Green microorganism colony detection filter membrane with printed grid lines and processing method thereof

Abstract

The application discloses a green microbial colony detection filter membrane with printed grid lines and a processing method thereof, and relates to the technical field of microbial detection consumables, which is composed of the following materials: 100-500 parts of bacterial cellulose green base material, 10-450 parts of phycocyanin-chitosan green color paste, 10-120 parts of colorant and 20-110 parts of treatment agent; wherein, the chitosan in the phycocyanin-chitosan green color paste serves as a positively charged bridging agent and is fixed in the negatively charged bacterial cellulose nanofiber network through electrostatic adsorption, and the phycocyanin is anchored in the nanofiber network pores. The application can effectively avoid the problems of pigment falling off and discoloration, and can be widely used in microbial colony detection scenes such as water quality, food and environment.

Description

Technical Field

[0001] This invention relates to the field of microbial detection consumables technology, specifically to a green microbial colony detection filter membrane with printed grid lines and its processing method. Background Technology

[0002] Filtration is the mainstream technology for microbial colony detection in water quality, food, and environmental fields. It is suitable for screening trace microorganisms and detecting large-volume samples, and features high sensitivity and wide applicability. As a core testing consumable, the performance of the filter membrane substrate, color contrast, and positioning structure directly affect the microbial retention effect, colony counting accuracy, and detection repeatability.

[0003] Currently, most commercially available microbial detection membranes use cellulose ester-based chemical synthetic materials, which have several technical drawbacks. Conventional membranes are mostly white or transparent, with little color difference from most microbial colonies, resulting in low identification of tiny colonies and a high risk of missed or false detections. To improve counting difficulties, existing technologies add grids through imprinting or spraying, but these grids have weak adhesion to the membrane and are prone to detachment and blurring during cultivation and filtration, losing their positioning function. Furthermore, relying solely on a single grid for positioning, without the aid of a contrasting background color, still results in poor identification of light-colored and semi-transparent colonies, leading to significant counting errors.

[0004] Furthermore, traditional filter membrane modification processes are mostly simple physical adhesion processes, where coloring and functional components are easily precipitated and lost. This not only leads to filter membrane fading and performance degradation but may also inhibit microbial growth, resulting in false negative test results. Traditional filter membrane substrates have poor pore stability and hydrophilicity, allowing microorganisms to easily spread and grow, causing colony adhesion and overlap, further reducing detection accuracy and repeatability. Simultaneously, chemically synthesized filter membranes have poor biocompatibility and degradability, and some additives are biotoxic, making them unsuitable for high-safety-level testing scenarios such as food and drinking water, and easily causing environmental pollution after disposal. To address these issues, we propose a green microbial colony detection filter membrane with printed grid lines and its processing method. Summary of the Invention

[0005] To address the aforementioned technical problems, a green microbial colony detection filter membrane with printed grid lines and its processing method are provided. This technical solution solves the problems of poor identification effect for light-colored and semi-transparent colonies, large counting errors, and reduced detection accuracy and repeatability.

[0006] To achieve the above objectives, the technical solution adopted by this invention is: a green microbial colony detection filter membrane with printed grid lines, composed of the following materials: 100-500 parts of bacterial cellulose green substrate, 10-450 parts of phycocyanin-chitosan green color paste, 10-120 parts of colorant and 20-110 parts of treatment agent; In the phycocyanin-chitosan green color paste, chitosan acts as a positively charged bridging agent, which is fixed in the negatively charged bacterial cellulose nanofiber network by electrostatic adsorption, thereby anchoring phycocyanin in the pores of the nanofiber network. The filter membrane surface is provided with printed grid lines, which together with the green substrate form a dual-contrast positioning system; the green substrate provides visual contrast of the colony body, and the printed grid lines provide spatial distribution positioning of the colony.

[0007] Preferably, the bacterial cellulose green substrate is prepared by purifying a bacterial cellulose membrane, wherein the bacterial cellulose membrane has a thickness of 20-80 μm and a pore size of 0.1-0.8 μm; and the bacterial cellulose membrane is synthesized by fermentation of Acetobacter xylinum or Gluconobacterium xylinum.

[0008] Preferably, the mass ratio of phycocyanin to chitosan in the phycocyanin-chitosan green paste is 1:0.5 to 1:3; the phycocyanin is extracted from Spirulina or cyanobacteria and has a purity of ≥30%; the degree of deacetylation of the chitosan is ≥85% and the molecular weight is 5 to 50 kDa.

[0009] Preferably, the colorant is a dark water-soluble colorant selected from one or more of food-grade carbon black, dark green chlorophyll copper sodium salt, and dark blue indigo; the colorant is used to print grid lines on the surface of the filter membrane, so that the grid lines form a color difference contrast with the green membrane.

[0010] Preferably, the treatment agent is a combination of a hydrophilic treatment agent and a crosslinking agent; the hydrophilic treatment agent is selected from one or more of glycerol, polyethylene glycol and Tween-80; the crosslinking agent is selected from glutaraldehyde or genipin.

[0011] A method for processing a green microbial colony detection filter membrane with printed grid lines, comprising the following steps: S1. Purify the bacterial cellulose membrane to obtain a bacterial cellulose green substrate; S2. Mix phycocyanin and chitosan in an aqueous phase to obtain phycocyanin-chitosan green paste; S3. After mixing the treatment agent with the phycocyanin-chitosan green color paste, control the pH of the system to be 3.5-5.5, and impregnate the bacterial cellulose green substrate under vacuum conditions, so that the chitosan in the protonated state acts as a positively charged bridging agent and is fixed in the negatively charged bacterial cellulose nanofiber network by electrostatic adsorption, thereby anchoring the phycocyanin in the pores of the nanofiber network. S4. The impregnated substrate is dried under heating conditions and a cross-linking reaction is initiated, so that the chitosan and bacterial cellulose nanofibers form chemical cross-links, which together with the electrostatic physical anchoring in S3 form a double fixation to obtain a green composite membrane. S5. Printing colorant on the surface of the green composite film to form a printing grid, so that the printing grid and the green substrate together constitute a dual contrast positioning system.

[0012] Preferably, the purification process described in S1 includes: placing the bacterial cellulose membrane in a 0.1-1 mol / L sodium hydroxide solution, treating it in a water bath at 70-90°C for 2-4 hours to remove bacterial cells and culture medium residues, and washing it with water until neutral to obtain a green bacterial cellulose substrate. The degree of deacetylation of the chitosan described in S2 is 85%–95%, and the mass ratio of phycocyanin to chitosan is 1:0.5–3.

[0013] Preferably, the vacuum condition described in S3 has a vacuum degree of -0.06 to -0.09 MPa, an impregnation temperature of 25 to 45°C, and an impregnation time of 30 to 120 min.

[0014] Preferably, the heating conditions in S4 are 50-80°C and the drying time is 1-4 hours; the crosslinking reaction is that the aldehyde group of the crosslinking agent reacts with the free amino group of the chitosan chain to form Schiff base crosslinking, while the other end of the crosslinking agent condenses with the hydroxyl groups on the surface of bacterial cellulose nanofibers to establish a covalent bridge between chitosan and bacterial cellulose.

[0015] Preferably, the printing in S5 is carried out using any one of screen printing, inkjet printing and transfer printing; the line width of the printed grid is 0.05 to 0.3 mm, the grid spacing is 1 to 5 mm, and the grid color is dark green, black or white.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses a specific ratio of bacterial cellulose green substrate, phycocyanin-chitosan green pigment, colorant, and treatment agent as raw materials. It utilizes positively charged chitosan as a bridging agent, anchoring phycocyanin to the negatively charged bacterial cellulose nanofiber pores through electrostatic adsorption. This is combined with a cross-linking reaction to form a dual-fixation structure of electrostatic anchoring and chemical cross-linking, effectively avoiding pigment shedding and fading, ensuring uniform and stable membrane color. The filter membrane constructs a dual-contrast positioning system through a green substrate and dark printed grid lines. The green substrate clearly distinguishes light-colored colonies, and the high-precision grid accurately achieves spatial positioning and counting of colonies, significantly reducing detection errors. Simultaneously, the substrate has regular pore size and excellent retention performance. Combined with a hydrophilic treatment agent, the effect is enhanced. Furthermore, the raw materials have good biocompatibility and do not affect microbial growth. This invention features controllable process parameters, high product consistency, and is suitable for mass production. It can be widely used in water quality, food, environmental, and other microbial colony detection scenarios. Attached Figure Description

[0017] Figure 1 This is a flowchart of the preparation steps of the present invention. Detailed Implementation

[0018] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0019] Example 1: Standard Example The green microbial colony detection filter membrane with printed grid lines in this embodiment is composed of the following components: 250 parts of bacterial cellulose green substrate, 200 parts of phycocyanin-chitosan green color paste, 50 parts of colorant and 60 parts of treatment agent.

[0020] Processing methods S1: Preparation of bacterial cellulose green substrate The bacterial cellulose membrane (synthesized by fermentation of Acetobacter xylinum, with a thickness of 50 μm and a pore size of 0.4 μm) was placed in a 0.5 mol / L NaOH solution and purified in a water bath at 80 °C for 3 h. After thorough washing with water until neutral, the purified bacterial cellulose green substrate was obtained after drying.

[0021] S2: Preparation of phycocyanin-chitosan green pigment paste Chitosan with a degree of deacetylation of 90% and a molecular weight of 20 kDa was prepared into a 1% (w / v) dilute acetic acid solution. Spirulina phycocyanin powder with a purity of 40% was prepared into a 1% (w / v) aqueous solution. The chitosan solution and phycocyanin solution were mixed at a mass ratio of 1:1 and stirred at 300 rpm for 2 hours on a magnetic stirrer to ensure complete binding of the phycocyanin and chitosan, yielding a phycocyanin-chitosan green paste. The mass ratio of phycocyanin to chitosan was 1:1.

[0022] S3: Vacuum impregnation / penetration A treatment agent was added to the phycocyanin-chitosan green pigment paste. The hydrophilic treatment agent was a mixture of glycerol (30 parts) and polyethylene glycol (PEG-400, 20 parts), and the crosslinking agent was glutaraldehyde (10% aqueous solution, 10 parts). The pH of the system was adjusted to 4.5, and the bacterial cellulose green substrate was immersed in the pigment paste under a vacuum of -0.08 MPa at a temperature of 35°C for 60 minutes. Under these pH conditions, the amino groups of chitosan (… (pKa≈6.3) is in a protonated state ( Phycocyanin (isoelectric point pI≈4.8) is negatively charged under this pH condition and can be anchored in the pores of the nanofiber network through the bridging effect of chitosan.

[0023] S4: Crosslinking fixation and drying The impregnated substrate was removed and dried at 60°C for 2.5 hours. During this process, the aldehyde group (-CHO) of glutaraldehyde reacts with the amino group (-CHO) of chitosan. The Schiff base reaction occurs, forming carbon-nitrogen double bonds (C=N). Simultaneously, covalent bridging between chitosan and bacterial cellulose nanofibers is achieved, forming a dual fixation mechanism of physical electrostatic anchoring and chemical cross-linking, resulting in a green composite membrane.

[0024] S5: Formation of Printing Grid Lines A screen printing method is used to print colorant onto the surface of the green composite film to form a grid pattern. The colorant is a mixture of food-grade dark green sodium copper chlorophyll and dark blue indigo (mass ratio 1:1). The printed lines are 0.3 mm wide and 4 mm apart. After printing, the color difference between the grid lines and the film is ΔE ≥ 10, forming a dual-contrast positioning system with the green substrate.

[0025] Product Performance The filter membrane prepared in this embodiment has an overall thickness of 60 μm, a porosity of 72%, a pore size of 0.4 μm, and is stable in a green color within the pH range of 4.0 to 8.0, with a color difference ΔE ≤ 3 after 48 hours. The upper surface of the filter membrane is the colony detection surface, and the lower surface is in contact with the culture medium.

[0026] Example 2: High-performance example The green microbial colony detection filter membrane with printed grid lines in this embodiment is composed of the following components: 300 parts of bacterial cellulose green substrate, 280 parts of phycocyanin-chitosan green color paste, 80 parts of colorant and 85 parts of treatment agent.

[0027] Processing methods S1: Preparation of bacterial cellulose green substrate A bacterial cellulose membrane (synthesized by fermentation of *Gastrodia elata*, 70 μm thick, 0.3 μm pore size) was placed in 0.8 mol / L NaOH solution and purified in a water bath at 85 °C for 3.5 h. After thorough washing with water until neutral, the membrane was dried to obtain the purified bacterial cellulose green substrate. Optimizing the pore size to 0.3 μm facilitates clearer edge contrast during subsequent colony counting.

[0028] S2: Preparation of phycocyanin-chitosan green pigment paste A 1.2% (w / v) dilute acetic acid solution was prepared using chitosan with a degree of deacetylation of 95% and a molecular weight of 15 kDa. A 1.2% (w / v) aqueous solution was prepared using spirulina phycocyanin powder with a purity of 50%. The chitosan solution and phycocyanin solution were mixed at a mass ratio of 1:1.5 and stirred at 400 rpm for 2.5 h on a magnetic stirrer to obtain a phycocyanin-chitosan green pigment. High-degree-of-deacetylation chitosan (≥85%) provides more protonated amino sites, which is beneficial for enhancing the electrostatic anchoring effect; the pigment retention rate after 48 h is ≥85%.

[0029] S3: Vacuum impregnation / penetration A treatment agent was added to the phycocyanin-chitosan green pigment paste. The hydrophilic treatment agent was a mixture of glycerol (25 parts), polyethylene glycol (PEG-600, 25 parts), and Tween-80 (5 parts), and the crosslinking agent was genipin (10% solution, 30 parts). The pH of the system was adjusted to 4.0, and the bacterial cellulose green substrate was immersed in the pigment paste under a vacuum of -0.085 MPa at a temperature of 40°C for 90 minutes. Genipin was used as a natural crosslinking agent, which is environmentally friendly and has better biocompatibility. The vacuum negative pressure drove the pigment paste to penetrate deep into the substrate, increasing the number of anchoring points.

[0030] S4: Crosslinking fixation and drying The impregnated substrate was removed and dried at 70°C for 3 hours. During this process, genipin, acting as a crosslinking agent, reacted with chitosan and bacterial cellulose. Simultaneously, the low-temperature, long-duration drying facilitated the full progress of the crosslinking reaction, resulting in a denser crosslinked network structure and a significant increase in wet tensile strength.

[0031] S5: Formation of Printing Grid Lines Inkjet printing is used to print colorant onto the surface of the green composite film to form a grid pattern. The colorant is a mixture of dark green sodium copper chlorophyll salt (60 parts) and food-grade carbon black (20 parts). Precise control via an inkjet printhead results in a line width of 0.15 mm, a spacing of 3 mm, and a color difference ΔE ≥ 12 between the grid pattern and the film. The colorant, printed on the grid area, forms a layered distribution structure with the pigment penetrating into the substrate. The colorant provides high-contrast grid markings, while the pigment provides a uniform green background.

[0032] Product Performance The filter membrane prepared in this embodiment has an overall thickness of 85 μm, a porosity of 68%, a pore size of 0.3 μm, and is stable in a green color within the pH range of 4.0–8.0, with a color difference ΔE ≤ 2.5 after 48 hours. The upper surface of the filter membrane is the colony detection surface, with a green background and printed grid lines creating a dual contrast; the lower surface is in contact with the culture medium. The wet tensile strength reaches 8.5 MPa, significantly better than that of Example 1.

[0033] Example 3: Alternative Solution Example Option A: Examples of Different Microbial Sources S1: A bacterial cellulose membrane synthesized by fermentation with Acetobacter xylinum, with a thickness of 40 μm and a pore size of 0.6 μm; treated with 0.3 mol / L NaOH solution in a 75℃ water bath for 2.5 h, and washed with water until neutral; S2: Take chitosan with a deacetylation degree of 85% and a molecular weight of 35kDa, and mix it with phycocyanin extracted from cyanobacteria with a purity of 35% at a mass ratio of 1:2 to prepare phycocyanin-chitosan green paste. S3: pH 5.0, vacuum degree -0.07MPa, impregnation temperature 30℃, impregnation time 80min. The treatment agent is 20 parts glycerol and 15 parts glutaraldehyde (8% solution); S4: Dry at 55℃ for 3.5h to initiate the Schiff base crosslinking reaction; S5: Screen printing is used, the colorant is dark indigo, the printing line width is 0.4mm, and the spacing is 5mm.

[0034] Product performance: Thickness 45μm, porosity 78%, pore size 0.6μm, color difference ΔE≤2.8 after 48h, phycocyanin retention rate 86% after 48h.

[0035] Option B: Examples of different colorants S1: Bacterial cellulose membrane synthesized by fermentation of Acetobacter xylinum, with a thickness of 55 μm and a pore size of 0.35 μm; treated with 0.6 mol / L NaOH solution in a water bath at 82℃ for 3 h; S2: Chitosan (92% deacetylation, 25kDa) and Spirulina phycocyanin (45% purity) are mixed at a mass ratio of 1:1.2; S3: pH 4.2, vacuum degree -0.08MPa, impregnation temperature 38℃, impregnation time 75min. The treatment agent is 30 parts of polyethylene glycol (PEG-400), 8 parts of Tween-808, and 20 parts of glutaraldehyde (10% solution); S4: Dry at 65℃ for 2.8 hours; S5: Utilizes transfer printing with a single food-grade carbon black colorant. The printing line width is 0.2mm, and the spacing is 3.5mm. The carbon black colorant provides high-contrast black grid lines, creating a striking visual contrast with the green substrate.

[0036] Product performance: Thickness 62μm, porosity 70%, pore size 0.35μm, color difference ΔE ≤ 2.6 after 48h, color difference ΔE between grid lines and film ≥ 15.

[0037] Option C: Examples of different printing methods S1: Bacterial cellulose membrane synthesized by fermentation of *Gastrodia elata*, with a thickness of 45 μm and a pore size of 0.5 μm; treated with 0.4 mol / L NaOH solution in a 78℃ water bath for 2.8 h; S2: Chitosan (88% deacetylation, 40kDa) and spirulina phycocyanin (38% purity) are mixed at a mass ratio of 1:2.5; S3: pH 4.8, vacuum degree -0.075MPa, impregnation temperature 32℃, impregnation time 100min. The treatment agent is 35 parts glycerol and 25 parts genipin (8% solution); S4: Dry at 58℃ for 4 hours.

[0038] S5: It adopts inkjet printing, and the colorant is dark green chlorophyll copper sodium salt. The printing line width is 0.1mm and the spacing is 4.5mm. Fine line width printing is suitable for high-density colony detection scenarios. The line width error is controlled within ±0.02mm.

[0039] Product performance: Thickness 52μm, porosity 75%, pore size 0.5μm, color difference ΔE≤3 after 48h, light-colored colony detection resolution reaches 0.8mm.

[0040] Comparative Example 1: Chitosan-free bridging Method: Spirulina phycocyanin (40% purity) was directly prepared into a 1% (w / v) aqueous solution without adding chitosan. A treatment agent (25 parts glycerol, 15 parts 10% glutaraldehyde solution) was added, and the pH was adjusted to 7.0 (neutral conditions). The bacterial cellulose membrane was immersed in the color paste for 2 hours under normal pressure, then removed and dried at 60℃ for 2 hours before printing grid lines (0.3 mm line width).

[0041] Results Analysis: Without chitosan as a bridging agent, phycocyanin could not be fixed to the bacterial cellulose nanofiber network via electrostatic adsorption. Test data showed that the phycocyanin retention rate after 48 hours was only 42%, significantly lower than the over 88% in the embodiments of this invention. The color difference ΔE reached 8.5 after 48 hours, indicating severe fading of the green substrate. In the chitosan-free system, phycocyanin adhered to the fiber surface via physical adsorption, resulting in weak binding force and easy desorption and migration in an aqueous environment. The grid line wash resistance rate reached 35%, indicating that stable fixation cannot be achieved solely through physical drying.

[0042] Comparative Example 2: Impregnation under normal pressure Method: The component formulation of Example 1 of this invention was used, but the vacuum impregnation step was omitted. The bacterial cellulose membrane was immersed in the color paste under normal pressure, and the impregnation time was extended to 180 min (equivalent permeation). The remaining steps were the same. The pH was controlled at 4.5, and the treatment agent was 30 parts glycerol, 20 parts polyethylene glycol, and 10 parts glutaraldehyde (10% solution).

[0043] Results Analysis: During atmospheric pressure impregnation, atmospheric pressure was insufficient to effectively drive the pigment to penetrate deep into the bacterial cellulose membrane. Test data showed that the phycocyanin retention rate after 48 hours was only 58%, lower than the over 88% in the vacuum impregnation example. The membrane pore size distribution was uneven, with an average pore size of 0.6 μm, and localized impermeable dead zones existed. The detection resolution of light-colored colonies was only 1.5 mm, significantly worse than the 0.5 mm resolution of the present invention's embodiment. This is because the pigment only remained on the surface layer and shallow pores of the substrate, resulting in insufficient coloring layer thickness and poor visual contrast for light-colored colonies.

[0044] Comparative Example 3: No cross-linking reaction Method: The component formulation of Example 1 of this invention was used, but the drying temperature was reduced to 35°C and the drying time was shortened to 1 hour in the S4 drying step, and no crosslinking agent was used (the treatment agent contained only 50 parts of glycerol and no glutaraldehyde). The printing grid parameters were the same as in Example 1.

[0045] Results Analysis: Relying solely on physical drying to evaporate the solvent, no chemical cross-linking occurred between chitosan and bacterial cellulose nanofibers. Test data showed that the phycocyanin retention rate after 48 hours was 51%, significantly lower than the over 88% of the previous embodiment. The wet tensile strength was only 2.8 MPa, only 54% of that in Example 1 (5.2 MPa). The grid line wash-resistant shedding rate reached 28%. This indicates that electrostatic physical anchoring alone is insufficient to maintain long-term stability, and chemical cross-linking is a key element in achieving dual fixation. The color difference ΔE reached 7.2 after 48 hours, indicating insufficient green stability.

[0046] Performance test data The comprehensive performance test data of each embodiment and comparative example are summarized below. Specifically, the phycocyanin retention rate at 48 h was determined by high-performance liquid chromatography (HPLC) (temperature 25℃, humidity 60%, pH 7.0 phosphate buffer soaking); the grid line washout rate was determined by colorimeter after magnetic stirring in deionized water at 100 r / min for 30 min; the membrane pore size was determined by the bubble point method (average of 5 times); the color difference between the grid lines and the membrane was determined by CIELAB color difference value (ΔE); the resolution for detecting light-colored colonies was determined by statistically analyzing the smallest distinguishable colony diameter of Candida albicans ATCC10231 (diameter 0.5–3 mm); porosity was determined by mercury intrusion porosimetry; and the wet tensile strength was determined using a universal testing machine (gauge length 50 mm, loading rate 10 mm / min, deionized water soaking for 24 h).

[0047] Table 1 below is a summary table of the comprehensive performance test data of different embodiments and comparative examples. Table 1 Chitosan electrostatic bridging and anchoring mechanism Bacterial cellulose nanofibers contain a large number of carboxyl groups (-COOH) and hydroxyl groups (-OH) on their surface, which become negatively charged after dissociation in water. Chitosan molecules contain abundant amino groups (…). When chitosan dissolves in a dilute aqueous acetic acid solution, the amino groups are protonated to form... This causes chitosan to carry a positive charge.

[0048] This invention utilizes this characteristic to achieve electrostatic bridging and anchoring under pH conditions of 3.5–5.5. The specific mechanism is as follows: Bacterial cellulose nanofibers (negative charge) + chitosan (positive charge) → electrostatic adsorption complex This electrostatic interaction creates an alternating arrangement of "negatively charged fibers - positively charged chitosan" at the molecular scale, with the chitosan molecular chains acting as "molecular bridges" connecting adjacent bacterial cellulose nanofibers.

[0049] Under these pH conditions (isoelectric point pI≈4.7~5.0), phycocyanin molecules carry a negative charge and can be anchored in the pores of the nanofiber network through secondary electrostatic adsorption of chitosan, forming a three-dimensional spatial positioning: negatively charged phycocyanin + positively charged chitosan → electrostatic adsorption and anchoring.

[0050] pH 3.5~5.5 window control principle This invention precisely controls the pH of the impregnation system within the range of 3.5 to 5.5, based on the following three scientific considerations: Chitosan protonation conditions: The pKa of chitosan amino groups is approximately 6.3. When pH < 6.3, the degree of amino protonation increases with decreasing pH. Within the pH range of 3.5–5.5, the degree of protonation of chitosan is 50%–99%, ensuring sufficient... Positively charged sites bind to bacterial cellulose nanofibers.

[0051] Phycocyanin negative charge conditions: The isoelectric point of spirulina phycocyanin is approximately 4.7–5.0. When pH > pI, phycocyanin carries a negative charge. Within the pH range of 4.8–5.5, phycocyanin can acquire a negative potential of -30 to -10 mV, which is beneficial for anchoring via chitosan bridging.

[0052] Phycocyanin acid stability limit: Phycocyanin will denature and become inactive under strongly acidic conditions (pH < 3.0), manifested as a decrease in absorbance and fluorescence quenching. pH 3.5 serves as the lower limit, ensuring the structural stability of phycocyanin.

[0053] Considering all the above factors, pH 3.5–5.5 is the optimal window for achieving a balance between chitosan protonation, phycocyanin negative charge conversion, and stability.

[0054] Vacuum impregnation driving mechanism Vacuum impregnation is a physical process that uses negative pressure to drive pigment penetration into a porous substrate. Its mechanism can be described by Poiseuille's law: Where Q is the volumetric flow rate, r is the pore radius, ΔP is the pressure difference, η is the fluid viscosity, and L is the permeation path length.

[0055] This invention employs a vacuum condition of -0.06 to -0.09 MPa to create a negative pressure environment inside the substrate. Driven by this pressure difference, the phycocyanin-chitosan pigment is forcibly injected into the nanoscale pores of the bacterial cellulose membrane, significantly increasing the penetration depth. Compared to atmospheric pressure impregnation, vacuum impregnation can increase the pigment penetration depth by 2 to 3 times, effectively improving the thickness of the colored layer and the number of anchoring points.

[0056] Schiff's dual fixation mechanism of base crosslinking and covalent bridging This invention employs chemical cross-linking fixation technology to simultaneously complete two types of chemical reactions during the S4 heating and drying step: Schiff base crosslinking reaction: Glutaraldehyde molecules contain two aldehyde groups (-CHO), which can react with chitosan amino groups ( An addition condensation reaction occurs to form a Schiff base: Genipin, as a natural crosslinking agent, has a similar reaction mechanism: the epoxy groups of genipin undergo a ring-opening addition reaction with the amino groups of chitosan.

[0057] Covalent bridging reaction: Under heating conditions, the hydroxyl groups (-OH) of chitosan can undergo a dehydration condensation reaction with the hydroxyl groups (-OH) of bacterial cellulose nanofibers to form ether bonds for covalent bridging. The synergistic effect of the dual fixation mechanisms: electrostatic physical anchoring in step S3 provides rapid initial fixation, while chemical cross-linking in step S4 provides lasting stability. Together, these mechanisms enable phycocyanin to achieve a retention rate of over 88% in the filter membrane after 48 hours.

[0058] The visual contrast principle of dual contrast positioning system The dual contrast positioning system proposed in this invention comprises two levels: First layer: Background contrast of the green substrate. The bacterial cellulose green substrate is colored with phycocyanin, forming a uniform green background. When light-colored colonies (such as Candida albicans and Staphylococcus aureus) grow on the filter membrane surface, the green background and the light-colored colonies create a hue difference, and the colonies themselves appear as light-colored or white spots, forming the first visual contrast with the green substrate.

[0059] The second layer: Spatial positioning of the printed grid lines. The printed grid lines use a dark colorant (sodium copper chlorophyllin, indigo, or carbon black) to create a color difference of ΔE ≥ 10 with the green substrate. The printed grid lines form a grid-like positioning mark, dividing the detection surface into several independent counting areas. When colonies grow within the areas enclosed by the grid lines, the grid boundaries provide clear area division, enabling precise positioning and counting of the spatial distribution of colonies.

[0060] The synergistic effect of dual contrast: the green substrate solves the "visibility" problem (colony identification), while the printed grid lines solve the "accuracy" problem (spatial distribution location). The combination of the two results in a higher colony detection accuracy than a single green background or a single grid line.

[0061] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A green microbial colony detection filter membrane with printed grid lines, characterized in that, It is composed of the following materials: 100-500 parts of bacterial cellulose green substrate, 10-450 parts of phycocyanin-chitosan green color paste, 10-120 parts of colorant and 20-110 parts of treatment agent; In the phycocyanin-chitosan green color paste, chitosan acts as a positively charged bridging agent, which is fixed in the negatively charged bacterial cellulose nanofiber network by electrostatic adsorption, thereby anchoring phycocyanin in the pores of the nanofiber network. The filter membrane surface is provided with printed grid lines, which together with the green substrate form a dual-contrast positioning system; the green substrate provides visual contrast of the colony body, and the printed grid lines provide spatial distribution positioning of the colony.

2. The green microbial colony detection filter membrane with printed grid lines according to claim 1, characterized in that: The bacterial cellulose green substrate is prepared by purifying a bacterial cellulose membrane. The bacterial cellulose membrane has a thickness of 20-80 μm and a pore size of 0.1-0.8 μm. The bacterial cellulose membrane is synthesized by fermentation of Acetobacter xylinum or Gluconobacterium xylinum.

3. The green microbial colony detection filter membrane with printed grid lines according to claim 1, characterized in that: The mass ratio of phycocyanin to chitosan in the phycocyanin-chitosan green paste is 1:0.5 to 1:3; the phycocyanin is extracted from Spirulina or cyanobacteria with a purity ≥30%; the degree of deacetylation of the chitosan is ≥85%, and the molecular weight is 5 to 50 kDa.

4. The green microbial colony detection filter membrane with printed grid lines according to claim 1, characterized in that: The colorant is a dark water-soluble colorant selected from one or more of food-grade carbon black, dark green chlorophyll copper sodium salt, and dark blue indigo; the colorant is used to print grid lines on the surface of the filter membrane, so that the grid lines form a color difference contrast with the green membrane.

5. The green microbial colony detection filter membrane with printed grid lines according to claim 1, characterized in that: The treatment agent is a combination of a hydrophilic treatment agent and a crosslinking agent; the hydrophilic treatment agent is selected from one or more of glycerol, polyethylene glycol and Tween-80; the crosslinking agent is selected from glutaraldehyde or genipin.

6. A method for processing a green microbial colony detection filter membrane with printed grid lines, applied to the green microbial colony detection filter membrane with printed grid lines as described in any one of claims 1 to 5, characterized in that, The preparation steps are as follows: S1. Purify the bacterial cellulose membrane to obtain a bacterial cellulose green substrate; S2. Mix phycocyanin and chitosan in an aqueous phase to obtain phycocyanin-chitosan green paste; S3. After mixing the treatment agent with the phycocyanin-chitosan green color paste, control the pH of the system to be 3.5-5.5, and impregnate the bacterial cellulose green substrate under vacuum conditions, so that the chitosan in the protonated state acts as a positively charged bridging agent and is fixed in the negatively charged bacterial cellulose nanofiber network by electrostatic adsorption, thereby anchoring the phycocyanin in the pores of the nanofiber network. S4. The impregnated substrate is dried under heating conditions and a cross-linking reaction is initiated, so that the chitosan and bacterial cellulose nanofibers form chemical cross-links, which together with the electrostatic physical anchoring in S3 form a double fixation to obtain a green composite membrane. S5. Printing colorant on the surface of the green composite film to form a printing grid, so that the printing grid and the green substrate together constitute a dual contrast positioning system.

7. The processing method of a green microbial colony detection filter membrane with printed grid lines according to claim 6, characterized in that: The purification process described in S1 includes: placing the bacterial cellulose membrane in a 0.1-1 mol / L sodium hydroxide solution, treating it in a water bath at 70-90°C for 2-4 hours to remove bacterial cells and culture medium residues, and washing it with water until neutral to obtain a green bacterial cellulose substrate. The degree of deacetylation of the chitosan described in S2 is 85%–95%, and the mass ratio of phycocyanin to chitosan is 1:0.5–3.

8. The processing method of a green microbial colony detection filter membrane with printed grid lines according to claim 6, characterized in that: The vacuum conditions described in S3 are as follows: vacuum degree -0.06 to -0.09 MPa, immersion temperature 25 to 45°C, and immersion time 30 to 120 min.

9. The processing method of a green microbial colony detection filter membrane with printed grid lines according to claim 6, characterized in that: The heating conditions described in S4 are 50–80°C and the drying time is 1–4 h. The cross-linking reaction is that the aldehyde group of the cross-linking agent reacts with the free amino group of the chitosan chain to form Schiff base cross-linking, while the other end of the cross-linking agent condenses with the hydroxyl groups on the surface of bacterial cellulose nanofibers to establish a covalent bridge between chitosan and bacterial cellulose.

10. The processing method of a green microbial colony detection filter membrane with printed grid lines according to claim 6, characterized in that: The printing described in S5 adopts any one of screen printing, inkjet printing and transfer printing; the line width of the printed grid is 0.05 to 0.3 mm, the grid spacing is 1 to 5 mm, and the grid color is dark green, black or white.

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