Preparation method for efficiently preparing novel carboxymethyl pachymaran derivative

Abstract

The invention belongs to the field of microorganisms, and particularly relates to a method for efficiently preparing a novel carboxymethyl pachymaran derivative. The method comprises the following steps: firstly, inoculating poria cocos bacteria CGMCC 5.78 for first-stage fermentation; then, lysinibacillus fusiformis CGMCC 1.10295 is inoculated, a selenium source is added, second-stage co-culture is carried out, and the stage comprises a key high-temperature stimulation step and a subsequent pH adjusting step; and finally, separating and purifying the fermentation liquor to obtain the selenized carboxymethyl pachymaran. By means of the specific technology, the yield of the selenized carboxymethyl pachymaran and the selenium element conversion rate are remarkably increased, and the selenized carboxymethyl pachymaran has good DPPH free radical scavenging capacity and in-vitro anti-tumor activity. The process is green and efficient, and a new way is provided for development of the functional selenium polysaccharide.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a highly efficient method for preparing novel carboxymethyl Poria cocos polysaccharide derivatives. Background Technology

[0002] Poria cocos polysaccharides are the main bioactive components extracted from the traditional Chinese medicine Poria cocos, possessing various pharmacological activities such as immunomodulation, antitumor activity, and antioxidant activity. However, natural Poria cocos polysaccharides suffer from poor water solubility and low bioavailability, limiting their further development and application. Chemical modification, such as carboxymethylation, can significantly improve their water solubility and bioactivity, yielding carboxymethyl Poria cocos polysaccharides. However, currently, carboxymethyl Poria cocos polysaccharides are mainly derived from chemical modification after extraction from Poria cocos fruiting bodies, resulting in limited yields.

[0003] Selenium plays a vital role in the metabolic activities of organisms and is essential for human health. Selenium-containing proteins exhibit activities such as activating anti-cancer factors, inhibiting cancer cell proliferation, preventing cardiovascular disease, anti-inflammation, and anti-oxidation. Furthermore, selenium is the active center of the glutathione peroxidase (GSH-Px) family and thioredoxin reductases (TrxRs), possessing antioxidant properties in vivo. However, selenium in nature mainly exists in inorganic forms such as selenate and selenite. Naturally occurring inorganic selenium is enriched and transformed into various organic forms, including selenoamino acids and selenoprotein polysaccharides, by organisms. Selenium obtained through biotransformation is more easily absorbed and utilized by the human body than naturally occurring inorganic selenium. Therefore, how to utilize organisms for selenium enrichment and the screening of selenium-enriched organisms have become research hotspots.

[0004] The preparation of selenized polysaccharides using microbial fermentation has attracted attention due to its mild and environmentally friendly conditions. However, current research mainly focuses on single-strain fermentation systems, which often have limited selenium conversion efficiency and polysaccharide yield. Utilizing multi-strain co-culture systems to efficiently synthesize selenized polysaccharides through synergistic interactions between microorganisms remains to be explored. Therefore, developing an efficient and environmentally friendly method based on multi-strain staged co-culture to prepare high-selenium-content carboxymethyl Poria cocos polysaccharide derivatives has significant research value and application prospects. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention explores obtaining carboxymethyl Poria cocos polysaccharide through fermentation, and further investigates obtaining selenized carboxymethyl Poria cocos polysaccharide through a green biotransformation method using a dual-strain co-fermentation model. Based on this, the fermentation process is further optimized, and a green and efficient method for preparing selenized carboxymethyl Poria cocos polysaccharide is developed through staged fermentation with different fermentation conditions at different stages, providing a new technical solution for the high-value utilization of Poria cocos polysaccharide.

[0006] On one hand, the present invention provides a method for preparing selenized carboxymethyl Poria cocos polysaccharide, the method comprising the following steps: First stage fermentation: Wolfiporia cocos was inoculated into the fermentation medium for the first stage fermentation. The preservation number of Wolfiporia cocos was CGMCC 5.78. Second-stage co-cultivation: After the first-stage fermentation is completed, *Lysinibacillus fusiformis* is inoculated into the fermentation system, and a selenium source is added for the second-stage co-cultivation. The preservation number of *Lysinibacillus fusiformis* is CGMCC 1.10295. The second-stage co-cultivation includes a high-temperature stimulation step and a pH adjustment step. Fermentation broth treatment: After co-culture, selenized carboxymethyl poria polysaccharide was isolated and purified from the fermentation broth.

[0007] Furthermore, in the method, the second stage of co-cultivation specifically includes: After inoculating with Bacillus fusiformis and adding selenium source, cultured with shaking at 27-28℃ for 1 day; The temperature was then raised to 36-37°C, and the culture was subjected to high-speed shaking for 1-2 hours to complete the high-temperature stimulation. Then lower the temperature to 25-26℃ and adjust the pH of the fermentation broth to 6.8-7.0, and continue to culture with shaking for 2-3 days.

[0008] Furthermore, in the method, the inoculum amount of Wolfiporia cocos in the first stage of fermentation is 5%-6% of the total mass of the fermentation medium.

[0009] Furthermore, in the method, the inoculum size of *Lysinibacillus fusiformis* in the second stage co-culture is 8%-12% of the fermentation broth volume in the first stage.

[0010] Furthermore, in the method, the selenium source is sodium selenite, and its addition amount in the second stage co-culture is 0.8%-1.2% of the total volume of the first stage fermentation broth, and the concentration of the sodium selenite is 5mM.

[0011] Secondly, selenized carboxymethyl poria cocos polysaccharide prepared by the method described in this invention is also provided.

[0012] Furthermore, the selenium content in the selenized carboxymethyl Poria cocos polysaccharide is not less than 150 μg / g.

[0013] Thirdly, the present invention provides the application of selenized carboxymethyl poria cocos polysaccharide in the preparation of antioxidants.

[0014] Finally, a fermentation medium for use in the method described in this invention is also provided, which comprises sodium carboxymethyl cellulose.

[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (1) This invention innovatively adopts a time-sequential, phased co-culture strategy of "Poria cocos bacteria and Bacillus fusiformis", combining the key steps of "high temperature stimulation" and "later pH adjustment" to stimulate metabolic synergy between the two bacteria and achieve synergistic effect.

[0016] (2) The selenium content of the product prepared by the present invention is 168.13-315.04 μg / g, which is more than 23 times that of the selenium content of the single strain fermentation product (13.27 μg / g), indicating that the specific process sequence of the present invention significantly promotes the efficient conversion of inorganic selenium to organic selenium polysaccharide.

[0017] (3) The selenized carboxymethyl poria cocos polysaccharide prepared in this invention exhibits a DPPH free radical scavenging rate comparable to that of the standard antioxidant vitamin C at a concentration of 4 mg / mL, demonstrating extremely strong free radical scavenging ability. Furthermore, at concentrations of 0.5 mg / mL and above, the product of this invention shows a significantly higher inhibition rate against human lung cancer cells A549 than that of unselenized carboxymethyl poria cocos polysaccharide, indicating that selenization modification enhances the in vitro antitumor activity of carboxymethyl poria cocos polysaccharide.

[0018] (4) This invention uses biological fermentation throughout the process, avoiding the toxic reagents used in chemical synthesis. The conditions are mild, making it an environmentally friendly green production process. At the same time, this method realizes the high-value development and utilization of the traditional Chinese medicine Poria cocos, providing an efficient technical approach for the development of functional selenium polysaccharides.

[0019] In summary, this invention achieves efficient and green preparation of selenized carboxymethyl Poria cocos polysaccharide through a unique combination of strains, specific high-temperature stimulation, and pH adjustment process control during the post-fermentation stage. The resulting product exhibits significant advantages in terms of yield, selenium content, and bioactivity, and has good market application value. Attached Figure Description

[0020] Figure 1 Figure 1 shows the results of DPPH free radical scavenging rate determination for selenized carboxymethyl Poria cocos polysaccharide prepared by different fermentation methods.

[0021] Figure 2 Figure 1 shows the results of the inhibition rate of selenized carboxymethyl poria cocos polysaccharide prepared by different fermentation methods on human lung cancer cells A549. Detailed Implementation

[0022] 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.

[0023] 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 in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0024] 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.

[0025] 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.

[0026] Lysinibacillus fusiformis, accession number CGMCC 1.10295, was purchased from the China General Microbiological Culture Collection Center and is hereinafter referred to as Lysinibacillus fusiformis CGMCC1.10295.

[0027] Example 1 This example describes the preparation of a seed culture medium for Poria cocos CGMCC 5.78.

[0028] 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.

[0029] Example 2 This example describes the preparation of seed culture of Bacillus fusiformis CGMCC 1.10295.

[0030] Bacillus fusiformis CGMCC 1.10295 was taken out of the -80℃ freezer, streaked on LB solid medium for activation, and cultured upside down at 30℃ for 1 day. One loopful of activated Bacillus fusiformis CGMCC 1.10295 was picked up with an inoculation loop and inoculated into 250 mL of LB liquid medium. The medium was cultured at 30℃ with shaking at 180 r / min for 1 day to obtain the seed culture of Bacillus fusiformis CGMCC 1.10295.

[0031] Example 3 This embodiment describes the preparation of selenized carboxymethyl poria cocos polysaccharide by staged co-culturing of Poria cocos CGMCC 5.78 and Bacillus fusiformis CGMCC 1.10295.

[0032] Fermentation medium: 20g glucose, 3.5g yeast extract, 3.5g peptone, 1g K2HPO4, 0.5g MgSO4·7H2O, 20g sodium carboxymethyl cellulose, 1L distilled water, initial pH 5.4.

[0033] First stage of fermentation: 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 3 days to obtain the first stage fermentation broth.

[0034] Second stage of fermentation: The *Bacillus fusiformis* CGMCC 1.10295 seed culture prepared in Example 2 was inoculated at 8% of the first-stage fermentation broth volume. Simultaneously, 5 mM sodium selenite was added at 1.0% of the total first-stage fermentation broth volume. The mixture was co-cultured at 27°C with shaking at 160 rpm for 1 day, then the temperature was increased to 36°C and co-cultured with shaking at 200 rpm for 1 hour. Afterward, the temperature was lowered to 25°C, and the pH of the fermentation broth was adjusted to 7.0 with sodium hydroxide. The mixture was then co-cultured with shaking at 160 rpm for 2 days to obtain the co-culture fermentation broth. This broth was labeled as #1 co-culture fermentation broth.

[0035] Example 4 This embodiment describes the preparation of selenized carboxymethyl poria cocos polysaccharide by staged co-culturing of Poria cocos CGMCC 5.78 and Bacillus fusiformis CGMCC 1.10295.

[0036] Fermentation medium: 30g glucose, 3.5g yeast extract, 3.5g peptone, 1g K2HPO4, 0.5g MgSO4·7H2O, 15g sodium carboxymethyl cellulose, 1L distilled water, initial pH 5.6.

[0037] First stage of fermentation: The seed culture broth 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. The medium was cultured at 28°C with shaking at 180 rpm for 4 days to obtain the first stage fermentation broth. This broth was labeled as #2 co-culture fermentation broth.

[0038] Second stage of fermentation: The seed culture of *Bacillus fusiformis* CGMCC 1.10295 prepared in Example 2 was inoculated at 10% of the volume of the first-stage fermentation broth. At the same time, 5 mM sodium selenite was added at 0.8% of the total volume of the first-stage fermentation broth. The culture was co-cultured at 28°C and 160 r / min for 1 day with shaking. Then the temperature was raised to 37°C and co-cultured at 220 r / min for 1.5 h with shaking. After that, the temperature was lowered to 26°C, the pH of the fermentation broth was adjusted to 6.9 with sodium hydroxide, and the culture was co-cultured at 160 r / min for 3 days with shaking to obtain the co-culture fermentation broth.

[0039] Example 5 This embodiment describes the preparation of selenized carboxymethyl poria cocos polysaccharide by staged co-culturing of Poria cocos CGMCC 5.78 and Bacillus fusiformis CGMCC 1.10295.

[0040] Fermentation medium: 40g glucose, 3.5g yeast extract, 3.5g peptone, 1g K2HPO4, 0.5g MgSO4·7H2O, 15g sodium carboxymethyl cellulose, 1L distilled water, initial pH 5.5.

[0041] First stage of fermentation: 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 28°C and 180 r / min for 5 days to obtain the first stage fermentation broth.

[0042] Second stage of fermentation: The *Bacillus fusiformis* CGMCC 1.10295 seed culture prepared in Example 2 was inoculated at 12% of the first-stage fermentation broth volume. Simultaneously, 5 mM sodium selenite was added at 1.2% of the total first-stage fermentation broth volume. The mixture was co-cultured at 28°C with shaking at 160 rpm for 1 day, then the temperature was increased to 37°C and co-cultured with shaking at 220 rpm for 2 hours. Afterward, the temperature was lowered to 26°C, and the pH of the fermentation broth was adjusted to 6.8 with sodium hydroxide. The mixture was then co-cultured with shaking at 170 rpm for 2 days to obtain the co-culture fermentation broth. This broth was labeled as co-culture fermentation broth #3.

[0043] Comparative Example 1 The difference between this comparative example and Example 5 is that only a single strain of *Poria cocos* CGMCC 5.78 was used for staged culture, and the seed culture of *Bacillus fusiformis* CGMCC 1.10295 in the second stage of fermentation was replaced with the seed culture of *Poria cocos* CGMCC 5.78. All other conditions were the same as in Example 5. The prepared fermentation broth was labeled as #1 control fermentation broth.

[0044] Comparative Example 2 The difference between this comparative example and Example 5 is that only a single strain of *Bacillus fusiformis* CGMCC 1.10295 was used for staged culture, and the *Poria cocos* CGMCC 5.78 seed culture in the first stage of fermentation was replaced with *Bacillus fusiformis* CGMCC 1.10295 seed culture. All other conditions were the same as in Example 5. The prepared fermentation broth was labeled as #2 control fermentation broth.

[0045] Comparative Example 3 The difference between this comparative example and Example 5 is that the second stage of fermentation does not involve high-temperature stimulation. The second stage of fermentation is specifically as follows: The *Bacillus fusiformis* CGMCC 1.10295 seed culture prepared in Example 2 was inoculated at 12% of the first-stage fermentation broth volume. Simultaneously, 5 mM sodium selenite was added at 1.2% of the total first-stage fermentation broth volume. The mixture was co-cultured at 28°C with shaking at 160 rpm for 1 day, followed by co-culture at 28°C with shaking at 220 rpm for 2 hours. The temperature was then lowered to 26°C, and the pH of the fermentation broth was adjusted to 6.8 with sodium hydroxide. The mixture was then co-cultured at 170 rpm for 2 days to obtain the co-culture fermentation broth. This broth was labeled as control fermentation broth #3.

[0046] Comparative Example 4 The difference between this comparative example and Example 5 is that the pH value is not adjusted in the second stage of fermentation. The second stage of fermentation is as follows: The *Bacillus fusiformis* CGMCC 1.10295 seed culture prepared in Example 2 was inoculated at 12% of the first-stage fermentation broth volume. Simultaneously, 5 mM sodium selenite was added at 1.2% of the total first-stage fermentation broth volume. The mixture was co-cultured at 28°C with shaking at 160 rpm for 1 day, followed by co-culture at 28°C with shaking at 220 rpm for 2 hours. Then, the temperature was lowered to 26°C and co-cultured at 170 rpm for 2 days to obtain the co-culture fermentation broth. This broth was labeled as control fermentation broth #4.

[0047] Example 6 This embodiment extracts selenized carboxymethyl poria polysaccharide from different fermentation broths.

[0048] 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, selenized carboxymethyl poria cocos polysaccharide was extracted from each test group.

[0049] 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 volume, then sonicate on ice for 30 min (ultrasonic disruption power 120W). Subsequently, centrifuge at 8000 r / min, 4℃ for 15 min, and collect the supernatant from each group.

[0050] 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. Ten 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 selenized carboxymethyl poria cocos polysaccharide was obtained. Sevag reagent (chloroform:n-butanol = 4:1) was added to the crude selenized carboxymethyl poria cocos polysaccharide to remove proteins. This process was repeated three times. The supernatant polysaccharide solution was carefully aspirated and dialyzed against distilled water for 48 hours (with 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 selenized carboxymethyl poria cocos polysaccharide. The mass of selenized carboxymethyl poria cocos polysaccharide from each group was weighed, and the yield of selenized carboxymethyl poria cocos polysaccharide from each group was calculated. The measurement results are shown in Table 1.

[0051] Table 1 shows that no selenized carboxymethyl pachymansia was obtained from the fermentation of the single strain *Bacillus fusiformis* CGMCC 1.10295, which is in line with expectations. The yield of selenized carboxymethyl pachymansia from the fermentation of the single strain *Poria cocos* CGMCC 5.78 was 5.3 g / L, which is related to the *Poria cocos*'s ability to convert selenium. The yield of selenized carboxymethyl pachymansia in the co-culture broths of strains 1 and 3 was significantly higher than that of the single-strain fermentation group, suggesting that the staged co-culture of the two strains can synergistically induce the synthesis and secretion of selenized carboxymethyl pachymansia. The yield of selenized carboxymethyl pachymansia in the co-culture broths of strains 1 and 3 was nearly twice that of the control broth of strain 3, and more than twice that of the control broth of strain 4, indicating that high temperature stimulation and pH adjustment in the later stages of fermentation have a synergistic effect in promoting the production of selenized carboxymethyl pachymansia in the staged co-culture process of the two strains.

[0052] The selenium content in selenized carboxymethyl poria cocos polysaccharide in each group was determined according to the method in GB 5009.93-2017 National Food Safety Standard - Determination of Selenium in Food (since the selenized carboxymethyl poria cocos polysaccharide in the No. 2 fermentation broth prepared in Comparative Example 2 was extremely low, its selenium content was not measured). The results are shown in Table 2.

[0053]

[0054] Table 2 shows that the selenium content in the selenized carboxymethyl pachymansia polysaccharide of the co-culture fermentation broths #1-#3 was significantly higher than that in the control fermentation broths #1, #3, and #4. The selenium content in the co-culture fermentation broth of selenized carboxymethyl pachymansia polysaccharide of #3 was 315.04 μg / g, approximately 23.7 times that in the control fermentation broth #1, and 3.05 times that in the control fermentation broth #4. The selenium content in the co-culture fermentation broths of #1 and #2 was 236.52 μg / g and 168.13 μg / g, respectively, both significantly higher than that in the single-strain and simultaneous fermentation groups. The selenium content in selenized carboxymethyl poria cocos polysaccharide in the No. 1 co-culture broth was extremely low (13.27 μg / g), indicating that although *Poria cocos* (CGMCC 5.78) can produce selenized carboxymethyl poria cocos polysaccharide when fermented alone, its ability to integrate selenium is limited. This suggests that the staged co-fermentation of *Bacillus fusiformis* CGMCC 1.10295 and *Poria cocos* CGMCC 5.78 promotes the production of selenized carboxymethyl poria cocos polysaccharide and improves the biotransformation of selenium. Furthermore, the selenium content in selenized carboxymethyl poria cocos polysaccharide in the No. 1 co-culture broth and the No. 3 co-culture broth was 2-3 times higher than that in the No. 3 and No. 4 co-culture broths, suggesting that high-temperature stimulation and pH adjustment in the later stages of fermentation also have a synergistic effect on selenium transformation in the staged co-culture process with two strains. This may be because short-term high temperatures can act as a "stress signal," activating the stress metabolic pathway of Bacillus spindleii, thereby greatly accelerating the selenium conversion process and producing a large number of active selenium intermediates.

[0055] High temperatures may temporarily alter the permeability of the *Poria cocos* cell membrane, promoting the transfer and uptake of active selenium intermediates from *Bacillus* to *Poria cocos*. Simultaneously, stress may also stimulate the primary and secondary metabolism of *Poria cocos*, making it more "eager" to utilize these selenium precursors to synthesize selenized polysaccharides with stress-resistant functions to protect itself. Furthermore, *Poria cocos* and *Bacillus* have different optimal growth pH values. Initial acidity favors the growth of *Poria cocos*, while a later shift to neutrality may simultaneously optimize the enzyme activities of both bacteria, particularly the activities of selenotransferases or related synthetic enzymes responsible for the final covalent attachment of active selenium precursors to the polysaccharide chain. Moreover, the later, more neutral environment is more conducive to the stability of selenized polysaccharides.

[0056] Example 7 This embodiment is for determining the bioactivity of selenized carboxymethyl poria cocos polysaccharide.

[0057] The selenized carboxymethyl poria polysaccharide prepared from the co-culture fermentation broth of 1#-3# was mixed and then formulated into different concentrations, which were labeled as selenized carboxymethyl poria polysaccharide of different concentrations, for later use.

[0058] (1) Monitoring of antioxidant activity of selenium-carboxymethyl poria cocos polysaccharide A 30 mmol / L DPPH solution was prepared using anhydrous ethanol. The test solutions of different concentrations were thoroughly mixed with the prepared DPPH solution at a ratio of 1:30. The mixture was reacted in the dark for 30 min, and the absorbance was measured at 517 nm. Vitamin C was used as a positive control (Vc's DPPH free radical scavenging rate was 100%). The DPPH free radical scavenging rate (%) was calculated using the following formula:

[0059] In the formula, A1 represents the absorbance of the mixture of the test solution and DPPH. A2: Absorbance of the mixture of the test solution and anhydrous ethanol A0: Absorbance of the DPPH and ultrapure water mixture The measurement results are as follows Figure 1 As shown, the concentration of selenized carboxymethyl poria cocos polysaccharide prepared by the co-culture fermentation broths of 1#-3# exhibits a dose-dependent relationship on the DPPH free radical scavenging rate within the range of 0.1-2 mg / mL. When the concentration of selenized carboxymethyl poria cocos polysaccharide prepared by the co-culture fermentation broths of 1#-3# reaches 4 mg / mL, the DPPH free radical scavenging rate of selenized carboxymethyl poria cocos polysaccharide prepared by the co-culture fermentation broths of 1#-3# reaches the maximum, and is almost the same as the DPPH free radical scavenging ability of Vc, indicating that the selenized carboxymethyl poria cocos polysaccharide prepared by the method of the present invention has extremely strong free radical scavenging activity.

[0060] (2) Detection of in vitro antitumor activity of selenized carboxymethyl poria cocos polysaccharide Human lung cancer cells (A549) were cultured in DMEM high-glucose medium supplemented with 10% (v / v) fetal bovine serum and 1% penicillin / streptomycin (100 U / mL penicillin and 100 mg / mL streptomycin). The in vitro antitumor activity of selenized carboxymethyl pachymansia polysaccharide prepared from the co-culture fermentation broth (cells 1-3) was assessed using a CCK-8 assay. Carboxymethyl pachymansia polysaccharide (CMP, 90% purity, 0.5% carboxymethyl substitution) served as a positive control. Cells were treated with 0.05-1.0 mg / mL selenized carboxymethyl pachymansia polysaccharide and CMP for 48 h, respectively, followed by incubation at 37°C for 1 h at 20 μL of CCK-8 solution. The absorbance at 450 nm was then measured. The cancer cell inhibition rate was calculated.

[0061]

[0062] In the formula, A0 represents the absorbance of the culture medium without the sample. A1: Absorbance of the culture medium in the sample A2: Absorbance without cell culture medium The measurement results are as follows Figure 2All samples showed concentration-dependent inhibition; within the concentration range of 0.05 mg / mL to 0.75 mg / mL, the inhibition rate against cancer cells significantly increased with increasing concentration. The positive control CMP reached a plateau after 0.1 mg / mL, maintaining an inhibition rate of approximately 70%, and no longer significantly increased with concentration. At low concentrations (0.05-0.1 mg / mL), unselenized CMP showed a higher inhibition rate. However, at concentrations of 0.5 mg / mL and above, the selenized carboxymethyl poria cocos polysaccharide prepared from the co-culture fermentation broths of cells 1#-3# showed a significantly higher inhibition rate against human lung cancer cells (A549) than CMP, demonstrating a stronger inhibition rate against A549 cells than CMP. This indicates that at low concentrations, selenization may alter the mechanism of action of the polysaccharide or its initial interaction with cells, resulting in a slightly lower initial activity than CMP, while at medium to high concentrations, selenization significantly enhanced the in vitro antitumor activity of carboxymethyl poria cocos polysaccharide.

[0063] 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 preparing selenized carboxymethyl Poria cocos polysaccharide, characterized in that, Includes the following steps: First stage fermentation: Wolfiporia cocos was inoculated into the fermentation medium for the first stage fermentation. The preservation number of Wolfiporia cocos is: CGMCC 5.

78. Second-stage co-cultivation: After the first-stage fermentation is completed, *Lysinibacillus fusiformis* is inoculated into the fermentation system, and a selenium source is added for the second-stage co-cultivation. The preservation number of *Lysinibacillus fusiformis* is CGMCC 1.10295. The second-stage co-cultivation includes a high-temperature stimulation step and a pH adjustment step. Fermentation broth treatment: After co-culture, selenized carboxymethyl poria polysaccharide was isolated and purified from the fermentation broth.

2. The method according to claim 1, characterized in that, The second phase of co-training specifically includes: After inoculating with Bacillus fusiformis and adding a selenium source, the bacteria were cultured with shaking at 27-28℃ for 1 day. The temperature was then raised to 36-37°C, and the culture was subjected to high-speed shaking for 1-2 hours to complete the high-temperature stimulation. Then lower the temperature to 25-26℃ and adjust the pH of the fermentation broth to 6.8-7.0, and continue to culture with shaking for 2-3 days.

3. The method according to claim 1 or 2, characterized in that, The inoculum size of *Wolfiporia cocos* in the first stage of fermentation is 5%-6% of the total mass of the fermentation medium.

4. The method according to claim 1 or 2, characterized in that, The inoculum size of *Lysinibacillus fusiformis* in the second stage of co-culture is 8%-12% of the fermentation broth volume in the first stage.

5. The method according to claim 1 or 2, characterized in that, The selenium source is sodium selenite, which is added in the second stage of co-culture at a rate of 0.8%-1.2% of the total volume of the fermentation broth in the first stage, and the concentration of sodium selenite is 5 mM.

6. A selenized carboxymethyl poria cocos polysaccharide prepared by the method according to any one of claims 1-5.

7. The selenized carboxymethyl poria cocos polysaccharide according to claim 6, characterized in that, The selenium content of the selenized carboxymethyl poria polysaccharide is not less than 150 μg / g.

8. The use of the selenized carboxymethyl poria cocos polysaccharide according to claim 6 or 7 in the preparation of antioxidants.

9. The use of a fermentation medium in the method according to any one of claims 1-5, characterized in that, The fermentation medium contains sodium carboxymethyl cellulose.

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