Application of deacetylated poly-β-(1,6)-N-acetylglucosamine dPNAG in the preparation of antibacterial and antioxidant products

Abstract

This invention relates to the application of deacetylated poly-β-(1,6)-N-acetylglucosamine (dPNAG) in the preparation of antibacterial and antioxidant products, belonging to the field of biotechnology. Using PNAG supernatant obtained from recombinant microbial fermentation as raw material, this invention prepares dPNAG with a free amino structure through deproteinization, alcohol precipitation, desalting, and alkaline deacetylation treatment. Furthermore, this invention is the first to discover that dPNAG possesses excellent antibacterial and antioxidant properties; therefore, dPNAG has promising application prospects and industrialization potential in antibacterial materials, functional biomaterials, food, pharmaceuticals, and cosmetics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of deacetylated poly-β-(1,6)-N-acetylglucosamine dPNAG in the preparation of antibacterial and antioxidant products. Background Technology

[0002] Poly-β-(1,6)-N-acetylglucosamine (PNAG, also known as poly-β-(1,6)-N-acetylglucosamine) is a structural polysaccharide widely found in various microbial biofilms, playing an important role in bacterial adhesion, biofilm stability maintenance, and immune escape. PNAG is a water-insoluble polysaccharide composed of N-acetylglucosamine (GlcNAc; NAG) polymerized in tandem via β1-6 glycosidic bonds. Existing research indicates that PNAG is an important component of biofilms in various pathogenic bacteria and has a key influence on the occurrence of pathogenic bacterial infections. Currently, related research mainly focuses on the biological functions of PNAG in pathogenic mechanisms, biofilm formation regulation, and as a candidate vaccine antigen.

[0003] Because PNAG is almost insoluble in water and uncharged, it exists in a biologically inert state. One possible way to activate PNAG is by removing its acetyl groups, giving it a positive charge. For example, studies have shown that the deacetylation of PNAG plays a crucial role in stimulating its participation in bacterial adhesion, biofilm formation, and host immune recognition, while PNAG without acetylation exhibits significant limitations in these functions. Therefore, deacetylated PNAG (dPNAG) is considered an important structural form with practical biological functions. Furthermore, low-degree deacetylated dPNAG (around 45% moderate deacetylation) can serve as an antigen, strongly stimulating the human immune response. Consequently, there are reports of developing moderately deacetylated dPNAG into novel glycoprotein vaccines that can induce broad-spectrum protective antibodies.

[0004] Unlike previous studies, this patent is the first to discover that dPNAG with a degree of deacetylation higher than 60% has strong direct antibacterial activity and significant antioxidant activity, and has good application prospects and industrialization potential in the fields of antibacterial materials, functional biomaterials, food, medicine and cosmetics. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the lack of research on dPNAG in the prior art.

[0006] To address the aforementioned technical problems, this invention provides the application of deacetylated poly-β-(1,6)-N-acetylglucosamine (dPNAG) in the preparation of antibacterial and antioxidant products. This invention uses PNAG supernatant obtained from recombinant microbial fermentation as raw material, and through deproteinization, alcohol precipitation, desalting, and alkaline deacetylation treatment, prepares dPNAG with a free amino structure. Furthermore, this invention is the first to discover that dPNAG possesses excellent antibacterial and antioxidant effects, and has broad application value in in vitro antibacterial and antioxidant processes. Specifically, dPNAG exhibits a significant growth inhibitory effect on Staphylococcus aureus and can be used in the preparation of antibacterial products, especially in vitro antibacterial products. In addition, dPNAG has good scavenging ability against DPPH radicals, ABTS radicals, and hydroxyl radicals (·OH), and also has antioxidant capacity against reducing iron ions, making it applicable in the preparation of antioxidant products.

[0007] The first objective of this invention is to provide the application of deacetylated poly-β-(1,6)-N-acetylglucosamine dPNAG in the preparation of antibacterial products.

[0008] Further, the degree of deacetylation of the deacetylated poly-β-(1,6)-N-acetylglucosamine dPNAG is 60-100%. Preferably, the degree of deacetylation of the deacetylated poly-β-(1,6)-N-acetylglucosamine dPNAG is 60-80%.

[0009] Further, the deacetylated poly-β-(1,6)-N-acetylglucosamine is prepared by the following method:

[0010] S1. Construct recombinant Corynebacterium glutamicum expressing IcaA, IcaC, IcaD, GlmS, GlmM and GlmU, ferment the recombinant Corynebacterium glutamicum to obtain a fermentation culture broth, and centrifuge the fermentation culture broth for the first time to obtain the fermentation supernatant;

[0011] S2. Add 2% sodium hydroxide (by mass) to the fermentation supernatant of S1 for deproteinization treatment to obtain the first treatment liquid;

[0012] S3. Add ethanol to the first treatment solution for the first alcohol precipitation, and after washing, obtain poly-β-(1,6)-N-acetylglucosamine precipitate.

[0013] S4. The poly-β-(1,6)-N-acetylglucosamine precipitate of S3 is dissolved in an alkaline solution for deacetylation treatment, and then ethanol is added for a second alcohol precipitation. After purification, the deacetylated poly-β-(1,6)-N-acetylglucosamine is obtained.

[0014] Further, in step S2, the temperature of the deproteinization treatment is 60-80℃; in step S4, the temperature of the deacetylation treatment is 30-80℃.

[0015] Furthermore, the alkaline solution includes sodium hydroxide.

[0016] A second objective of the present invention is to provide an antimicrobial product comprising deacetylated poly-β-(1,6)-N-acetylglucosamine dPNAG.

[0017] Furthermore, the antibacterial product inhibits bacterial growth.

[0018] Furthermore, the bacteria include Staphylococcus aureus.

[0019] Furthermore, the antibacterial product can be used for in vitro antibacterial activity.

[0020] A third objective of this invention is to provide the application of deacetylated poly-β-(1,6)-N-acetylglucosamine dPNAG in the preparation of antioxidant products.

[0021] Furthermore, the antioxidant product works by scavenging free radicals.

[0022] A fourth objective of this invention is to provide an antioxidant product comprising deacetylated poly-β-(1,6)-N-acetylglucosamine dPNAG.

[0023] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0024] This invention reveals that dPNAG possesses excellent antibacterial and antioxidant properties. Specifically, dPNAG exhibits a significant growth-inhibiting effect on Staphylococcus aureus and can be used in the preparation of antibacterial products, particularly in vitro antibacterial products. Furthermore, dPNAG demonstrates good scavenging ability against DPPH radicals, ABTS radicals, and hydroxyl radicals (·OH), and also possesses antioxidant capacity against reduced iron ions, making it suitable for the preparation of antioxidant products. Attached Figure Description

[0025] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0026] Figure 1 This is a flowchart of the preparation method of dPNAG;

[0027] Figure 2 This is a molecular weight diagram of dPNAG;

[0028] Figure 3It is a dPNAG one-dimensional proton NMR spectrum ( 1 H NMR structural analysis diagram;

[0029] Figure 4 It is a dPNAG Fourier transform infrared spectrum;

[0030] Figure 5 This is a graph showing the antibacterial activity of dPNAG;

[0031] Figure 6 This is a graph showing the antioxidant activity of dPNAG. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0033] (a) Strains and plasmids

[0034] Strains: Corynebacterium glutamicum and Staphylococcus aureus, with Corynebacterium glutamicum serving as the host strain for the fermentation preparation of PNAG, and Staphylococcus aureus serving as the indicator strain for evaluating antimicrobial activity.

[0035] Plasmids: Commercial vectors that stably express exogenous genes in Corynebacterium glutamicum, such as pXMJ19 and pEC-XK99E.

[0036] (II) Culture medium and solution formulation

[0037] LB medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L. Solid medium contains 20 g / L agar.

[0038] BHI: Brain and heart extract 37g / L, sorbitol 91g / L.

[0039] Fermentation medium: glucose 40g / L, corn syrup 20g / L, (NH4)2SO4 20g / L, KH2PO4 1g / L.

[0040] K2HPO4 1g / L, MgSO4 0.25g / L, MOPS (3-morpholinopropanesulfonic acid) 42g / L, pH 7.0.

[0041] MH medium: MH broth 21 g / L

[0042] dPNAG solution: dPNAG 5.12 g / L, 0.2M sodium chloride, pH 6.0-7.0.

[0043] (III) Antibacterial activity assay

[0044] The antibacterial activity of dPNAG was evaluated using the 96-well plate microbroth dilution method, in accordance with the relevant standards published by the Clinical and Laboratory Standards Institute (CLSI), and its minimum inhibitory concentration (MIC) was determined.

[0045] After culturing the indicator strain to the logarithmic growth phase, a standardized bacterial suspension was prepared and diluted to a working concentration suitable for the microbroth dilution method (1 × 10⁻⁶ viable cells). 5 CFU / mL). The above dPNAG solution was serially diluted, and the resulting dPNAG solutions of different concentrations were mixed with the working bacterial culture and cultured. Bacterial culture control wells (positive control) and culture medium blank control wells (negative control) were also set up. After the reaction system was incubated at 37°C with shaking for a certain period of time, the absorbance (OD) of the bacterial culture at 600 nm was measured. 600 To evaluate the inhibitory effect of dPNAG on bacterial growth.

[0046] The lowest concentration of dPNAG that can completely inhibit visible bacterial growth under the above experimental conditions is taken as its minimum inhibitory concentration.

[0047] (iv) Antioxidant activity assay

[0048] (1) Determination of DPPH free radical scavenging ability

[0049] The antioxidant capacity of dPNAG was evaluated using the DPPH radical scavenging method. DPPH reagent was a commercially available analytical grade reagent, and the DPPH working solution was prepared in-house using standard methods. Different concentrations of dPNAG solutions were mixed with the DPPH working solution, reacted under light-protected conditions, and the absorbance changes were measured. The radical scavenging rate was then calculated.

[0050] (2) Determination of ABTS free radical scavenging ability

[0051] The ABTS radical scavenging ability of dPNAG was determined using a commercially available ABTS antioxidant assay kit. Following the kit's instructions, the dPNAG solution was mixed with the ABTS working system, and the absorbance change was measured under specified conditions. The ABTS radical scavenging ability of dPNAG was evaluated using the calculation method provided in the kit.

[0052] (3) Determination of hydroxyl radical (·OH) scavenging ability

[0053] The ·OH scavenging ability of dPNAG was evaluated using a commercially available hydroxyl radical scavenging assay kit. Following the kit instructions, the scavenging effect of dPNAG on hydroxyl radicals was calculated by measuring changes in the absorbance of the reaction system.

[0054] (4) FRAP antioxidant capacity determination

[0055] The reducing power of dPNAG was determined using a commercially available ferric reduction antioxidant capacity (FRAP) assay kit. The dPNAG sample solution was added to the kit's reaction system, and the absorbance change was measured after a specified reaction time. The ferric reduction capacity of dPNAG was evaluated using the standard system provided in the kit as a reference.

[0056] Example 1: Preparation of dPNAG

[0057] (1) Fermentation preparation of PNAG

[0058] The recombinant bacterial strain and fermentation recombinant strain were constructed according to the method described in patent publication number CN120082492A. Using *Corynebacterium glutamicum* as the host, a recombinant strain capable of synthesizing PNAG was constructed. The recombinant strain expressed the synthase subunits IcaA, IcaC, and IcaD required for PNAG synthesis, and simultaneously expressed the enzymes GlmS, GlmM, and GlmU related to the UDP-N-acetylglucosamine synthesis pathway. The construction of the recombinant strain and the disclosure of related genes are described in patent publication number CN120082492A.

[0059] The recombinant Corynebacterium glutamicum was inoculated into a fermentation medium and cultured in shake flasks at 28-30°C for 24-72 h. After the culture was completed, the bacterial cells were removed by centrifugation, and the fermentation supernatant was collected. The fermentation supernatant contained PNAG in extracellular form.

[0060] (2) Deproteinization and preliminary separation of PNAG in fermentation supernatant

[0061] Add an alkaline solution (2% (m / v) NaOH) to the PNAG fermentation supernatant to make the system alkaline, and treat it at 60-80℃ for 2 hours to remove residual protein components in the fermentation system and prevent them from forming complexes with PNAG.

[0062] After deproteinization, the treatment solution was adjusted to a slightly alkaline state (pH 7.5-8.5), followed by the addition of 4-5 volumes of pre-cooled ethanol. The solution was then allowed to stand at -20°C for 8-12 hours to allow PNAG to precipitate. The precipitate was collected after centrifugation to obtain crude PNAG precipitate.

[0063] (3) Desalination and decolorization treatment of PNAG

[0064] The crude PNAG precipitate was resuspended in ethanol solution and washed repeatedly by centrifugation to remove inorganic salts, small molecule impurities, and pigments until the washing solution was clear. After this treatment, a wet PNAG precipitate was obtained, which was used for subsequent deacetylation reactions.

[0065] (4) Preparation of dPNAG by alkaline deacetylation treatment of PNAG

[0066] The wet PNAG precipitate was dispersed in an alkaline solution and reacted at 60-80℃ for 3 hours to remove the N-acetyl group from the PNAG molecule, generating dPNAG containing a free amino group.

[0067] After the reaction was completed, the reaction solution was cooled, and pre-cooled ethanol was added for a second alcohol precipitation, causing dPNAG to precipitate. The precipitate was collected by centrifugation to obtain crude dPNAG.

[0068] (5) Purification and drying of dPNAG

[0069] The crude dPNAG precipitate was repeatedly resuspended and washed with ethanol solution until the washing solution was nearly neutral to further remove residual alkaline substances and small molecule impurities. After washing, the precipitate was collected and freeze-dried to obtain the dried dPNAG solid product.

[0070] Example 2: Structural Identification of dPNAG

[0071] Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 The structure of dPNAG obtained in Example 1 was identified by 1H NMR and Fourier transform infrared spectroscopy (FT-IR). The results showed that the N-acetyl-related signal in dPNAG was significantly weakened, the signal complexity in the 3.0-5.0 ppm region corresponded to the hydrogen atom signal on the glucose ring, and characteristic signals related to amino groups were also present, indicating that PNAG had successfully undergone deacetylation. Further analysis of... 1 By integrating the relevant characteristic peaks in the 1H NMR spectrum, the degree of deacetylation of the obtained dPNAG was determined to be 60-80%. Furthermore, the molecular weight of the product was determined by gel permeation chromatography (GPC), and the results showed that the molecular weight of the obtained dPNAG was 500-3000 Da.

[0072] Example 3: Antibacterial activity of dPNAG

[0073] The dPNAG obtained in Example 1 was used for antibacterial activity testing. The experimental results showed that dPNAG exhibited significant growth inhibition against Gram-positive Staphylococcus aureus (ATCC 25923), with a minimum inhibitory concentration of 256 μg / mL, indicating that it has good antibacterial activity.

[0074] Example 4: Antioxidant activity of dPNAG

[0075] The antioxidant activity of dPNAG obtained in Example 1 was tested using a variety of in vitro antioxidant evaluation methods, including DPPH radical scavenging ability, ABTS radical scavenging ability, hydroxyl radical (·OH) scavenging ability, and ferric reduction antioxidant capacity (FRAP).

[0076] Experimental results showed that the dPNAG obtained in Example 1 exhibited certain free radical scavenging and reducing abilities in the various antioxidant evaluation systems mentioned above. The optimal scavenging ability of dPNAG against DPPH free radicals was 54% at a mother liquor concentration of 5 mg / mL; the optimal scavenging ability against ABTS free radicals was 23% at a mother liquor concentration of 5 mg / mL; the optimal scavenging ability against hydroxyl radicals (·OH) was 8% at a mother liquor concentration of 2 mg / mL; and the optimal FRAP (ferric reduction antioxidant capacity) was 0.22 mmol Fe. 2+ The concentration of the mother liquor was 1 mg / mL. This indicates that dPNAG has good in vitro antioxidant activity, providing experimental evidence for its application in functional materials, food, and cosmetics.

[0077] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Application of deacetylated poly-β-(1,6)-N-acetylglucosamine dPNAG in the preparation of antibacterial products.

2. The application according to claim 1, characterized in that, The degree of deacetylation of the deacetylated poly-β-(1,6)-N-acetylglucosamine dPNAG is 60-100%.

3. The application according to claim 1, characterized in that, The deacetylated poly-β-(1,6)-N-acetylglucosamine was prepared by the following method: S1. Construct a recombinant Corynebacterium glutamicum expressing IcaA, IcaC, IcaD, glutamine-fructose-6-phosphate aminotransferase GlmS, glucose-6-phosphate mutase GlmM, and UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase bifunctional enzyme GlmU. Ferment the recombinant Corynebacterium glutamicum to obtain a fermentation culture broth. Centrifuge the fermentation culture broth for the first time to obtain the fermentation supernatant. S2. Add an alkaline solution to the fermentation supernatant of S1 to remove protein and obtain the first treatment liquid; S3. Add ethanol to the first treatment solution for the first alcohol precipitation, and after washing, obtain poly-β-(1,6)-N-acetylglucosamine precipitate. S4. The poly-β-(1,6)-N-acetylglucosamine precipitate of S3 is dissolved in an alkaline solution for deacetylation treatment, and then ethanol is added for a second alcohol precipitation. After purification, the deacetylated poly-β-(1,6)-N-acetylglucosamine is obtained.

4. The application according to claim 3, characterized in that, In step S2, the temperature for the deproteinization treatment is 60-80℃; in step S4, the temperature for the deacetylation treatment is 30-80℃.

5. The application according to claim 3, characterized in that, The alkaline solution includes sodium hydroxide.

6. The application according to claim 1, characterized in that, The antibacterial product inhibits bacterial growth.

7. An antibacterial product, characterized in that, The antimicrobial product includes deacetylated poly-β-(1,6)-N-acetylglucosamine dPNAG.

8. Application of deacetylated poly-β-(1,6)-N-acetylglucosamine dPNAG in the preparation of antioxidant products.

9. The application according to claim 8, characterized in that, The antioxidant product works by scavenging free radicals.

10. An antioxidant product, characterized in that, The antioxidant products include deacetylated poly-β-(1,6)-N-acetylglucosamine dPNAG.

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