Functional polysaccharide extracted from waste microbial culture medium as well as extraction method and application of functional polysaccharide

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to functional polysaccharide extracted from a waste microorganism culture medium and an extraction method and application thereof. According to the method, the functional polysaccharide in the waste microbial culture medium can be well extracted, separated and purified, and the functional polysaccharide has an anti-oxidation effect and also has the capability of inhibiting the formation of a biological membrane. Meanwhile, the beneficial functional polysaccharide components are extracted from the microorganism waste culture medium for the first time, a new thought is provided for recycling of a waste culture base, and the method has important significance on resource saving.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a functional polysaccharide extracted from waste microbial culture medium, its extraction method, and its application. Background Technology

[0002] Culture media, as nutrient-rich substrates for bacterial culture, are indispensable in modern microbiology. They support the cultivation of a wide variety of microorganisms, driving the development of industries such as biomedical research, industrial fermentation, and biomanufacturing.

[0003] Microbial culture generates a large amount of waste culture media. Waste culture media from industrial production typically contains residual bacteria, unused media, and metabolic byproducts. Some waste culture media may also contain antibiotic residues, heavy metals, or high salt content. These components are not only complex and pose a high risk of biotoxicity, but are also difficult to handle. The disposal of these waste culture media not only occupies valuable space resources but also poses a potential risk of environmental biopollution. Therefore, developing effective recycling and reuse strategies is crucial for reducing waste culture media, alleviating the environmental burden, and improving resource efficiency.

[0004] It is worth noting that discarded culture media are rich in many bioactive substances, such as polysaccharides or enzymes, and have great application potential in the fields of biosynthesis, drug development, and biomaterials. However, methods for extracting beneficial polysaccharides from discarded culture media and exploring and applying their bioactivity have not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to provide a functional polysaccharide extracted from waste microbial culture medium, its extraction method and application. The method can extract functional polysaccharides with antioxidant and anti-biofilm activities from waste microbial culture medium, thereby realizing the efficient resource utilization of waste biological resources.

[0006] This invention provides a method for extracting functional polysaccharides using waste microbial culture medium, comprising the following steps: The waste microbial culture medium was subjected to a first solid-liquid separation to obtain the liquid phase; The polysaccharide in the liquid phase was extracted by alcohol precipitation to obtain crude polysaccharide; The protein in the crude polysaccharide was removed, and the resulting supernatant was dialyzed to remove impurities, yielding a polysaccharide solution. The polysaccharide solution was purified by chromatography to obtain the functional polysaccharide; The waste microbial culture medium is waste Escherichia coli culture medium.

[0007] Preferably, the waste Escherichia coli culture medium is the fermentation broth from Escherichia coli cultured in LB medium to the stationary phase.

[0008] Preferably, the step of extracting polysaccharides from the liquid phase using alcohol precipitation includes: concentrating the liquid phase, mixing the concentrated supernatant with 2-3 times the volume of anhydrous ethanol, and precipitating at 4°C for 16-24 h to obtain an alcohol precipitation mixture; The alcohol-precipitated mixture was subjected to a second solid-liquid separation, and the precipitate was collected to obtain the crude polysaccharide.

[0009] Preferably, the step of removing protein from crude polysaccharide includes: dissolving the crude polysaccharide in ultrapure water, mixing the obtained crude polysaccharide solution with 1 / 2-1 / 4 volume of Sevag solution, shaking for 10-30 min, performing a third solid-liquid separation on the obtained mixture, and taking the supernatant.

[0010] Preferably, the dialysis purification step includes: dialyzing the supernatant in a dialysis bag with a molecular weight cutoff of 3500 Da for 2-3 days to obtain a polysaccharide solution.

[0011] Preferably, the chromatographic purification step includes: lyophilizing the polysaccharide solution, dissolving the lyophilized polysaccharide in a mixture of two times its weight of ultrapure water, and then loading the mixture onto a DEAE-52 cellulose ion exchange column for gradient elution to obtain the functional polysaccharide.

[0012] Preferably, the eluent used in the gradient elution includes NaCl solutions with concentrations of 0.1 M, 0.3 M, 0.4 M, 0.7 M, and 1.0 M.

[0013] The present invention also provides a functional polysaccharide, which is extracted using the method described above; the functional polysaccharide includes one or more of PS1, PS2, PS3, PS4 and PS5; PS1, PS2, PS3, PS4 and PS5 are obtained by elution and separation using NaCl solutions with concentrations of 0.1 M, 0.3 M, 0.4 M, 0.7 M and 1.0 M, respectively.

[0014] Preferably, the PS1 is composed of monosaccharides including mannose, N-acetylglucosamine and glucose.

[0015] The present invention also provides the application of the method described in the above technical solution or the functional polysaccharide described in the above technical solution in the preparation of products with antioxidant capacity and / or anti-biofilm function.

[0016] Beneficial effects: This invention provides a method for extracting functional polysaccharides from waste microbial culture medium, comprising the following steps: performing a first solid-liquid separation on the waste microbial culture medium to obtain a liquid phase; extracting polysaccharides from the liquid phase using alcohol precipitation to obtain crude polysaccharides; removing proteins from the crude polysaccharides; dialysis the obtained supernatant to remove impurities to obtain a polysaccharide solution; and purifying the polysaccharide solution by chromatography to obtain the functional polysaccharides; wherein the waste microbial culture medium is waste *E. coli* culture medium. This invention can effectively extract, separate, and purify functional polysaccharides from waste microbial culture media. These functional polysaccharides possess antioxidant capabilities and exhibit good resistance to biofilm formation, inhibiting biofilm formation. Simultaneously, it broadens the functional activity range of these polysaccharides, achieving efficient resource utilization of waste, significantly increasing added value, reducing environmental pollution, and possessing significant economic, environmental, and social benefits, with broad prospects for industrial application. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The process preparation diagrams are shown in Examples 1-2; Figure 2 This is the elution curve of the DEAE-52 cellulose ion exchange column in Example 2; Figure 3 This is a graph showing the activity screening results of PS in Example 3. Different letters indicate significant differences between treatments. P＜ 0.05 ; Figure 4 This is a graph showing the monosaccharide composition determination of PS1 in Example 4; Figure 5 The graph shows the DPPH antioxidant effect of different concentrations of PS1 in Example 5; Figure 6 The graph shows the antioxidant effect of ABTS at different concentrations of PS1 in Example 5. Figure 7 The graph shows the FRAP antioxidant effect of different concentrations of PS1 in Example 5; Figure 8 The diagram shows the effect of different concentrations of PS1 on the anti-Staphylococcus aureus biofilm in Example 6. Figure 9 The image shows the effect of different concentrations of PS1 on the anti-fluorescent Pseudomonas biofilm in Example 6. Detailed Implementation

[0019] This invention provides a method for extracting functional polysaccharides using waste microbial culture medium, comprising the following steps: The waste microbial culture medium was subjected to a first solid-liquid separation to obtain the liquid phase; The polysaccharide in the liquid phase was extracted by alcohol precipitation to obtain crude polysaccharide; The protein in the crude polysaccharide was removed, and the resulting supernatant was dialyzed to remove impurities, yielding a polysaccharide solution. The polysaccharide solution was purified by chromatography to obtain the functional polysaccharide; The waste microbial culture medium is waste Escherichia coli culture medium.

[0020] This invention performs a first solid-liquid separation on waste microbial culture medium to obtain a liquid phase. In one embodiment, the waste *E. coli* culture medium is the fermentation supernatant obtained after culturing *E. coli* in LB medium to the stationary phase; that is, the liquid after separating the bacterial cells from the *E. coli* cultured in LB medium to the stationary phase, containing unused culture medium and metabolic byproducts. In one embodiment, the first solid-liquid separation is performed by centrifugation; in another embodiment, the centrifugation speed is 8000 × g; in another embodiment, the centrifugation time is 15-30 min; in yet another embodiment, the centrifugation time is 15-20 min.

[0021] After obtaining the liquid phase, the present invention uses alcohol precipitation to extract polysaccharides from the liquid phase to obtain crude polysaccharides. As one embodiment, the step of extracting polysaccharides from the liquid phase using alcohol precipitation includes: concentrating the liquid phase; mixing the concentrated supernatant with 2-3 times its volume of anhydrous ethanol; precipitating at 4°C for 16-24 h to obtain an alcohol precipitation mixture; subjecting the alcohol precipitation mixture to a second solid-liquid separation, collecting the precipitate, and obtaining the crude polysaccharides. As one embodiment, the concentration can be vacuum concentration; as one embodiment, the vacuum concentration temperature is 45-60°C; as another embodiment, the vacuum concentration temperature is 55°C. As one embodiment, the liquid phase is concentrated to one-third of its volume. As one embodiment, the precipitation time is 16-20 h. The present invention does not specifically limit the method of the second solid-liquid separation; conventional solid-liquid separation parameters in the art can be used.

[0022] After obtaining the crude polysaccharide, the present invention removes the protein from the crude polysaccharide and dialyzes the obtained supernatant to remove impurities, obtaining a polysaccharide solution. As one embodiment, the step of removing the protein from the crude polysaccharide includes: dissolving the crude polysaccharide in ultrapure water, mixing the obtained crude polysaccharide solution with 1 / 2-1 / 4 volume of Sevag solution, shaking for 10-30 min, performing a third solid-liquid separation on the resulting mixture, and collecting the supernatant. As one embodiment, the Sevag solution is 1 / 3-1 / 4 of the volume of the crude polysaccharide solution. As one embodiment, the shaking time can be 10-20 min. As one embodiment, the third solid-liquid separation is performed by centrifugation; as another embodiment, the centrifugation speed is 6000-8000 × g; as another embodiment, the centrifugation speed is 6000 × g; as one embodiment, the centrifugation time can be 5-15 min; as another embodiment, the centrifugation time is 8 min. As one implementation method, in the process of removing proteins from the crude polysaccharide, the steps of removing proteins from the crude polysaccharide described above are repeated until no protein precipitation occurs.

[0023] In one embodiment, the dialysis purification step includes: dialyzing the supernatant in a dialysis bag with a molecular weight cutoff of 3500 Da for 2-3 days to obtain a polysaccharide solution. In one embodiment, the dialysis time is 2 days.

[0024] After obtaining the polysaccharide solution, the present invention performs chromatographic purification on the polysaccharide solution to obtain the functional polysaccharide. As one embodiment, the chromatographic purification step is as follows: the polysaccharide solution is lyophilized; the lyophilized polysaccharide is dissolved by mixing with twice its weight of ultrapure water and then loaded onto a DEAE-52 cellulose ion exchange column for gradient elution to obtain the functional polysaccharide. As one embodiment, the eluent used for the gradient elution includes NaCl solutions with concentrations of 0.1 M, 0.3 M, 0.4 M, 0.7 M, and 1.0 M.

[0025] As one embodiment, after obtaining the functional polysaccharide, the present invention further includes using a Sephadex G-100 gel column to perform further non-gradient elution of the functional polysaccharide obtained by elution with a 0.1 M NaCl solution, for further desalting and purification.

[0026] This invention also provides a functional polysaccharide, extracted using the method described above; the functional polysaccharide includes one or more of PS1, PS2, PS3, PS4, and PS5; PS1, PS2, PS3, PS4, and PS5 are obtained by elution and separation using NaCl solutions with concentrations of 0.1 M, 0.3 M, 0.4 M, 0.7 M, and 1.0 M, respectively. As one embodiment, the functional polysaccharide of this invention can be PS1 and / or PS2. As one embodiment, PS1 is composed of a monosaccharide comprising mannose, N-acetylglucosamine, and glucose.

[0027] This invention also provides the application of the method described in the above-mentioned technical solutions or the functional polysaccharides described in the above-mentioned technical solutions in the preparation of products with antioxidant capacity and / or anti-biofilm function. As one embodiment, the biofilm is a biofilm of Gram-positive bacteria and / or Gram-negative bacteria; as one embodiment, the Gram-positive bacteria is Staphylococcus aureus; the Gram-negative bacteria is Pseudomonas fluorescens. As one embodiment, the product can be a pharmaceutical and / or a health food.

[0028] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1 Preparation of crude polysaccharides, and the extraction process of crude polysaccharides as follows: Figure 1 As shown, the steps are as follows: 1) Place the waste microbial culture medium into a centrifuge tube and centrifuge at 8000 × g for 15 min; 2) Add the supernatant to a round-bottom flask and concentrate it to one-third of its volume in a rotary evaporator under reduced pressure at 55°C; 3) While stirring, add 3 times the volume of anhydrous ethanol to the concentrated supernatant, seal with plastic wrap, and precipitate at 4°C for 16 h. 4) Discard the supernatant, retain the precipitate, add an appropriate amount of ultrapure water to redissolve it, and obtain crude polysaccharide; 5) Add 1 / 4 volume of Sevag solution to the crude polysaccharide solution, shake vigorously for 10 min to fully denature the protein, centrifuge at 6000 × g for 8 min to remove the denatured protein, repeat the process until no protein precipitate is found, and determine the content of crude polysaccharide in the supernatant.

[0030] Crude polysaccharides were extracted from waste culture media of *E. coli*, waste culture media of *Bacillus subtilis*, and unused waste culture media using steps 1)-5), respectively. The content of crude polysaccharides in the supernatants obtained from the extraction using the three types of microbial waste culture media was determined. The waste culture media of *E. coli* was the fermentation supernatant obtained after isolating *E. coli* cells from LB medium after culturing them to the stationary phase; the waste culture media of *Bacillus subtilis* was the fermentation supernatant obtained after isolating *Bacillus subtilis* cells from LB medium after culturing them to the stationary phase; and the unused waste culture media was LB medium.

[0031] The results showed that the crude polysaccharide contents in the supernatants obtained from E. coli waste culture medium, Bacillus subtilis waste culture medium, and unused waste culture medium were 3.75 g / L, 3.49 g / L, and 1.18 g / L, respectively. Further purification and activity studies of the crude polysaccharides were conducted using E. coli waste culture medium as a raw material.

[0032] Example 2 The purification process of the crude polysaccharide extracted in Example 1 is as follows: Figure 1 As shown, the steps are as follows: The crude polysaccharide obtained from the waste culture of E. coli in Example 1, extracted according to steps 1)-4), was subjected to the following purification steps: 1) Add 1 / 4 volume of Sevag solution to the crude polysaccharide solution, shake vigorously for 10 min to fully denature the protein, centrifuge at 6000 × g for 8 min to remove the denatured protein, and repeat the process until no protein precipitate is found. 2) Collect the supernatant and perform dialysis for 2 days using a 3500 Da dialysis bag; 3) Freeze-dry the polysaccharide liquid obtained in the previous step; 4) Weigh the polysaccharide obtained in the previous step, dissolve it in twice the mass of ultrapure water, and use a DEAE-52 cellulose ion exchange column for gradient elution to separate the crude polysaccharide; 4.1) Sample loading: The polysaccharide solution was uniformly added dropwise to the surface of the DEAE-52 cellulose ion exchange column, and the crude polysaccharide was purified by gradient elution. 4.2) Elution with 0.1 M NaCl solution: The presence of sugar in the effluent was detected using the phenol-sulfuric acid method. The sample was then freeze-dried and labeled as PS1. 4.3) Elution with 0.3 M NaCl solution: The presence of sugar in the effluent was detected using the phenol-sulfuric acid method. The sample was then freeze-dried and labeled as PS2. 4.4) Elution with 0.4 M NaCl solution: The presence of sugar in the effluent was detected using the phenol-sulfuric acid method. The sample was then freeze-dried and labeled as PS3. 4.5) Elution with 0.7 M NaCl solution: The effluent was tested for sugar content using the phenol-sulfuric acid method, then freeze-dried and labeled as PS4. 4.6) Elution with 1.0 M NaCl solution: The presence of sugar in the effluent was detected using the phenol-sulfuric acid method, and the sample was freeze-dried and labeled as PS5; 5) Weigh the largest component PS1, dissolve it in 2 times its mass of ultrapure water, and elute it using a Sephadex G-100 gel column to further desalt and purify the PS1 polysaccharide.

[0033] Depend on Figure 1-2 It can be concluded that five polysaccharide components, PS1, PS2, PS3, PS4 and PS5, were eluted using a DEAE-52 cellulose ion exchange column. The contents and percentages of PS1, PS2, PS3, PS4 and PS5 were 0.93 g / L and 24.75%, 0.26 g / L and 7.02%, 0.50 g / L and 13.30%, 0.53 g / L and 14.04%, and 0.29 g / L and 7.76%, respectively.

[0034] Further purification using a Sephadex G-100 gel column yielded PS1 with high purity, containing 0.71 g / L and 18.84% purity.

[0035] Example 3 Screening of the activity of the purified PS polysaccharide obtained in Example 2 1) Adult AB strain zebrafish were raised under the following environmental conditions: temperature 28.0℃, and photoperiod of 14 h light / 10 hr darkness; 2) Embryos are obtained through natural mating and cultured at 28.5℃. The E3 culture medium (containing methylene blue) is changed daily, and the methylene blue is removed 3 days after fertilization (days post-fertilization, dpf). 3) At 4 dpf, zebrafish fry were randomly transferred to 12-well plates, with 10 fry per well; 4) Add E3 culture water for each of the five isolated PS samples (80 μg / mL, PS1-PS5) to each well, controlling the total volume to 2 mL, with 3 wells for each concentration; 5) Add Staphylococcus aureus to bring the concentration in the E3 culture water to 10. 7 CFU / mL; 6) Do not change the water or feed the fish during the experiment. Record the mortality of the juvenile fish after 2 days and clean up the dead fish in time.

[0036] The results are as follows Figure 3As shown, PS1 and PS2 have significant protective effects on zebrafish juveniles, resisting infections caused by pathogens and reducing mortality, with PS1 showing the best effect.

[0037] Example 4 Identification of PS1 components purified in Example 2 The monosaccharide composition of PS1 was analyzed using PMP pre-column derivatization-high performance liquid chromatography.

[0038] 1) Place 2 mg of PS1 sample in an ampoule, add an appropriate amount of 2.0 mol / L trifluoroacetic acid, mix well, fill with nitrogen, and seal the ampoule with an alcohol burner; 2) Place the sealed sample tubes in a 115℃ oven for 6-8 hours to hydrolyze completely; 3) After cooling to room temperature, use a nitrogen blower to dry the trifluoroacetic acid, add methanol and then blow dry again. Repeat this process at least three times to ensure that all trifluoroacetic acid is removed. 4) Dissolve in ultrapure water, centrifuge to remove precipitate, and keep the supernatant for later use; 5) Mix the monosaccharide samples in equimolar amounts to prepare a 2.0 mmol / L standard solution; 6) Take 450 μL of the standard mixture, monosaccharide samples (mannose, rhamnose, glucuronic acid (GlcA), galacturonic acid (GalA), ribose (Rib), N-acetylglucosamine (GlcNAc), glucose (Glc), xylose (Xyl), galactose (Gal), arabinose (Ara), and PS1 hydrolysate, respectively. Then add 450 μL of 0.5 mol / L PMP-methanol solution and 450 μL of 0.3 mol / L NaOH solution, mix well, and incubate in a 70℃ water bath for 30 min. 7) After cooling, add 450 μL of 0.3 mol / L HCl for neutralization; 8) Extract with chloroform, repeat the extraction three times, and then take the upper aqueous layer and pass it through a 0.45 μm water film for HPLC analysis; 9) Liquid Chromatography Conditions: Promosil C18 column (4.6 × 250 mm, 5 μm), UV detector 250 nm, flow rate 1.0 mL / min, column temperature 25℃, injection volume 5 µL, run time 40 min. Mobile phase A: 15% (v / v) acetonitrile + 0.05 mol / L phosphate buffer (KH₂PO₄-NaOH, pH=6.9). Mobile phase B: 40% (v / v) acetonitrile + 0.05 mol / L phosphate buffer (KH₂PO₄-NaOH, pH=6.9). Gradient mode: time gradient 0 min → 10 min → 30 min → 35 min → 40 min, corresponding solvent B concentration gradient 0 → 15% → 25% → 0.

[0039] The results are as follows Figure 4 As shown, PS1 is mainly composed of mannose, N-acetylglucosamine and glucose.

[0040] Example 5 The antioxidant activity of PS1 in Example 2 was determined by the following steps: 1) The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity is composed of the following steps: 1.1) Sample preparation: Weigh an appropriate amount of PS1 sample and positive control ascorbic acid (Vc), dissolve them in ultrapure water, and dilute to the corresponding concentration; 1.2) Mix PS1 or Vc solution with freshly prepared DPPH solution in the same volume, and use sterile PBS solution as a blank control; 1.3) All samples were reacted at room temperature in the dark for 30 min, and then centrifuged at 8000 × g for 10 min; 1.4) Subsequently, take the supernatant and analyze it at OD... 517nm (C) 517 Measure absorbance at ( ) point; 1.5) Calculate the clearance rate (E1): ; 2) The radical scavenging activity of 2,2'-azidobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) is composed of the following steps: 2.1) Mix 20 mL of 7 mmol / L ABTS solution with 358 μL of 140 mmol / L potassium persulfate solution, and then incubate at room temperature in the dark for 14 hours to obtain the working solution; 2.2) Dilute the working solution 20 times with ultrapure water before use; 2.3) Equal volumes of ABTS· + The working solution is added to the PS or Vc solution, and sterile PBS solution is used as a blank. 2.4) All samples were reacted at room temperature in the dark for 6 min, and then centrifuged at 8000 × g for 10 min; 2.5) Take the supernatant and measure the OD. 734nm (C) 734 Absorbance at ( ) 2.6) Calculate the clearance rate (E2): ; 3) The determination of ferric reducing antioxidant capacity (FRAP) consists of the following steps: 3.1) The working solution was prepared by mixing 300 mmol / L acetate buffer (pH 3.5), 10 mmol / L 2,4,6-tripyridyl-S-triazine solution (TPTZ) and 20 mmol / L FeCl3 solution in a ratio of 10:1:1 (V / V / V). 3.2) Mix the PS or Vc solution with an equal volume of FRAP working solution and react at 37°C in the dark for 30 min; 3.3) At the end of the reaction, all samples were centrifuged at 8000×g for 10 min; 3.4) Take the supernatant and measure the OD. 593nm (C) 593 Absorbance at ( ) 3.5) Calculate the relative total reduction (R, %): ; The results are as follows Figure 5-7 As shown, the scavenging rate of PS1 is generally lower than that of Vc, but both have a certain scavenging ability, and the scavenging rate gradually increases with increasing concentration. Regarding the iron ion reduction capacity, PS1 is closest to Vc; when PS1 > 500 μg / mL, the reduction capacity reverses that of Vc and approaches 100%. Based on linear fitting, PS1 shows good reduction performance against DPPH and ABTS. + Half-inhibitory concentration (IC50) of free radical scavenging 50 The concentrations were 1258.93 μg / mL and 319.89 μg / mL, respectively.

[0041] Example 6 The determination of the anti-biofilm activity of PS1 in Example 2 was performed as follows: 1) Adjust the activated strains (Staphylococcus aureus and Pseudomonas fluorescens) to 10 9 CFU / mL, and inoculated at a rate of 10% (v / v) into fresh LB medium (or different concentrations of PS, 0.25 mg / mL, 0.5 mg / mL and 1 mg / mL); 2) Take 200 μL of inoculation culture medium and add it to a 96-well plate. Set up 5 parallel wells in each group. Add 200 μL of ultrapure water to the outer wells and seal with a sealing film. 3) Incubate the 96-well plate in an incubator at 37°C for 24 h; 4) Remove the 96-well plate for crystal violet staining analysis; 5) After the biofilm forms, discard the bacterial solution and wash three times with ultrapure water; 6) Add methanol to fix for 10 min, then discard the liquid in each well; 7) Add 180 μL of 1% crystal violet solution to each well and stain for 10 min. Then discard the liquid in each well and rinse thoroughly. 8) Add 200 μL of 33% glacial acetic acid solution to each well, incubate at 37℃ for 30 min, and finally measure the OD in each well. 570nm value.

[0042] The results are as follows Figure 8-9 It is evident that PS1 exhibits anti-biofilm activity against both pathogens. Within the range of 0.25-1 mg / mL, PS1 inhibits biofilm formation of Staphylococcus aureus and Pseudomonas fluorescens in a dose-dependent manner. At a PS1 concentration of 1 mg / mL, the reduction in biofilm formation is statistically significant, with an inhibition rate of 40.86% against Staphylococcus aureus and 29.04% against Pseudomonas fluorescens. P<0.05 ).

[0043] The above examples show that PS1 extracted and purified from waste microbial culture medium has good antioxidant activity and the ability to inhibit the formation of pathogen biofilms.

[0044] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting functional polysaccharides using waste microbial culture medium, characterized in that, Includes the following steps: The waste microbial culture medium was subjected to a first solid-liquid separation to obtain the liquid phase; The polysaccharide in the liquid phase was extracted by alcohol precipitation to obtain crude polysaccharide; The protein in the crude polysaccharide was removed, and the resulting supernatant was dialyzed to remove impurities, yielding a polysaccharide solution. The polysaccharide solution was purified by chromatography to obtain the functional polysaccharide; The waste microbial culture medium is waste Escherichia coli culture medium.

2. The method according to claim 1, characterized in that, The discarded Escherichia coli culture medium is the fermentation broth from Escherichia coli cultured in LB medium to the stationary phase.

3. The method according to claim 1, characterized in that, The step of extracting polysaccharides from the liquid phase using alcohol precipitation includes: concentrating the liquid phase, mixing the concentrated supernatant with 2-3 times the volume of anhydrous ethanol, and precipitating at 4°C for 16-24 h to obtain an alcohol precipitation mixture; The alcohol-precipitated mixture was subjected to a second solid-liquid separation, and the precipitate was collected to obtain the crude polysaccharide.

4. The method according to claim 1, characterized in that, The step of removing proteins from crude polysaccharides includes: dissolving the crude polysaccharides in ultrapure water, mixing the resulting crude polysaccharide solution with 1 / 2 to 1 / 4 volume of Sevag solution, shaking for 10 to 30 minutes, performing a third solid-liquid separation on the resulting mixture, and taking the supernatant.

5. The method according to claim 1 or 4, characterized in that, The dialysis purification step includes: dialyzing the supernatant in a dialysis bag with a molecular weight cutoff of 3500 Da for 2-3 days to obtain a polysaccharide solution.

6. The method according to claim 1, characterized in that, The chromatography purification steps include: lyophilizing the polysaccharide solution, dissolving the lyophilized polysaccharide in a mixture of 2 times its weight of ultrapure water, loading the mixture onto a DEAE-52 cellulose ion exchange column for gradient elution to obtain the functional polysaccharide.

7. The method according to claim 6, characterized in that, The eluent used in the gradient elution includes NaCl solutions with concentrations of 0.1 M, 0.3 M, 0.4 M, 0.7 M, and 1.0 M.

8. A functional polysaccharide, characterized in that, The polysaccharide is extracted using the method described in any one of claims 1 to 7; the functional polysaccharide includes one or more of PS1, PS2, PS3, PS4 and PS5; PS1, PS2, PS3, PS4 and PS5 are obtained by elution and separation using NaCl solutions with concentrations of 0.1 M, 0.3 M, 0.4 M, 0.7 M and 1.0 M, respectively.

9. The functional polysaccharide according to claim 8, characterized in that, The PS1 is composed of monosaccharides including mannose, N-acetylglucosamine and glucose.

10. The use of the method according to any one of claims 1 to 7 or the functional polysaccharide according to claim 8 or 9 in the preparation of products with antioxidant capacity and / or anti-biofilm function.

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