C, the survival rate of Volvariella volvacea was only 20%.

[0050] (2) The low temperature stress tolerance test showed that the V23 fruiting bodies treated with DGP at a final concentration of 300 μM could maintain their mushroom shape, dry surface, and no water seepage during 24 h of low temperature storage (Figure 3).

[0051] (3) The polysaccharide content of the fruiting body of Volvariella volvacea treated with 300 μM DGP was 31.34 mg·g -1 , slightly higher than the fruiting bodies treated with control water (30.31 mg·g -1 ), which was significantly lower than that of the fruiting body of Volvariella volvacea with a final concentration of 200 μM DGP (42.273 mg·g -1 )(Figure 9).

[0052] Those skilled in the art can conceive and make various other corresponding changes and deformations based on the technical solutions described above, and all of these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. A method for increasing the polysaccharide content of straw mushroom fruiting bodies, characterized in that: During the sowing period of straw mushrooms, spray a 200 μM glucose hexaphosphate disodium solution.

2. The method for increasing the polysaccharide content of the fruiting body of Volvariella volvacea according to claim 1, characterized in that: Spray 10-20 ml of 200 μM glucose hexaphosphate disodium solution per square meter of cultivation material.

3. The method for increasing the polysaccharide content of the fruiting body of Volvariella volvacea according to claim 1, characterized in that: Each bed of 1.3 m × 1.0 m was sprayed with 20 ml of 200 μM disodium glucose hexaphosphate solution.

4. A method for screening a compound for increasing the polysaccharide content of a straw mushroom fruiting body, characterized in that The steps include: The compounds to be screened were added to PDA medium and cultured on plates of Volvariella volvacea V23, and then subjected to low temperature stress at 4°C, and the survival rate was calculated; Based on the survival rate, antifreeze compounds were selected and then subjected to fruiting body antifreeze experiments; Collect antifreeze phenotypes, conduct transcriptome analysis, and analyze the antifreeze mechanism; The obtained antifreeze compound was used to spray different concentrations of Volvariella volvacea at the sowing and watering stages, with water treatment used as a control. Then, the egg-shaped fruiting bodies were collected. During the straw mushroom sowing and watering period, spray antifreeze compounds; The harvested fruiting bodies are tested for agronomic traits such as cohesion, damaged area, brittleness, resilience, hardness, gelatinity, elasticity, chewiness, and stickiness; The polysaccharide content of the fruiting body of the Volvariella volvacea is detected, and compounds that can increase the polysaccharide content of the fruiting body of the Volvariella volvacea are screened.

5. The method for screening a compound for increasing the polysaccharide content in the fruiting body of Volvariella volvacea according to claim 4, characterized in that: During the sowing and watering period of the straw mushroom, spray 200 μM disodium glucose hexaphosphate solution at an interval of 5 days, spray 20 ml per bed of 1.3 m×1.0 m, and then harvest the fruiting bodies in the egg-shaped stage.

6. The method for screening a compound for increasing the polysaccharide content in the fruiting body of Volvariella volvacea according to claim 4, 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 culture medium, respectively; while no exogenous compounds were added to the PDA culture medium of the control group. Next, 20 mL of PDA culture medium was added to a 90 mm plastic plate, and then 1 cm diameter Volvariella V23 clods were inoculated with an inoculation spatula. After inoculation, the culture medium was first cultured at 32°C for 24 h, then taken out and placed in a low-temperature environment of 4°C for 24 h, and then continued to be cultured at 32°C for 72 h.

7. Application of disodium glucose hexaphosphate in the preparation of products for increasing the polysaccharide content in straw mushroom fruiting bodies.

Images