Preparation method and application of functionalized bacterial cellulose nanofiber membrane for specific detection of microorganisms

Abstract

The invention discloses a preparation method and application of a functional bacterial cellulose nanofiber membrane for specific detection of microorganisms. The preparation method comprises the following steps: firstly, taking a carboxyl-containing glucose derivative as a functional carbon source monomer, and blending the carboxyl-containing glucose derivative with a glucose monomer according to a certain proportion; a surface carboxyl modified bacterial cellulose nanofiber membrane is obtained through acetobacter xylinum catalytic biological copolymerization, and then functional sugar molecules containing amido and carboxyl chemical bonds on the surface of the membrane are coupled through a chemical catalytic coupling reaction, so that the surface glycosyl modified bacterial cellulose nanofiber membrane is obtained. The glycosylated bacterial cellulose nanofiber membrane obtained by the method disclosed by the invention has a remarkable specific affinity adhesion characteristic on specific bacteria, has an excellent non-specific adhesion resistance characteristic on other bacteria, and can be successfully used for bacteria specificity detection by a filter membrane method. The preparation method of the glycosylated bacterial cellulose nanofiber membrane is simple and green, the surface glycosyl grafting amount is controllable, and the glycosylated bacterial cellulose nanofiber membrane has excellent biological nonspecific adhesion resistance, has remarkable specific adhesion and affinity enrichment characteristics on specific bacteria, and has wide application prospects in the field of efficient detection of bacteria specificity through a filter membrane method.

Description

Technical Field

[0001] The invention belongs to the field of bio-based nanomaterials and bacteria-specific detection, and specifically relates to a preparation method and application of a glycosylated bacterial cellulose nanofiber membrane for bacteria-specific detection. Background Art

[0002] Microbial contamination in drinking water and food poses a serious threat to human life and health. Therefore, the detection of microorganisms plays a vital role in water quality safety and the prevention and control of foodborne diseases. In recent years, a new method for microbial detection by membrane filtration has been established based on polymer microfiltration membrane filtration technology. Compared with other microbial detection methods, the membrane filtration method has many advantages such as simple operation, low cost, high sensitivity, and applicability to large volume sample detection. It has been widely used in water quality safety, beverage testing, environmental monitoring, cosmetics and pharmaceutical industry quality control, and the electronics industry. The pharmacopoeias of countries such as the United States, Europe and Japan, and organizations such as the EPA and FDA have long listed it as a standard test method for microbial detection. my country has gradually listed the membrane filtration method as a national standard method for detecting specific microorganisms (e.g. coli, enterococci and Clostridium perfringens, etc.) in multiple industries, such as the national "Standard Test Method for Drinking Water" (GB / T5750-2023), "National Food Safety Standard-Test Method for Drinking Natural Mineral Water" (GB8538-2022), "Chinese Pharmacopoeia" 2015 edition and "Hospital Disinfection and Hygiene Standards" (GB 15982-2012).

[0003] However, there are still bottleneck problems in the actual application of the membrane filter method: there is no specific interaction between the membrane material and specific microorganisms, and the indiscriminate enrichment of microorganisms by the membrane filter will seriously interfere with the counting accuracy of specific colonies. It is also necessary to go through cumbersome secondary separation verification to obtain the results. For example, in order to accurately detect Escherichia coli (E.Coli), one of the important indicator microorganisms, the national standard "Standard Test Methods for Drinking Water" (GB / T 5750-2023) clearly stipulates that the membrane filter method needs to be verified in two steps. First, the total coliform group positive samples are determined by the membrane filter method, and then the membrane filter method is used again to determine Escherichia coli (E.Coli) in the total coliform group positive samples. Therefore, if an affinity membrane material with specific affinity enrichment characteristics for typical target bacteria can be constructed, it can provide new ideas for solving the bottleneck problem of microbial detection by the membrane filter method, and it has important practical significance for building a convenient, fast, low-cost, highly sensitive and accurate microbial efficient detection technology.

[0004] Among the many candidate functional membrane materials, glycosylated bacterial cellulose nanofiber membrane has unique advantages, which combines the biospecific recognition function of sugar molecules with the three-dimensional porous filamentous structure of bacterial cellulose nanofiber membrane. On the one hand, there is a specific recognition effect between sugar molecules and microbial surface lectin proteins, which can give the material the specificity of bacterial adhesion and enrichment; on the other hand, bacterial cellulose nanofiber membrane has a three-dimensional porous mesh nanofiber structure with a high specific surface area, which can promote the multivalent interaction between microorganisms and membrane materials, and enrich them on the membrane surface with enhanced affinity and efficient affinity adhesion; in addition, the asymmetric pore size structure on both sides of the bacterial cellulose nanofiber membrane can promote unidirectional mass transfer of nutrients, thereby promoting the recovery of enriched microbial activity and accelerating cell growth, providing an important bionic material platform for efficient detection of microorganisms by filtration membrane method.

[0005] However, there are significant defects in the existing preparation technology of glycosylated bacterial cellulose nanofiber membranes: the positively charged amine groups introduced on the surface of the bacterial cellulose nanofiber membrane by biopolymerization in the first step cannot react 100% in the subsequent chemical catalytic coupling reaction, resulting in a certain number of positively charged amine groups remaining on the membrane surface (for example, the method for preparing glycosylated bacterial cellulose disclosed in CN118324934A). The residual positively charged amine groups interact with microorganisms through electrostatic attraction of positive and negative charges, thereby having obvious nonspecific bioadhesion to microbial cells, which seriously affects the affinity enrichment specificity of the membrane material for specific microorganisms, and ultimately affects the specificity and accuracy of microbial detection by the filter membrane method based on glycosylated bacterial cellulose nanofiber membranes. Summary of the invention

[0006] The present invention develops a method for preparing a functionalized bacterial cellulose nanofiber membrane for specific detection of microorganisms with significant resistance to nonspecific biological adhesion and its application. First, a functionalized bacterial cellulose nanofiber membrane with negatively charged carboxyl groups on the surface is prepared by microbial catalytic biopolymerization technology, and then the surface carboxyl groups of the functionalized bacterial cellulose nanofiber membrane obtained above are reacted with amino-containing sugar derivatives under the action of chemical catalytic coupling, so that the sugar molecules are modified and fixed to the surface of the bacterial cellulose nanofiber membrane to prepare a glycosylated bacterial cellulose nanofiber membrane. The preparation process of this method is simple, green, controllable and efficient. The obtained glycosylated bacterial cellulose nanofiber membrane has obvious resistance to nonspecific adhesion to other microorganisms, and has specific affinity enrichment characteristics for specific microorganisms, and can be effectively applied to the efficient detection of microorganisms by membrane filtration method.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A method for preparing a functionalized bacterial cellulose nanofiber membrane for specific microbial detection comprises obtaining a functionalized bacterial cellulose nanofiber membrane with negatively charged carboxyl groups on its surface through microbial catalytic biopolymerization technology, and then subjecting the obtained functionalized bacterial cellulose nanofiber membrane to amidation reaction with an amino-containing sugar derivative under the action of chemical catalytic coupling to modify the sugar molecules on the surface of the bacterial cellulose nanofiber membrane, thereby preparing a glycosylated bacterial cellulose nanofiber membrane.

[0009] The specific steps include:

[0010] 1) Preparation of functionalized bacterial cellulose nanofiber membrane with negatively charged carboxyl groups on the surface by biopolymerization:

[0011] The Acetobacter xylinum bacterial liquid is inoculated into a fermentation medium containing carboxyl sugar derivatives such as gluconic acid, lactobionic acid, maltobionic acid, etc., and cultured at 30° C. for 1 to 7 days to obtain functionalized bacterial cellulose with surface carboxyl modified.

[0012] 2) Preparation of glycosylated bacterial cellulose by chemical catalytic coupling:

[0013] A certain amount of amino-containing functional sugar molecules such as glucosamine, amino-polyethylene glycol-glucose, amino-polyethylene glycol-galactose, amino-polyethylene glycol-mannose, and EDC / NHS are added to a reaction bottle, and a sodium bicarbonate (pH=5.5, 50mM) buffer solution is added thereto. After the solid is dissolved, the functional bacterial cellulose containing surface carboxyl groups obtained in step (1) is added to the bottle, and the reaction is carried out at a constant temperature of 2 to 4° C. and shaken for 1 to 24 hours. The material is taken out, washed with pure water, and freeze-dried to obtain glycosylated bacterial cellulose.

[0014] Step 1) Acetobacter xylinum is activated and seed cultured to obtain Acetobacter xylinum bacterial liquid, which is inoculated into a fermentation medium at an inoculation rate of 1% to 20% by volume.

[0015] The activation of Acetobacter xylinum: inoculating Acetobacter xylinum into a HS culture medium test tube, and culturing in a shaker at 30° C. and 150 rpm for 24 hours;

[0016] Seed liquid culture: 1% activated Acetobacter xylinum suspension was inoculated into a HS medium flask and cultured in a shaker at 30°C and 150 rpm for 24 h.

[0017] The HS culture medium comprises: 20 g / L glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 2.7 g / L disodium hydrogen phosphate, and 0.5% w / v 95% ethanol.

[0018] Step 1) The fermentation medium is: adding 0-20% glucose, 0-20% carboxyl sugar derivative molecules, 0.001-10% peptone, 0.001-10% yeast powder, 0.001-10% citric acid, 0.001-10% disodium hydrogen phosphate, 95% ethanol 0.001-10% w / v to pure water according to weight percentage, and sterilizing at 115° C. for 20 minutes;

[0019] The total carbon source concentration including glucose and carboxyl-containing glucose derivative molecules is 40 g / L.

[0020] Further preferably, 0-20% glucose, 0-20% carboxyl glucose derivative molecules, 1% peptone, 1% yeast powder, 0.2% citric acid, 0.5% disodium hydrogen phosphate, 95% ethanol 1% w / v, sterilized at 115°C for 20 min.

[0021] The carboxyl-containing glucose derivative molecule is one or more of gluconic acid, lactobionic acid, maltobionic acid and the like.

[0022] The weight percentage of glucose and carboxyl-containing glucose derivative molecules is (0-2): (0-2); for example, it can be 2:0 (all glucose); 1.5:0.5; 1:1; 0.5:1.5; 0:2 (all carboxyl-containing glucose derivative molecules).

[0023] Step 1) After obtaining the functionalized bacterial cellulose product containing carboxyl groups on the surface, rinse it with water for 0.5 to 2 hours, then soak it in a boiling 1% to 5% NaOH solution for 1 to 5 hours, continue to soak it in a 75% ethanol solution for 2 to 6 hours, finally take it out and rinse it repeatedly with pure water until the eluate is neutral, and vacuum freeze-dry it to constant weight.

[0024] The Acetobacter xylinum described in step 1) has the ability to biosynthesize cellulose. The present invention can use common Acetobacter xylinum disclosed in the prior art or Acetobacter xylinum purchased from commercial channels, for example, Acetobacter xylinum 1.1812, Acetobacter xylinum ATCC23767, Acetobacter xylinum CCTCC M 207163, etc.

[0025] In step 2), the amount of the amino-containing sugar derivative added is 1 to 1000 times the molar amount of carboxyl groups on the membrane surface, and the molar ratio of EDC and NHS to the amino-containing sugar derivative is 1 to 100: 1 to 100: 0.1 to 20.

[0026] Further preferably, the ratio of glucosamine, amino-polyethylene glycol-glucose, amino-polyethylene glycol-galactose, amino-polyethylene glycol-mannose and other amino-group-containing functional glucose molecules, EDC, and NHS is as follows: the concentration of glucosamine, amino-polyethylene glycol-glucose, amino-polyethylene glycol-galactose, amino-polyethylene glycol-mannose and other amino-group-containing functional glucose molecules is 1 to 1000 times the molar concentration of carboxyl groups on the membrane surface, and the molar ratio of EDC and NHS to the amount of glucosamine, amino-polyethylene glycol-glucose, amino-polyethylene glycol-galactose, amino-polyethylene glycol-mannose and other amino-group-containing functional sugar molecules is 7:3:1. Further preferably, the concentration of glucosamine, amino-polyethylene glycol-glucose, amino-polyethylene glycol-galactose, amino-polyethylene glycol-mannose and other amino-group-containing functional grape molecules is 100 times the molar concentration of carboxyl groups on the membrane surface.

[0027] The concentration of the solution of the amino group-containing sugar derivative is 0 to 100 μmol / mL.

[0028] The glycosylated bacterial cellulose nanofiber membrane is prepared by any of the methods described above.

[0029] A glycosylated bacterial cellulose nanofiber membrane, wherein the sugar group is used as a functional group to modify and fix on the surface of a carboxyl-modified bacterial cellulose membrane, wherein the bacterial cellulose nanofiber membrane is a nanofiber membrane material with a three-dimensional porous filamentous structure, and the sugar group is glucose, galactose or mannose.

[0030] The average diameter of the glycosylated bacterial cellulose is 35±2nm, there are relatively large pores between the nanofibers, and the porosity is 80-90%.

[0031] The glycosylated bacterial cellulose nanofiber membrane is used in the specific and efficient detection of bacteria.

[0032] The glycosylated bacterial cellulose nanofiber membrane, whose surface is modified with glucose, mannose or galactose, has specific recognition properties for Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), and can be used for specific affinity enrichment detection of Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa).

[0033] The glycosylated bacterial cellulose nanofiber membrane was immersed in Escherichia coli (E.coli), Pseudomonas aeruginosa (P.aeruginosa) or Staphylococcus aureus (S.aureus) solution and affinity enriched for 10 to 300 minutes at 25°C. The glycosylated bacterial cellulose nanofiber membrane specifically affinity enriched Escherichia coli (E.coli) and Pseudomonas aeruginosa (P.aeruginosa).

[0034] The glycosylated bacterial cellulose nanofiber membrane of the present invention is also suitable for affinity enrichment detection of corresponding bacteria in ultra-low concentration Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) solutions, and the concentration is as low as 1 CFU / ml.

[0035] A preparation method and application of a functionalized bacterial cellulose nanofiber membrane for microbial specific detection, comprising the following steps:

[0036] 1) Preparation of surface carboxyl-modified bacterial cellulose nanofiber membrane:

[0037] The seed liquid of Acetobacter xylinum is inoculated into a fermentation medium containing 0-20% glucose and 0-20% amino functional sugar molecules (one or more of glucosamine, amino-polyethylene glycol-glucose, amino-polyethylene glycol-galactose, and amino-polyethylene glycol-mannose) at an inoculation rate of 1%-30% by volume, and cultured at 30°C for 1-7 days. After the product is taken out, it is washed with water for 1-4 hours, and then immersed in a boiling 1%-5% NaOH solution for 2-6 hours, and then immersed in a 75% ethanol solution for 1-4 hours. Finally, it is taken out and repeatedly washed with pure water until the eluate is neutral, and vacuum freeze-dried to constant weight.

[0038] 2) Preparation of glycosylated bacterial cellulose nanofiber membrane

[0039] Weigh 1 to 1000 times of the molar equivalent of the carboxyl groups on the membrane surface, glucosamine or amino-polyethylene glycol-glucose or amino-polyethylene glycol-galactose or amino-polyethylene glycol-mannose, 7 times the molar equivalent of EDC, and 3 times the molar equivalent of NHS in a flask, and add 5 mL of sodium bicarbonate (pH=5.5, 50 mM) buffer solution thereto, and after the solid is dissolved, add the surface carboxyl-containing bacterial cellulose nanofiber membrane prepared in step 1) to the flask, and react at 4°C for 1 to 24 hours with constant temperature oscillation, take out the membrane material, wash it with pure water for 6 times in sequence, and freeze-dry it to obtain a glycosylated bacterial cellulose nanofiber membrane. The concentration of the glucosamine or amino-polyethylene glycol-glucose or amino-polyethylene glycol-galactose or amino-polyethylene glycol-mannose is 0 to 100 μmol / mL.

[0040] The present invention also claims to protect the use of the glycosylated bacterial cellulose nanofiber membrane prepared by the above method in the specific affinity enrichment of Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) and the efficient detection by filtration membrane method.

[0041] Both mannose-glycosylated bacterial cellulose nanofiber membranes and glucose-modified glycosylated bacterial cellulose nanofiber membranes have specific affinity adhesion capture effects on Escherichia coli (E. coli), but compared with glucose-glycosylated bacterial cellulose nanofiber membranes, mannose-glycosylated bacterial cellulose nanofiber membranes have stronger affinity adhesion capture effects on Escherichia coli (E. coli), and mannose-glycosylated bacterial cellulose nanofiber membranes have more sensitive specific affinity enrichment detection effects on Escherichia coli (E. coli). In addition, galactose-glycosylated bacterial cellulose nanofiber membranes have specific adhesion enrichment detection effects on Pseudomonas aeruginosa (P. aeruginosa). The present invention firstly adopts microbial catalytic biopolymerization to prepare a bacterial cellulose nanofiber membrane with negatively charged carboxyl groups on the surface. According to the principle of intermolecular electrostatic repulsion, the negatively charged carboxyl groups introduced on the surface of the bacterial cellulose nanofiber membrane have an electrostatic repulsion effect on negatively charged microorganisms, so that the material has excellent antimicrobial nonspecific adhesion properties; then, an EDC / NHS-mediated chemical catalytic amidation coupling reaction is used to modify amino-functional sugar molecules on the surface of the carboxyl-modified bacterial cellulose nanofiber membrane. Sugar molecules can not only show specific recognition effects on specific microorganisms through the specific interaction of "sugar-microbial lectin", but also the polyhydroxyl hydrophilic properties of sugar molecules also give the material surface a thick hydration layer structure. The presence of the hydration layer and the synergistic effect of the residual negatively charged carboxyl groups further effectively inhibit the nonspecific biological adhesion of other microorganisms on the membrane surface. The new strategy for preparing glycosylated bacterial cellulose nanofiber membranes can effectively inhibit the nonspecific biological adhesion of membrane materials, so that the specific affinity enrichment performance of membrane materials for specific microorganisms is significantly enhanced, which provides important technical support for further use in the specific and efficient detection of microorganisms by the filter membrane method. The anti-nonspecific bioadhesion inhibition effect on microorganisms is mainly achieved through the introduction of negatively charged carboxyl electrostatic repulsion on the surface of the membrane material and the synergistic effect of the highly hydrophilic barrier of the sugar group; while the specific affinity enrichment effect on specific microorganisms is mainly achieved through the synergistic effect of the special biological effect of "sugar-microbial lectin" specific recognition and the three-dimensional network nano-bionic structure of bacterial cellulose.

[0042] The average diameter of the glycosylated bacterial cellulose nanofiber membrane prepared by the present invention is 35±2nm, the porosity is 80-90%, and due to the introduction of negatively charged carboxyl groups on the surface of the bacterial cellulose nanofiber membrane during the interfacial static fermentation biopolymerization process, the electrostatic repulsion between the same charges presents larger pores, so that the two sides of the membrane have a significant asymmetric pore structure. This structural feature is more conducive to promoting unidirectional mass transfer of nutrients, thereby promoting the recovery of enriched microbial activity and accelerating cell growth, providing an important material platform and technical support for the efficient detection of microorganisms by the filter membrane method, and has a broad and important practical application prospect.

[0043] Beneficial effects:

[0044] The present invention innovatively introduces negatively charged carboxyl groups and sugar groups on the surface of bacterial cellulose nanofiber membranes by combining biopolymerization and post-chemical catalytic functionalization modification, thereby preparing glycosylated bacterial cellulose nanofiber membrane functional materials. The preparation method of the present invention is green, simple, efficient, and effectively inhibits the nonspecific adhesion of microorganisms on the surface of the material. In addition, while the glycosylated bacterial cellulose nanofiber membrane does not change the bulk properties of the material, it gives the material excellent microorganism-specific affinity enrichment efficacy, and has broad application prospects in the field of efficient detection of microorganisms by filtration membrane method. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The drawings described herein are used to provide further understanding of the present invention and constitute a part of this application. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0046] In the attached picture:

[0047] Figure 1 : The carboxyl density on the membrane surface changes with the feed ratio of gluconic acid to glucose;

[0048] Figure 2 :Effect of carboxyl content on the amount of sugar grafted on the membrane surface;

[0049] Figure 3 :The effect of EDC / NHS catalytic coupling time on the amount of glycosyl grafted onto the membrane surface;

[0050] Figure 4 : Total reflection infrared spectra of the membrane surface before and after each step of reaction;

[0051] Figure 5 : XPS spectra of the membranes before and after each step of the reaction (a: blank bacterial cellulose nanofiber membrane; b: carboxyl-modified bacterial cellulose nanofiber membrane; c: glucose-modified glycosylated bacterial cellulose nanofiber membrane);

[0052] Figure 6 : SEM images of the membranes before and after each step of the reaction (a: blank bacterial cellulose nanofiber membrane; b: carboxyl-modified bacterial cellulose nanofiber membrane; c: glucose-modified glycosylated bacterial cellulose nanofiber membrane);

[0053] Figure 7 :The change of bacterial adhesion amount of functionalized bacterial cellulose nanofiber membrane over time;

[0054] Figure 8 : SEM and CLSM images of bacteria captured by different bacterial cellulose nanofiber membranes (a: blank bacterial cellulose nanofiber membrane; b: carboxyl-modified bacterial cellulose nanofiber membrane; c: glucose-modified glycosylated bacterial cellulose nanofiber membrane);

[0055] Fig. 9 : Highly sensitive bacterial capture at extremely low bacterial concentrations;

[0056] Fig.10 : SEM and CLSM images of highly sensitive bacterial capture at extremely low bacterial concentrations;

[0057] Fig.11 :Specific capture efficiency of Escherichia coli (E. coli) by glucose-modified glycosylated bacterial cellulose nanofiber membrane;

[0058] Fig.12 : Specific adhesion of functionalized bacterial cellulose membranes to mixed fluorescently labeled bacteria: (a) Capture of Escherichia coli (E. coli) by carboxyl-modified bacterial cellulose nanofiber membrane; (b) Capture of Pseudomonas aeruginosa (P. aeruginosa) by carboxyl-modified bacterial cellulose nanofiber membrane; (c) Capture of Escherichia coli (E. coli) by glycosylated bacterial cellulose nanofiber membrane; (d) Capture of Pseudomonas aeruginosa (P. aeruginosa) by glycosylated bacterial cellulose nanofiber membrane;

[0059] Fig.13 :Enrichment detection diagram of Escherichia coli (E. coli) based on glycosylated bacterial cellulose nanofiber membrane filtration method. DETAILED DESCRIPTION

[0060] The following will refer to the accompanying drawings to exemplify or describe in detail the technical solutions of the present invention by way of embodiments. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention.

[0061] The term "and / or" in this article is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist at the same time. The term " / and" in this article describes another type of association object relationship, indicating that there can be two relationships. For example, A / and B can mean: A exists alone, and A and B exist alone. In addition, the character " / " in this article generally indicates that the previous and next associated objects are in an "or" relationship.

[0062] Example 1

[0063] A method for preparing a functionalized bacterial cellulose nanofiber membrane for microbial specific detection, specifically comprising the following steps:

[0064] (1) Activation of Acetobacter xylinum

[0065] Acetobacter xylinum (Acetobacter xylinum ATCC23767) was inoculated into a 5 mL HS medium test tube sterilized at 115°C for 20 min, and cultured in a shaker at 30°C and 160 rpm for 24 h;

[0066] (2) Seed liquid culture

[0067] Take 1% activated Acetobacter xylinum suspension and inoculate it into 100 mL HS medium flask, and culture it in a shaker at 30°C and 150 rpm for 24 h;

[0068] The HS culture medium in steps (1) and (2) comprises: 20 g / L glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 2.7 g / L disodium hydrogen phosphate, and 0.5% w / v 95% ethanol.

[0069] (3) Preparation of fermentation medium

[0070] The fermentation medium was prepared by adding 2% glucose and gluconic acid, 0.5% peptone, 0.5% yeast powder, 0.115% citric acid, 0.27% disodium hydrogen phosphate, and 95% ethanol 0.5% w / v to pure water according to weight percentage, and sterilized at 115° C. for 20 min.

[0071] The total carbon source concentration including glucose and gluconic acid is 20 g / L.

[0072] The weight percentages of glucose and gluconic acid are 2:0, 1.5:0.5, 1:1, 0.5:1.5 and 0:2 respectively.

[0073] (4) Preparation of bacterial cellulose membrane containing carboxyl groups on the surface

[0074] The seed solution in step (2) is inoculated with 10% by volume into a fermentation medium of glucose and gluconic acid (the weight percentages are 2:0, 1.5:0.5, 1:1, 0.5:1.5, and 0:2, respectively), and cultured at 30° C. for 7 days. After taking out the product, rinse it with water for 2 hours, then soak it in a boiling 1% NaOH solution for 4 hours, continue to soak it in a 75% ethanol solution for 2 hours, finally take it out and rinse it repeatedly with pure water until the eluate is neutral, and vacuum freeze-dry it to constant weight; obtain a bacterial cellulose nanofiber membrane with different surface carboxyl density modified;

[0075] (5) Preparation of glycosylated bacterial cellulose membrane

[0076] Weigh 10 times the molar equivalent of the carboxyl group on the membrane surface of glucosamine, 7 times the molar equivalent of EDC of glucosamine, and 3 times the molar equivalent of NHS of glucosamine into a flask, and add 5 mL of sodium bicarbonate (pH = 5.5, 50 mM) buffer solution thereto. After the solid is dissolved, add the carboxyl-modified bacterial cellulose nanofiber membrane with different surface carboxyl densities prepared in step (4) into the flask, and react at a constant temperature of 4°C for 24 hours. Take out the material, wash it with pure water for 6 times in sequence, and freeze-dry it to obtain glycosylated bacterial cellulose with different surface glycosylation densities.

[0077] The surface carboxyl density of the carboxyl-modified bacterial cellulose obtained in Example 1 was measured, and the results were as follows: Figure 1 As shown. When the molar ratio of gluconic acid to glucose monomer increases, the amount of carboxyl groups on the membrane surface also increases, varying between 0 and 400 μmol / g. When all of them are gluconic acid monomers, the density of carboxyl groups on the membrane surface reaches a maximum value of 400 μmol / g. By adjusting the density of carboxyl groups on the membrane surface, the density of sugar grafting on the membrane surface can be effectively controlled. The results are shown in Figure 2 As shown in Figure 2, as the density of carboxyl groups on the membrane surface increases, the density of sugar grafting on the membrane surface also increases. When the density of carboxyl groups on the membrane surface reaches a maximum value of 400 μmol / g, the density of sugar groups on the membrane surface reaches a maximum value of 100 μmol / g.

[0078] Example 2

[0079] A method for preparing a functionalized bacterial cellulose nanofiber membrane for microbial specific detection, specifically comprising the following steps:

[0080] (1) Activation of Acetobacter xylinum

[0081] Acetobacter xylinum (Acetobacter xylinum ATCC23767) was inoculated into a 5 mL HS medium test tube sterilized at 115°C for 20 min, and cultured in a shaker at 30°C and 150 rpm for 24 h;

[0082] (2) Seed liquid culture

[0083] Take 1% activated Acetobacter xylinum suspension and inoculate it into 100 mL HS medium flask, and culture it in a shaker at 30°C and 160 rpm for 24 h;

[0084] The HS culture medium in steps (1) and (2) comprises: 20 g / L glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 2.7 g / L disodium hydrogen phosphate, and 0.5% w / v 95% ethanol.

[0085] (3) Preparation of fermentation medium

[0086] The fermentation medium was prepared by adding 1.0% glucose, 1.0% gluconic acid, 0.5% peptone, 0.5% yeast powder, 0.115% citric acid, 0.27% disodium hydrogen phosphate, and 95% ethanol 0.5% w / v to pure water according to weight percentage, and sterilized at 115° C. for 20 min.

[0087] The total carbon source concentration including glucose and gluconic acid is 20 g / L.

[0088] (4) Preparation of bacterial cellulose membrane containing carboxyl groups on the surface

[0089] The seed solution in step (2) is inoculated with 10% by volume into a fermentation medium of glucose and carboxyl sugar molecule derivative (weight percentage is 1.0:1.0), cultured at 30° C. for 7 days, and the product is taken out and rinsed with water for 2 hours, and then immersed in a boiling 1% NaOH solution for 4 hours, and then immersed in a 75% ethanol solution for 2 hours, and finally taken out and repeatedly rinsed with pure water until the eluate is neutral, and vacuum freeze-dried to constant weight; obtaining carboxyl-modified bacterial cellulose with a carboxyl density of 250 μmol / g;

[0090] (5) Preparation of glycosylated bacterial cellulose membrane

[0091] Weigh 50 times the molar equivalent of glucosamine, 7 times the molar equivalent of EDC, and 3 times the molar equivalent of NHS on the surface of the membrane into a flask, and add 5 mL of sodium bicarbonate (pH = 5.5, 50 mM) buffer solution thereto. After the solid is dissolved, add the surface carboxyl-containing bacterial cellulose prepared in step (4) into the flask, and react at 4°C with constant temperature oscillation for 0 h, 4 h, 8 h, 12 h, and 24 h, respectively. Take out the membrane after the reaction, wash it with pure water for 6 times in sequence, and freeze-dry it to obtain glycosylated bacterial cellulose with a glycosyl density of 0 to 60 μmol / g.

[0092] By adjusting the chemical catalytic coupling time, the density of sugar grafting on the membrane surface can be effectively controlled. Figure 3 As shown in Figure 2, as the chemical catalytic coupling time increases, the density of sugar grafting on the membrane surface also increases and reaches a maximum after 12 hours and then tends to equilibrium. The density of sugar grafting on the membrane surface reaches a maximum of 60 μmol / g.

[0093] Example 3

[0094] A method for preparing a functionalized bacterial cellulose nanofiber membrane for microbial specific detection, specifically comprising the following steps:

[0095] (1) Activation of Acetobacter xylinum

[0096] Acetobacter xylinum CCTCC M 207163 was inoculated into a 5 mL HS medium test tube sterilized at 115°C for 20 min and cultured in a shaker at 30°C and 160 rpm for 24 h;

[0097] (2) Seed liquid culture

[0098] Take 1% activated Acetobacter xylinum suspension and inoculate it into 100 mL HS medium flask, and culture it in a shaker at 30°C and 160 rpm for 24 h;

[0099] The HS culture medium in steps (1) and (2) comprises: 20 g / L glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 2.7 g / L disodium hydrogen phosphate, and 0.5% w / v 95% ethanol.

[0100] (3) Preparation of fermentation medium

[0101] The fermentation medium was prepared by adding 1.5% glucose, 0.5% gluconic acid, 0.5% peptone, 0.5% yeast powder, 0.115% citric acid, 0.27% disodium hydrogen phosphate, and 95% ethanol 0.5% w / v to pure water according to weight percentage, and sterilized at 115° C. for 20 min.

[0102] The total carbon source concentration including glucose and gluconic acid is 20 g / L.

[0103] The carboxyl-containing glucose derivative molecule is gluconic acid.

[0104] (4) Preparation of bacterial cellulose nanofiber membranes containing carboxyl groups on the surface

[0105] The seed solution in step (2) is inoculated with 10% by volume into a fermentation medium having a molar ratio of glucose to glucosamine of 1.5:0.5, and cultured at 30° C. for 7 days. After taking out the product, it is rinsed with water for 2 hours, and then immersed in a boiling 1% NaOH solution for 4 hours, and then immersed in a 75% ethanol solution for 2 hours, and finally taken out and repeatedly rinsed with pure water until the eluate is neutral, and vacuum freeze-dried to constant weight; carboxyl-modified bacterial cellulose with a carboxyl density of 325 μmol / g is obtained;

[0106] (5) Preparation of glycosylated bacterial cellulose membrane

[0107] Weigh 50 times the molar equivalent of the membrane surface carboxyl groups of amino-polyethylene glycol-glucose, 5 times the molar equivalent of EDC of amino-polyethylene glycol-glucose, and 3 times the molar equivalent of NHS of amino-polyethylene glycol-glucose into a flask, and add 5 mL of sodium bicarbonate (pH = 5.5, 50 mM) buffer solution thereto. After the solid is dissolved, add the surface carboxyl-containing bacterial cellulose prepared in step (4) into the flask, and react at a constant temperature of 4°C with shaking for 24 hours. Take out the material, wash it with pure water for 6 times in sequence, and freeze-dry it to obtain glycosylated bacterial cellulose with a glycosyl density of 90 μmol / g.

[0108] The surface infrared and XPS analysis of the obtained glycosylated bacterial cellulose are shown in Figure 4-5 Compared with the infrared spectrum of pure bacterial cellulose nanofiber membrane, the infrared spectrum after biopolymerization showed an obvious carboxyl infrared absorption peak at 1730cm-1, indicating the successful introduction of carboxyl groups on the membrane surface, while after glycosylation, a special infrared absorption peak of amide bonds appeared, indicating the successful introduction of sugar groups on the membrane surface. Similar to the infrared spectrum, the XPS fine peak analysis spectrum showed that the membrane surface after glycosylation modification showed significant XPS characteristic peaks of amide bonds after successful grafting of sugar groups, further indicating the successful introduction of sugar groups on the membrane surface.

[0109] Example 4

[0110] A method for preparing a functionalized bacterial cellulose nanofiber membrane for microbial specific detection, specifically comprising the following steps:

[0111] (1) Activation of Acetobacter xylinum

[0112] Acetobacter xylinum ATCC23767 was inoculated into a 5 mL HS medium test tube sterilized at 115°C for 20 min and cultured in a shaker at 30°C and 150 rpm for 24 h;

[0113] (2) Seed liquid culture

[0114] Take 1% activated Acetobacter xylinum suspension and inoculate it into 100 mL HS medium flask, and culture it in a shaker at 30°C and 160 rpm for 24 h;

[0115] The HS culture medium in steps (1) and (2) comprises: 20 g / L glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 2.7 g / L disodium hydrogen phosphate, and 0.5% w / v 95% ethanol.

[0116] (3) Preparation of fermentation medium

[0117] The fermentation medium was prepared by adding 1.0% glucose, 1.0% maltobionic acid, 0.5% peptone, 0.5% yeast powder, 0.115% citric acid, 0.27% disodium hydrogen phosphate, and 95% ethanol 0.5% w / v to pure water according to weight percentage, and sterilized at 115° C. for 20 min.

[0118] The total carbon source concentration including the glucose and maltobionic acid derivative molecules is 20 g / L.

[0119] (4) Preparation of bacterial cellulose membrane containing carboxyl groups on the surface

[0120] The seed solution in step (2) is inoculated with 8% by volume into a fermentation medium having a molar ratio of glucose to maltobionic acid of 1.0:1.0, and cultured at 30°C for 7 days. After the product is taken out, it is rinsed with water for 2 hours, and then immersed in a boiling 2% NaOH solution for 4 hours, and then immersed in a 75% ethanol solution for 2 hours. Finally, it is taken out and repeatedly rinsed with pure water until the eluate is neutral, and vacuum freeze-dried to constant weight to obtain carboxyl-modified bacterial cellulose with a carboxyl density of 200 μmol / g.

[0121] (5) Preparation of glycosylated bacterial cellulose membrane

[0122] Weigh 500 times the molar equivalent of the carboxyl groups on the membrane surface, 7 times the molar equivalent of EDC, and 3 times the molar equivalent of NHS on the membrane surface, and add 5 mL of sodium bicarbonate (pH = 5.5, 50 mM) buffer solution into a flask. After the solid is dissolved, add the surface carboxyl-containing bacterial cellulose prepared in step (4) into the flask, and perform a constant temperature oscillation chemical catalytic coupling reaction at 4°C for 24 hours. Take out the material and wash it with pure water for 6 times in sequence, and freeze-dry it to obtain a glycosylated bacterial cellulose with a saccharide density. The concentration of maltobionic acid is 50 μmol / g.

[0123] The morphology of the bacterial cellulose membrane was observed by scanning electron microscopy (SEM). Figure 6 As shown, the average diameter of the obtained galactose glycosylated bacterial cellulose nanofiber membrane is 35±2nm, and the porosity between nanofibers is 85%. Compared with pure bacterial cellulose, as carboxyl and sugar groups are introduced on the surface of bacterial cellulose, the fiber diameter of the obtained glycosylated bacterial cellulose decreases.

[0124] Example 5

[0125] A method for preparing a functionalized bacterial cellulose nanofiber membrane for microbial specific detection, specifically comprising the following steps:

[0126] (1) Activation of Acetobacter xylinum

[0127] Acetobacter xylinum ATCC23767 was inoculated into a 5 mL HS medium test tube sterilized at 115°C for 20 min and cultured in a shaker at 30°C and 150 rpm for 24 h;

[0128] (2) Seed liquid culture

[0129] Take 1% activated Acetobacter xylinum suspension and inoculate it into 100 mL HS medium flask, and culture it in a shaker at 30°C and 160 rpm for 24 h;

[0130] The HS culture medium in steps (1) and (2) comprises: 20 g / L glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 2.7 g / L disodium hydrogen phosphate, and 0.5% w / v 95% ethanol.

[0131] (3) Preparation of fermentation medium

[0132] The fermentation medium was prepared by adding 1% glucose, 1% maltobionic acid, 0.5% peptone, 0.5% yeast powder, 0.115% citric acid, 0.27% disodium hydrogen phosphate, and 95% ethanol 0.5% w / v to pure water according to weight percentage, and sterilized at 115° C. for 20 min.

[0133] The total carbon source concentration including glucose and maltobionate is 20 g / L.

[0134] (4) Preparation of bacterial cellulose membrane containing carboxyl groups on the surface

[0135] The seed solution in step (2) is inoculated with 10% by volume into a fermentation medium with a molar ratio of glucose to maltobionic acid of 1:1, and cultured at 30°C for 7 days. After taking out the product, it is rinsed with water for 2 hours, and then immersed in a boiling 1% NaOH solution for 4 hours, and then immersed in a 75% ethanol solution for 2 hours, and finally taken out and repeatedly rinsed with pure water until the eluate is neutral, and vacuum freeze-dried to constant weight. A carboxyl-modified bacterial cellulose membrane with a carboxyl density of 220 μmol / g is obtained.

[0136] (5) Preparation of glycosylated bacterial cellulose membrane

[0137] Weigh 800 times the molar equivalent of amino-polyethylene glycol-mannose on the membrane surface, 7 times the molar equivalent of EDC on the membrane surface, and 3 times the molar equivalent of NHS on the membrane surface, and add 5 mL of sodium bicarbonate (pH = 5.5, 50 mM) buffer solution thereto. After the solid is dissolved, add the surface carboxyl-containing bacterial cellulose prepared in step (4) to the flask, and conduct a coupling reaction at 4°C for 24 hours. Take out the material, wash it with pure water for 6 times in sequence, and freeze-dry it to obtain a glycosylated bacterial cellulose nanofiber membrane modified with surface mannose, with a glycosyl density of 50 μmol / g.

[0138] Specific adhesion of glycosylated bacterial cellulose nanofiber films to microorganisms at different times

[0139] E. coli was cultured in LB medium at 37°C and 180 rpm for 12 h. The bacterial solution was centrifuged, and the supernatant was discarded. The cell pellet was resuspended in PBS (0.01 M, pH = 7.2). Before bacterial capture, the OD of E. coli was adjusted by adjusting the volume of PBS solution. 600 The value was readjusted to 0.1, so that the concentration of E. coli bacteria was 1×10 8 CFU / mL.

[0140] Before attaching bacteria, the concentration of the bacterial solution was adjusted to 1×10 8 CFU / mL, dilute 10 5 Pipette 0.1 ml and spread on the plate to calculate the number of bacteria N1. 2 The glycosylated bacterial cellulose nanofiber membrane (mannose glycosyl density is 50 μmol / g), carboxyl-modified bacterial cellulose nanofiber (carboxyl content is 220 μmol / g), and pure bacterial cellulose nanofiber membrane prepared in Example 5 were sterilized by ultraviolet for 30 min, and then immersed in 1 mL of Escherichia coli (E. coli) solution and adsorbed at 25 ° C and 60 rpm for 15 min, 30 min, 45 min, 60 min, 90 min, and 120 min, respectively. After the adsorption was completed, the bacterial solution was shaken and 0.1 ml of the bacterial solution was taken from the sample bottle and diluted by 10 5 Then, 0.1 mL of bacterial solution was taken and spread on the plate to count the number of bacteria N2. Three parallel samples were collected each time. The amount of bacteria captured C (CFU / cm 2 ) is calculated according to the following formula:

[0141]

[0142] Where, C: number of bacteria captured (CFU / cm 2); N1: number of bacteria before capture (CFU); N2: number of bacteria before capture (CFU); S: membrane area (cm 2 ).

[0143] The bacterial adhesion properties of the glycosylated bacterial cellulose nanofiber membrane modified with surface mannose obtained in Example 5 were investigated, and the results are shown in Figure 7 Compared with pure bacterial cellulose nanofiber membrane and carboxyl-modified bacterial cellulose nanofiber membrane, the adhesion of glycosylated bacterial cellulose nanofiber membrane modified with surface mannose to Escherichia coli (E. coli) was significantly enhanced, and with the extension of time, the amount of E. coli adsorbed on the membrane surface gradually increased, and the adsorption amount reached saturation after 60 minutes.

[0144] Example 6

[0145] A preparation method and application of a functionalized bacterial cellulose nanofiber membrane for microbial specific detection, specifically comprising the following steps:

[0146] (1) Activation of Acetobacter xylinum

[0147] Acetobacter xylinum 1.1812 was inoculated into a 5 mL HS medium test tube sterilized at 115°C for 20 min and cultured in a shaker at 30°C and 150 rpm for 24 h;

[0148] (2) Seed liquid culture

[0149] Take 1% activated Acetobacter xylinum suspension and inoculate it into 100 mL HS medium flask, and culture it in a shaker at 30°C and 150 rpm for 24 h;

[0150] The HS culture medium in steps (1) and (2) comprises: 20 g / L glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 2.7 g / L disodium hydrogen phosphate, and 0.5% w / v 95% ethanol.

[0151] (3) Preparation of fermentation medium

[0152] The fermentation medium was prepared by adding 2.0% gluconic acid, 0.5% peptone, 0.5% yeast powder, 0.115% citric acid, 0.27% disodium hydrogen phosphate, and 95% ethanol 0.5% w / v to pure water according to weight percentage, and sterilized at 115° C. for 20 min.

[0153] The gluconic acid concentration is 20 g / L.

[0154] (4) Preparation of bacterial cellulose membrane containing carboxyl groups on the surface

[0155] The seed solution in step (2) was inoculated with 10% by volume into a fermentation medium with a molar ratio of glucose to gluconic acid of 0:2.0, and cultured at 30°C for 7 days. After the product was taken out, it was rinsed with water for 4 hours, and then immersed in a boiling 5% NaOH solution for 6 hours, and then immersed in a 75% ethanol solution for 3 hours, and finally taken out and repeatedly rinsed with pure water until the eluate was neutral, and vacuum freeze-dried to constant weight. The surface carboxyl density of the obtained carboxyl-modified membrane was 400 μmol / g.

[0156] (5) Preparation of glycosylated bacterial cellulose nanofiber membrane

[0157] Weigh 250 times the molar equivalent of amino-polyethylene glycol-glucose on the membrane surface carboxyl groups, 7 times the molar equivalent of EDC on the amino-polyethylene glycol-glucose, and 3 times the molar equivalent of NHS on the amino-polyethylene glycol-glucose into a flask, and add 5 mL of sodium bicarbonate (pH = 5.5, 50 mM) buffer solution thereto. After the solid is dissolved, add the surface carboxyl-containing bacterial cellulose prepared in step (4) into the flask, and react at a constant temperature of 4°C with shaking for 24 hours. Take out the material, wash it with pure water for 6 times in sequence, and freeze-dry it to obtain a glycosylated bacterial cellulose membrane.

[0158] (6) Specific adhesion of glycosylated bacterial cellulose nanofiber membrane to microorganisms

[0159] E. coli was cultured in LB medium at 37°C and 180 rpm for 12 h. The bacterial solution was centrifuged, and the supernatant was discarded. The cell pellet was resuspended in PBS (0.01 M, pH = 7.2). Before bacterial capture, the OD of E. coli was adjusted by adjusting the volume of PBS solution. 600 The value was readjusted to 0.1, so that the concentration of E. coli bacteria was 1×10 8 CFU / mL.

[0160] Before attaching bacteria, the concentration of the bacterial solution was adjusted to 1×10 8 CFU / mL, dilute 10 5 Pipette 0.1 ml and spread on the plate to calculate the number of bacteria N1. 2The glycosylated bacterial cellulose nanofiber membrane (glucose density is 100 μmol / g), carboxyl-modified bacterial cellulose nanofiber (carboxyl content is 400 μmol / g), and unmodified bacterial cellulose nanofiber membrane were sterilized by ultraviolet for 30 minutes, and then immersed in 1 mL of Escherichia coli (E. coli) solution for adsorption at 25°C and 60 rpm for 90 minutes. After the adsorption of bacteria, the membrane was fixed with 2.5% glutaraldehyde solution at 4°C for 12 hours, freeze-dried, and observed using SEM. In order to more intuitively evaluate the bacterial capture ability of glycosylated BC, fluorescent Escherichia coli (E. coli) was used to perform bacterial capture experiments on glycosylated bacterial cellulose nanofiber membranes. The captured membrane was immersed in 2.5% glutaraldehyde solution for low-temperature fixation for 12 hours and freeze-dried. The sample was observed using CLSM.

[0161] The results are as follows Figure 8 As shown in the figure, pure bacterial cellulose nanofiber membrane still has a small amount of non-specific adhesion to E. coli due to the nano effect, while the non-specific adhesion of carboxyl-modified bacterial cellulose nanofiber membrane to E. coli is effectively suppressed. Compared with the first two, the glycosylated bacterial cellulose nanofiber membrane modified with sugar groups shows significant affinity and adhesion properties to E. coli.

[0162] Example 7

[0163] A preparation method and application of a functionalized bacterial cellulose nanofiber membrane for microbial specific detection, specifically comprising the following steps:

[0164] (1) Activation of Acetobacter xylinum

[0165] Acetobacter xylinum 1.1812 was inoculated into a 5 mL HS medium test tube sterilized at 115°C for 20 min and cultured in a shaker at 30°C and 160 rpm for 24 h;

[0166] (2) Seed liquid culture

[0167] Take 1% activated Acetobacter xylinum suspension and inoculate it into 100 mL HS medium flask, and culture it in a shaker at 30°C and 160 rpm for 24 h;

[0168] The HS culture medium in steps (1) and (2) comprises: 20 g / L glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 2.7 g / L disodium hydrogen phosphate, and 0.5% w / v 95% ethanol.

[0169] (3) Preparation of fermentation medium

[0170] The fermentation medium was prepared by adding 0.5% glucose, 1.5% maltobionic acid, 0.5% peptone, 0.5% yeast powder, 0.115% citric acid, 0.27% disodium hydrogen phosphate, and 95% ethanol 0.5% w / v to pure water according to weight percentage, and sterilized at 115° C. for 20 min.

[0171] The total concentration of glucose and maltobionate is 20 g / L.

[0172] (4) Preparation of bacterial cellulose membrane containing carboxyl groups on the surface

[0173] The seed solution in step (2) was inoculated with 10% by volume into a fermentation medium with a glucose to maltobionic acid molar ratio of 0.5:1.5, and cultured at 30°C for 7 days. After the product was taken out, it was rinsed with water for 4 hours, and then immersed in a boiling 5% NaOH solution for 6 hours, and then immersed in a 75% ethanol solution for 2 hours. Finally, it was taken out and repeatedly rinsed with pure water until the eluate was neutral, and vacuum freeze-dried to constant weight. The resulting membrane surface carboxyl density was 120 μmol / g.

[0174] (5) Preparation of glycosylated bacterial cellulose membrane

[0175] Weigh 800 times the molar equivalent of the carboxyl groups on the membrane surface of glucosamine, 7 times the molar equivalent of the glucosamine of EDC, and 3 times the molar equivalent of the glucosamine of NHS into a flask, and add 5 mL of sodium bicarbonate (pH = 5.5, 50 mM) buffer solution thereto. After the solid is dissolved, add the surface carboxyl-containing bacterial cellulose prepared in step (4) into the flask, and react at a constant temperature of 4°C with shaking for 12 h. Take out the material, wash it with pure water for 6 times in sequence, and freeze-dry it to obtain glycosylated bacterial cellulose with a glycosyl density of 180 μmol / g on the membrane surface.

[0176] (6) Bacteria capture capability at extremely low bacterial concentrations

[0177] Before capturing bacteria, the concentration of E. coli was adjusted to 10 CFU / mL with PBS solution, and 0.1 mL of the bacterial solution with different bacterial counts was applied to the plate to calculate the number of bacteria N1. 100 μL of 10 CFU / mL bacterial solution was directly added to the surface of the glycosyl bacterial cellulose nanofiber membrane (sugar content 180 μmol / g) (the membrane area was 1 cm 2 ), and cultured on a shaker at 25°C and 60 rpm for 1 h, then washed with 900 μL of PBS, and all the washing solution was aspirated to apply to the plate for colony counting, and the number of bacteria N2 was calculated. The amount of captured bacteria was calculated according to the following formula.

[0178] C=N1-N2

[0179] Wherein, C: bacterial capture number (CFU); N1: bacterial number before capture (CFU); N2: bacterial number before capture (CFU).

[0180] The captured membrane was fixed with 2.5% glutaraldehyde solution at 4°C for 12h, freeze-dried, and observed using SEM. In order to more intuitively evaluate the bacterial capture ability of glycosylated BC at extremely low bacterial concentrations, fluorescent Escherichia coli (E. coli) was used to perform bacterial capture experiments on glycosylated BC (sugar content 180μmol / g). The captured membrane was immersed in 2.5% glutaraldehyde solution for 12h and freeze-dried. The sample was observed using CLSM.

[0181] The results are as follows Figure 9-10 As shown, the glucose-modified glycosylated bacterial cellulose nanofiber membrane can still effectively capture Escherichia coli (E. coli) at extremely low bacterial concentrations (1 CFU / mL). The material has sensitive bacterial affinity adhesion and enrichment capture properties.

[0182] Example 8

[0183] A preparation method and application of a functionalized bacterial cellulose nanofiber membrane for microbial specific detection, specifically comprising the following steps:

[0184] The specific steps are as follows:

[0185] (1) Activation of Acetobacter xylinum

[0186] Acetobacter xylinum 1.1812 was inoculated into a 5 mL HS medium test tube sterilized at 115°C for 20 min and cultured in a shaker at 30°C and 160 rpm for 24 h;

[0187] (2) Seed liquid culture

[0188] Take 1% activated Acetobacter xylinum suspension and inoculate it into 100 mL HS medium flask, and culture it in a shaker at 30°C and 160 rpm for 24 h;

[0189] The HS culture medium in steps (1) and (2) comprises: 20 g / L glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 2.7 g / L disodium hydrogen phosphate, and 0.5% w / v 95% ethanol.

[0190] (3) Preparation of fermentation medium

[0191] The fermentation medium was prepared by adding 1% glucose, 1% gluconic acid, 0.5% peptone, 0.5% yeast powder, 0.115% citric acid, 0.27% disodium hydrogen phosphate, and 95% ethanol 0.5% w / v to pure water according to weight percentage, and sterilized at 115° C. for 20 min.

[0192] The total carbon source concentration of glucose and maltobionic acid is 20 g / L.

[0193] (4) Preparation of bacterial cellulose membrane containing carboxyl groups on the surface

[0194] The seed solution in step (2) is inoculated with 10% by volume into a fermentation medium with a molar ratio of glucose to gluconic acid of 1:1, and cultured at 30°C for 7 days. After the product is taken out, it is rinsed with water for 3 hours, and then immersed in a boiling 3% NaOH solution for 5 hours, and then immersed in a 75% ethanol solution for 2 hours. Finally, it is taken out and repeatedly rinsed with pure water until the eluate is neutral, and vacuum freeze-dried to constant weight, and the surface carboxyl density is 220 μmol / g.

[0195] (5) Preparation of mannose-glycosylated bacterial cellulose membrane

[0196] Weigh 1000 times the molar equivalent of the membrane surface carboxyl group of amino-polyethylene glycol-mannose, 5 times the molar equivalent of EDC of amino-polyethylene glycol-mannose, and 3 times the molar equivalent of NHS of amino-polyethylene glycol-mannose into a flask, and add 5 mL of sodium bicarbonate (pH = 5.5, 50 mM) buffer solution thereto. After the solid is dissolved, add the surface carboxyl-containing bacterial cellulose prepared in step (4) into the flask, and react at 4°C with constant temperature shaking for 24 hours. Take out the material, wash it with pure water for 6 times in sequence, and freeze-dry it to obtain mannose glycosylated bacterial cellulose with a glycosyl density of 225 μmol / g.

[0197] (6) Preparation of Glucose Glycosylated Bacterial Cellulose Membrane

[0198] Weigh 1000 times the molar equivalent of the membrane surface carboxyl groups of amino-polyethylene glycol-glucose, 5 times the molar equivalent of amino-polyethylene glycol-glucose of EDC, and 3 times the molar equivalent of amino-polyethylene glycol-mannose of NHS into a flask, and add 5 mL of sodium bicarbonate (pH = 5.5, 50 mM) buffer solution thereto. After the solid is dissolved, add the surface carboxyl-containing bacterial cellulose prepared in step (4) into the flask, and react at a constant temperature of 4°C with shaking for 24 hours. Take out the material, wash it with pure water for 6 times in sequence, and freeze-dry it to obtain glucose-glycosylated bacterial cellulose with a glycosyl density of 225 μmol / g.

[0199] (7) Comparison of the affinity capture performance of two glycosylated membranes for Escherichia coli (E. coli)

[0200] Before affinity capture of bacteria, the concentration of E. coli was adjusted to 10,000 CFU / mL with PBS solution, diluted 10 times, and 100 μL was aspirated to apply to the plate for colony counting and calculation of the number of bacteria N1. 100 μL of 10,000 CFU / mL bacterial solution was directly added to the surface of two sugar-modified glycosylated membranes (sugar content 225 μmol / g) (the area of ​​the membrane was 1 cm 2 ), and cultured on a shaker at 25°C and 60 rpm for 1 hour, then washed with 900 μL of PBS, 100 μL of the washing solution was aspirated and applied to a plate for colony counting, and the number of bacteria N2 was calculated. The number of bacteria captured by affinity on the membrane surface N3 = N1-N2.

[0201] The results showed that the affinity capture numbers of Escherichia coli (E. coli) by mannose (glycosyl content 225 μmol / g) and glucose (glycosyl content 225 μmol / g) glycosylated bacterial cellulose membranes with the same surface glycosyl density were different. The affinity capture number of Escherichia coli (E. coli) by mannose glycosylated bacterial cellulose membrane was 99 CFU, and the affinity capture number of Escherichia coli (E. coli) by glucose glycosylated bacterial cellulose membrane was 91 CFU. Compared with glucose glycosylated bacterial cellulose nanofiber membrane, mannose glycosylated bacterial cellulose nanofiber membrane captured more Escherichia coli (E. coli) and had stronger affinity and adhesion to Escherichia coli (E. coli).

[0202] Example 9

[0203] A preparation method and application of a functionalized bacterial cellulose nanofiber membrane for microbial specific detection, specifically comprising the following steps:

[0204] (1) Activation of Acetobacter xylinum

[0205] Acetobacter xylinum 1.1812 was inoculated into a 5 mL HS medium test tube sterilized at 115°C for 20 min and cultured in a shaker at 30°C and 160 rpm for 24 h;

[0206] (2) Seed liquid culture

[0207] Take 1% activated Acetobacter xylinum suspension and inoculate it into 100 mL HS medium flask, and culture it in a shaker at 30°C and 160 rpm for 24 h;

[0208] The HS culture medium in steps (1) and (2) comprises: 20 g / L glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 2.7 g / L disodium hydrogen phosphate, and 0.5% w / v 95% ethanol.

[0209] (3) Preparation of fermentation medium

[0210] The fermentation medium was prepared by adding 1% glucose, 1% maltobionic acid, 0.5% peptone, 0.5% yeast powder, 0.115% citric acid, 0.27% disodium hydrogen phosphate, and 95% ethanol 0.5% w / v to pure water according to weight percentage, and sterilized at 115° C. for 20 min.

[0211] The total carbon source concentration of glucose and maltobionic acid is 20 g / L.

[0212] (4) Preparation of bacterial cellulose membrane containing carboxyl groups on the surface

[0213] The seed solution in step (2) is inoculated with 10% by volume into a fermentation medium with a molar ratio of glucose to maltobionic acid of 1:1, and cultured at 30°C for 7 days. After the product is taken out, it is rinsed with water for 3 hours, and then immersed in a boiling 3% NaOH solution for 5 hours, and then immersed in a 75% ethanol solution for 2 hours. Finally, it is taken out and repeatedly rinsed with pure water until the eluate is neutral, and vacuum freeze-dried to constant weight, and the surface carboxyl density is 200 μmol / g.

[0214] (5) Preparation of glycosylated bacterial cellulose membrane

[0215] Weigh 1000 times the molar equivalent of the membrane surface carboxyl group of amino-polyethylene glycol-mannose, 5 times the molar equivalent of amino-polyethylene glycol-mannose EDC, and 3 times the molar equivalent of amino-polyethylene glycol-mannose NHS into a flask, and add 5 mL of sodium bicarbonate (pH = 5.5, 50 mM) buffer solution thereto. After the solid is dissolved, add the surface carboxyl-containing bacterial cellulose prepared in step (4) into the flask, and react at a constant temperature of 4°C with shaking for 24 hours. Take out the material, wash it with pure water for 6 times in sequence, and freeze-dry it to obtain glycosylated bacterial cellulose with a glycosyl density of 210 μmol / g.

[0216] (6) Specific capture properties of glycosylated bacterial cellulose nanofiber membranes for bacteria

[0217] Before capturing bacteria, the concentration of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) was adjusted to 10,000 CFU / mL with PBS solution. After diluting 10 times, 100 μL was aspirated and plated for colony counting to calculate the number of bacteria N1. 100 μL of 10,000 CFU / mL bacterial solution was directly added to the surface of the glycosylated membrane (glycosylation content 210 μmol / g) and the surface carboxyl-modified membrane (carboxyl content 200 μmol / g) (the membrane area was 1 cm 2), and cultured on a shaker at 25°C and 60 rpm for 1 hour, then washed with 900 μL of PBS, 0.1 mL of the washing solution was aspirated and plated for colony counting, and the number of bacteria N2 was calculated. The bacterial capture efficiency C% was calculated according to the following formula.

[0218]

[0219] Wherein, C%: bacterial capture efficiency; N1: bacterial number before capture (CFU); N2: bacterial number before capture (CFU).

[0220] The results are as follows Fig.11 As shown in the figure, at a bacterial concentration of 1000 CFU / mL, the mannose glycosylated membrane has a stronger capture effect on Escherichia coli (E.coli), while the capture efficiency of the other two bacteria decreases and is lower than that of the carboxyl modified membrane. This is because the carboxyl group is negatively charged at a solution pH of 7.4, and bacteria are negatively charged at a solution pH of 7.4. Negative and negative charges repel each other, resulting in a certain repulsive effect of the carboxyl modified membrane on bacteria. After glycosylation, the membrane surface is more hydrophilic, and the surface hydration layer further hinders the nonspecific adhesion of bacteria.

[0221] Example 10

[0222] A preparation method and application of a functionalized bacterial cellulose nanofiber membrane for microbial specific detection, specifically comprising the following steps:

[0223] (1) Activation of Acetobacter xylinum

[0224] Acetobacter xylinum 1.1812 was inoculated into a 5 mL HS medium test tube sterilized at 115°C for 20 min and cultured in a shaker at 30°C and 160 rpm for 24 h;

[0225] (2) Seed liquid culture

[0226] Take 1% activated Acetobacter xylinum suspension and inoculate it into 100 mL HS medium flask, and culture it in a shaker at 30°C and 160 rpm for 24 h;

[0227] The HS culture medium in steps (1) and (2) comprises: 20 g / L glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 2.7 g / L disodium hydrogen phosphate, and 0.5% w / v 95% ethanol.

[0228] (3) Preparation of fermentation medium

[0229] The fermentation medium was prepared by adding 0.5% glucose, 1.5% galactose acid, 0.5% peptone, 0.5% yeast powder, 0.115% citric acid, 0.27% disodium hydrogen phosphate, and 95% ethanol 0.5% w / v to pure water according to weight percentage, and sterilized at 115° C. for 20 min.

[0230] The total carbon source concentration of glucose and galactonic acid is 20 g / L.

[0231] (4) Preparation of bacterial cellulose membrane containing carboxyl groups on the surface

[0232] The seed solution in step (2) is inoculated with 10% by volume into a fermentation medium with a glucose to galactonic acid molar ratio of 0.5:1.5, and cultured at 30°C for 7 days. After the product is taken out, it is rinsed with water for 3 hours, and then immersed in a boiling 3% NaOH solution for 5 hours, and then immersed in a 75% ethanol solution for 2 hours. Finally, it is taken out and repeatedly rinsed with pure water until the eluate is neutral, and vacuum freeze-dried to constant weight, and the surface carboxyl density is 300 μmol / g.

[0233] (5) Preparation of glycosylated bacterial cellulose membrane

[0234] Weigh 700 times the molar equivalent of the membrane surface carboxyl groups of amino-polyethylene glycol-glucose, 7 times the molar equivalent of EDC of amino-polyethylene glycol-glucose, and 3 times the molar equivalent of NHS of amino-polyethylene glycol-glucose into a flask, and add 5 mL of sodium bicarbonate (pH = 5.5, 50 mM) buffer solution thereto. After the solid is dissolved, add the surface carboxyl-containing bacterial cellulose prepared in step (4) into the flask, and react at a constant temperature of 4°C with shaking for 24 hours. Take out the material, wash it with pure water for 6 times in sequence, and freeze-dry it to obtain glycosylated bacterial cellulose with a glycosyl density of 250 μmol / g.

[0235] (6) Specific adsorption test of glycosylated membrane on mixed fluorescent bacteria

[0236] Before capturing bacteria, the concentration of green fluorescent Escherichia coli (E. coli) and red fluorescent Pseudomonas aeruginosa (P. aeruginosa) was adjusted to 1×10 8 CFU / mL, 0.5mL of fluorescent Escherichia coli (E. coli) and 0.5mL of Pseudomonas aeruginosa (P. aeruginosa) bacterial suspension were respectively mixed into 1mL of bacterial solution, and the glycosylated membrane (membrane area of ​​1cm 2 ) was added to the mixed bacterial solution and cultured on a shaker at 25°C and 60 rpm for 1 hour. After the reaction was completed, the membrane was washed and the bacteria were fixed with 2.5% glutaraldehyde. After the membrane was freeze-dried, it was placed under a fluorescent inverted microscope to observe the fluorescence.

[0237] The results are as follows Fig.12 As shown in the figure, compared with the carboxyl-modified membrane, the glucose-glycosylated membrane has a specific adsorption effect on green fluorescent Escherichia coli (E. coli), but only a small amount of non-specific adsorption on red Pseudomonas aeruginosa (P. aeruginosa). This further proves the specific adsorption performance of glucose-glycosylated bacterial cellulose nanofiber membrane on E. coli.

[0238] Embodiment 11

[0239] A preparation method and application of a functionalized bacterial cellulose nanofiber membrane for microbial specific detection, specifically comprising the following steps:

[0240] (1) Activation of Acetobacter xylinum

[0241] Acetobacter xylinum 1.1812 was inoculated into a 5 mL HS medium test tube sterilized at 115°C for 20 min and cultured in a shaker at 30°C and 160 rpm for 24 h;

[0242] (2) Seed liquid culture

[0243] Take 1% activated Acetobacter xylinum suspension and inoculate it into 100 mL HS medium flask, and culture it in a shaker at 30°C and 160 rpm for 24 h;

[0244] The HS culture medium in steps (1) and (2) comprises: 20 g / L glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 2.7 g / L disodium hydrogen phosphate, and 0.5% w / v 95% ethanol.

[0245] (3) Preparation of fermentation medium

[0246] The fermentation medium was prepared by adding 1.5% glucose, 0.5% maltobionic acid, 0.5% peptone, 0.5% yeast powder, 0.115% citric acid, 0.27% disodium hydrogen phosphate, and 95% ethanol 0.5% w / v to pure water according to weight percentage, and sterilized at 115° C. for 20 min.

[0247] The total carbon source concentration of glucose and maltobionic acid is 20 g / L.

[0248] (4) Preparation of bacterial cellulose membrane containing carboxyl groups on the surface

[0249] The seed solution in step (2) is inoculated with 10% by volume into a fermentation medium with a molar ratio of glucose to maltobionic acid of 1:1, and cultured at 30°C for 7 days. After the product is taken out, it is rinsed with water for 3 hours, and then immersed in a boiling 3% NaOH solution for 5 hours, and then immersed in a 75% ethanol solution for 2 hours. Finally, it is taken out and repeatedly rinsed with pure water until the eluate is neutral, and vacuum freeze-dried to constant weight, and the surface carboxyl density is 100 μmol / g.

[0250] (5) Preparation of glycosylated bacterial cellulose membrane

[0251] Weigh 300 times the molar equivalent of the membrane surface carboxyl groups of amino-polyethylene glycol-mannose, 7 times the molar equivalent of EDC of amino-polyethylene glycol-mannose, and 3 times the molar equivalent of NHS of amino-polyethylene glycol-mannose into a flask, and add 5 mL of sodium bicarbonate (pH = 5.5, 50 mM) buffer solution thereto. After the solid is dissolved, add the surface carboxyl-containing bacterial cellulose prepared in step (4) into the flask, and react at a constant temperature of 4°C with shaking for 24 hours. Take out the material, wash it with pure water for 6 times in sequence, and freeze-dry it to obtain glycosylated bacterial cellulose with a glycosyl density of 160 μmol / g.

[0252] (6) Detection of Escherichia coli (E. coli) by membrane filtration method

[0253] (6.1) Sterilization of membranes and filters

[0254] 1) Place a porous acetate cellulose membrane with a pore size of 0.45um and a diameter of 50mm and a glycosylated BC membrane with a diameter of 50mm in a beaker, add distilled water, and boil and sterilize in a boiling water bath for three times, each time for 15 minutes. After the first two boilings, change the water and wash 2-3 times to remove the residual solvent.

[0255] 2) Sterilize filters such as filtration bottles, funnels, and tweezers. Wrap the filters in tin foil and sterilize them under high pressure at 115°C steam for 20 minutes.

[0256] (6.2) Isolation and culture

[0257] 1) Filter the water sample. The bacterial concentration in the filtered water sample is about 150 CFU. Use sterile tweezers to clamp the edge of the sterilized filter membrane, with the rough surface facing up, and place it on the sterilized filter. Fix the filter securely, inject the water sample into the filter, cover it, open the filter valve, and filter. After the water sample is extracted, add an equal amount of sterile water to continue filtering, in order to rinse the bucket wall.

[0258] 2) Use sterile tweezers to grasp the edge of the filter membrane and move it onto the surface of the eosin-methylene blue (EMB) culture medium, with the bacteria-retaining side of the filter membrane facing up and the other side completely in contact with the culture medium. No bubbles should be left between the two. Repeat the above steps and inoculate two more plates. Invert the culture dish and culture at 37°C for 24 hours.

[0259] 3) Principle: The colonies of Escherichia coli (E. coli) grown on the surface of solid culture medium containing EMB are black.

[0260] (6.3) Observation and counting of bacterial colonies

[0261] 1) Observe the growth of bacterial colonies on the two membranes, take photos of the colonies on the plates at 0h, 6h, 12h, and 22h, and then select the colonies that meet the characteristics of Escherichia coli (E. coli) colonies for counting.

[0262] The results are as follows Fig.13 As shown, the positive control showed that the total number of E. coli in the total system was 35 CFU, the commercial cellulose acetate filter membrane detected 31 CFU E. coli, and the glycosylated bacterial cellulose nanofiber membrane detected 33 CFU E. coli. Compared with the commercial cellulose acetate filter membrane, the glycosylated bacterial cellulose nanofiber membrane-based filter membrane has higher sensitivity and accuracy in microbial detection.

[0263] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a functionalized bacterial cellulose nanofiber membrane for microbial specific detection, characterized in that: The preparation method comprises: Step 1), obtaining a carboxyl-modified bacterial cellulose membrane with negatively charged carboxyl groups on the surface through microbial catalytic biopolymerization; Step 2), the carboxyl-modified bacterial cellulose membrane obtained above is catalyzed by coupling reaction with an amino-containing sugar derivative, and the sugar molecules are chemically bonded and fixed to the surface of the bacterial cellulose nanofiber membrane to prepare a glycosylated bacterial cellulose nanofiber membrane.

2. The method for preparing a functionalized bacterial cellulose nanofiber membrane for microorganism-specific detection according to claim 1, characterized in that: Step 1) The microbial catalytic biopolymerization is to inoculate the microorganism into a fermentation medium of a carboxyl-containing sugar derivative and culture it at 30° C. for 1 to 7 days.

3. The method for preparing a functionalized bacterial cellulose nanofiber membrane for microorganism-specific detection according to claim 1, characterized in that: Step 2) The catalytic coupling reaction is to immerse the carboxyl-modified bacterial cellulose membrane in an amino-containing sugar derivative solution under the catalysis of EDC and NHS, and react with constant temperature oscillation at 0-25°C for 1-24 hours to obtain a glycosylated bacterial cellulose nanofiber membrane.

4. The method for preparing a functionalized bacterial cellulose nanofiber membrane for microorganism-specific detection according to claim 2, characterized in that: The fermentation medium is prepared by adding 0-20% glucose, 0-20% carboxyl-containing sugar derivatives, 0.001-10% peptone, 0.001-10% yeast powder, 0.001-10% citric acid, 0.001-10% disodium hydrogen phosphate, and 0.001-10% w / v 95% ethanol to pure water according to weight percentage.

5. The method for preparing a functionalized bacterial cellulose nanofiber membrane for microorganism-specific detection according to claim 3, characterized in that: The amino-containing sugar derivative is at least one of glucosamine, amino-polyethylene glycol-glucose, amino-polyethylene glycol-galactose, and amino-polyethylene glycol-mannose.

6. The method for preparing a functionalized bacterial cellulose nanofiber membrane for microorganism-specific detection according to claim 2 or 4, characterized in that: The carboxyl-containing sugar derivative is at least one of gluconic acid, lactobionic acid and maltobionic acid.

7. The method for preparing a functionalized bacterial cellulose nanofiber membrane for microorganism-specific detection according to claim 3, characterized in that: The feeding amount of the amino-containing sugar derivative is 1-1000 times the molar amount of the carboxyl group on the membrane surface, and the molar ratio of the amount of EDC and NHS to the amino-containing sugar derivative is 1-100:1-100:0.1-20.

8. Glycosylated bacterial cellulose nanofiber membrane for microorganism specific detection prepared by the method according to any one of claims 1 to 7.

9. Use of the glycosylated bacterial cellulose nanofiber membrane according to claim 8 in bacteria-specific detection.

10. Use of the glycosylated bacterial cellulose nanofiber membrane according to claim 8 in bacteria-specific identification and enrichment.

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