Composite nanofiber membrane, preparation method thereof and low-temperature-resistant wipe applicable to multiple scenes

Abstract

The invention belongs to the field of nanofiber membranes, and relates to a composite nanofiber membrane, a preparation method thereof and a low-temperature-resistant wipe applicable to multiple scenes. The composite nanofiber membrane is a polyvinyl alcohol-quaternized lignin nanofiber membrane and a polycaprolactone-quercetin nanofiber membrane which are blended with each other; the composite nanofiber membrane contains an N-halamine precursor. The invention develops a low-temperature-resistant composite nanofiber membrane, so that rapid inactivation of viruses is realized, the problems that a disinfectant is not suitable for a low-temperature environment and is not suitable for household use are solved, and the problem that a conventional antibacterial factor is long in acting time is also solved.

Description

Technical Field

[0001] The present invention belongs to the field of nanofiber membranes. Specifically, it relates to a composite nanofiber membrane and its preparation method, a chlorinated composite nanofiber membrane and its preparation method, the application of the nanofiber membrane, and a low-temperature resistant wipe suitable for multiple scenarios. Background Art

[0002] In 2018, the World Health Organization proposed "Disease X" and named the "priority pathogens" that may trigger an epidemic or pandemic as "Disease X", which has the opportunity to be triggered at any time from multiple sources and may claim millions of lives. Since 2019, the incidence of new or re-emerging viral epidemics such as Severe Acute Respiratory Syndrome (SARS) and Coronavirus (COVID-19) has been on the rise.

[0003] The food cold chain is an important way for fresh food during transportation, trading, and storage. The low temperature ensures the quality and freshness of the food. The development of food cold chain logistics has accelerated the global spread of "Disease X"; contamination of solid surfaces is the main route for virus and bacteria infection and transmission, seriously threatening the safety of the public. Viruses and foodborne pathogenic bacteria such as Escherichia coli and Staphylococcus aureus can attach to food packaging and maintain a high survival rate during storage and transportation, which also makes low-temperature foods and their outer packaging potentially become carriers of the virus, resulting in cross-contamination. For example, COVID-19 can retain its activity under low-temperature conditions, and goods contaminated with the virus may cause cross-contamination through person-to-person contact during handling, selling, trading, etc. To date, SARS-CoV-2 has continued to evolve, and new strains such as XEC, JN.1.7, and KP3.1.1 have emerged in an endless stream and are all constantly mutating, causing COVID-19 to sweep the globe. Therefore, an effective virus elimination method is needed to prevent the spread of virus particles through food packaging media.

[0004] China pointed out in the requirements for on-site disinfection evaluation during the epidemic that according to the resistance of the novel coronavirus to disinfection factors, Staphylococcus aureus (ATCC 6538) and Escherichia coli (8099) are selected as indicator microorganisms for chemical disinfection. Currently, common physical disinfection methods include autoclaving, ultraviolet sterilization, ionizing radiation sterilization, etc.; these physical disinfection methods are common in food production workshops, etc., and often require certain mechanical equipment to complete the disinfection process, which is not suitable for household use. Common chemical disinfection methods use ozone, peroxides, and alcohol-based disinfectants. The efficacy of traditional disinfectants is easily affected by many conditions, such as concentration, cleaning method, environmental conditions, etc. For example, the frequent use of alcohol-based disinfection has led to the production of highly alcohol-tolerant bacterial strains. When using traditional chemical disinfectants at low temperatures, they are prone to freezing, affecting the disinfectant concentration, prolonging the action time, and unable to effectively inactivate the virus. Quaternary ammonium compounds (QACs), as a new type of disinfectant, have low toxicity, good environmental stability, and low bacterial drug resistance, and have been used on many contact-active surfaces. Its mechanism of action is that the positively charged quaternary ammonium group can attract negatively charged bacteria and irreversibly damage the bacterial cell membrane through electrostatic and hydrophobic interactions, ultimately leading to bacterial death. Relevant studies have shown that quaternary ammonium groups can combine with natural polymers to improve the antibacterial properties of natural polymers. Such as chitosan, lignin, etc. The introduction of quaternary ammonium groups can improve the hydrophilicity of the compound and enhance its antibacterial performance.

[0005] The disinfection method of chemical disinfection is generally through spraying, soaking or wiping disinfection, but it is not suitable for low-temperature stored foods. At the same time, the overuse of disinfectants also poses a serious challenge to the ecosystem. Therefore, a new disinfection method is needed that is suitable for the disinfection of low-temperature foods and can also be used at home, with good safety. Bacteria and viruses are easily attached to the surface of objects such as food packaging, supermarket shopping cart handles, door handles, etc. Infected people leave the viruses or bacteria they carry on the surface, which in turn causes the next person who comes into contact to be infected, causing cross-infection to a certain extent. When avoiding infection, the simplest wiping disinfection can be selected. In dynamic wiping, shear force and compressive force are applied, which will help overcome the adhesion force between bacteria and the object surface and transfer bacteria from the object surface to the wiping cloth surface. Cellulose (including cotton and wood pulp), regenerated cellulose materials (such as viscose fiber), and synthetic polymers (such as polyester and polypropylene) are the most commonly used raw materials for commercial wiping cloth production at present. However, research has shown that choosing inappropriate textile materials may combine with the absorbed active ingredients, resulting in a reduction or complete inhibition of the antibacterial efficacy. Therefore, it is very important to select a suitable textile material in combination with an antibacterial agent. And conventional textile materials cannot be degraded, so not only can disinfection wipes not be used in low-temperature environments, but their large-scale use also brings a burden to the environment.

[0006] In summary, conventional disinfection methods are not applicable to low-temperature scenarios and household use. Bio-based disinfection products have a long action time, weak antiviral ability, and poor performance. There is an urgent need to develop a new type of product that can quickly kill bacteria and viruses and is resistant to low temperatures and suitable for multiple scenarios. Summary of the Invention

[0007] To overcome the above deficiencies, the present invention innovatively introduces haloamine precursor substances and PCL into the preparation of PQL-Cl / PQ composite nanofiber membranes, aiming to improve the performance of the nanofiber membranes in terms of wipeable and rapid antibacterial properties. At the same time, the PQL-Cl / PQ composite nanofiber membranes achieve biodegradation and reduce the burden on the ecosystem.

[0008] To achieve the above object, the present invention provides a composite nanofiber membrane, which is a blended polyvinyl alcohol-quaternized lignin nanofiber membrane and polycaprolactone-quercetin nanofiber membrane; the composite nanofiber membrane contains N-haloamine precursors.

[0009] The second aspect of the present invention provides a method for preparing the above composite nanofiber membrane, comprising the following steps:

[0010] (1) Mix an aqueous solution of quaternized lignin with polyvinyl alcohol and polyethylene oxide, and heat to dissolve thoroughly to obtain a stable polyvinyl alcohol-quaternized lignin electrospinning solution;

[0011] (2) Mix the polycaprolactone solution with quercetin evenly to obtain a stable polycaprolactone-quercetin electrospinning solution;

[0012] (3) Mix the N-haloamine precursor evenly with the polyvinyl alcohol-quaternized lignin electrospinning solution, and then co-electrospin with the polycaprolactone-quercetin electrospinning solution to obtain the composite nanofiber membrane.

[0013] According to a preferred embodiment of the present invention, in step (1), the concentration of quaternized lignin in the polyvinyl alcohol-quaternized lignin electrospinning solution is 0.2-0.8 wt%, and the concentration of polyvinyl alcohol is 6-10 wt%;

[0014] The conditions for thorough heating and dissolution include: continuously stirring at 85-95 °C for 1-3 h first, and then stirring overnight at room temperature.

[0015] According to a preferred embodiment of the present invention, in step (1), the quaternized lignin is prepared by a method comprising the following steps:

[0016] Under alkaline conditions, mix the lignosulfonate solution with (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride solution, and carry out a heating reaction. After the reaction is completed, dialysis purification is carried out to obtain the quaternized lignin.

[0017] Preferably, the alkaline condition is a pH of 11.5 to 12.5.

[0018] Preferably, the heating reaction conditions include: a temperature of 80 to 90 °C and a time of 3 to 6 h.

[0019] Preferably, the dialysis purification is carried out using a dialysis bag with a cut-off molecular weight of 800 to 1200 kDa in a solution with a pH of 11.5 to 12.5.

[0020] According to a preferred embodiment of the present invention, in step (2), the solvent of the polycaprolactone solution is N,N-dimethylformamide and dichloromethane, and the volume ratio of the two is 1:1 to 2;

[0021] The concentration of the polycaprolactone solution is 12 to 18 wt%, and the concentration of quercetin is 2 to 8 wt%.

[0022] According to a preferred embodiment of the present invention, in step (3), the addition amount of the N-halamine precursor is 0.1 to 1 wt% of the polyvinyl alcohol-quaternized lignin electrospinning solution, preferably 0.1 to 0.5 wt%, and more preferably 0.1 to 0.3 wt%.

[0023] The N-halamine precursor compound is one or more of 5,5-dimethylhydantoin, 2,2,6,6-tetramethylpiperidinol, and cyanuric acid.

[0024] The parameters of the electrospinning include: an 18-gauge blunt needle is selected as the spinning needle, the distance between the spinning needle and the roller collector is 15 to 17 cm, the rotation speed of the roller is 400 to 600 r / min, a 10 mL syringe is selected, the flow rate is 0.6 to 1.0 mL / h, the high-voltage power supply is set to 18 to 20 kV, the temperature is 25 ± 2 °C, and the relative air humidity is 50 ± 1%.

[0025] The third aspect of the present invention provides a chlorine-containing composite nanofiber membrane, which is obtained by chlorinating the above composite nanofiber membrane.

[0026] According to a preferred embodiment of the present invention, the chlorination treatment is to bring the composite nanofiber membrane into contact with chlorine gas.

[0027] Preferably, the method of the chlorination treatment includes: placing the composite nanofiber membrane close to the sodium hypochlorite-hydrochloric acid mixed solution but not in direct contact with the mixed solution, and at the same time enabling the gas generated by the sodium hypochlorite-hydrochloric acid mixed solution to contact the composite nanofiber membrane and chlorinate it; further preferably, the pH of the sodium hypochlorite-hydrochloric acid mixed solution is 4.5 to 5.5.

[0028] According to a specific embodiment of the present invention, the chlorination treatment method includes: preparing a sodium hypochlorite-hydrochloric acid mixed solution with a pH of 4.5 to 5.5 to enable uniform gas generation, placing the composite nanofiber membrane in a beaker, and separating it from the mixed solution using a polytetrafluoroethylene partition, and performing chlorination treatment under the condition that the sodium hypochlorite-hydrochloric acid mixed solution generates gas uniformly.

[0029] The fourth aspect of the present invention provides the application of the above composite nanofiber membrane or chlorine-containing composite nanofiber membrane in the preparation of disinfection products.

[0030] The fifth aspect of the present invention provides a low-temperature-resistant wipe applicable to multiple scenarios, which is made of the above composite nanofiber membrane or the above chlorine-containing composite nanofiber membrane, and specifically can adopt the conventional wipe preparation process in the art.

[0031] The multiple scenarios include: cardboard boxes (such as express packaging, whole-box fresh food packaging, etc.), plastic packaging (such as ice cream packaging, meat product packaging, etc.), stainless steel equipment, etc.

[0032] The present invention has developed a low-temperature-resistant composite nanofiber membrane to achieve rapid inactivation of viruses, which not only overcomes the problems that disinfectants are not applicable in low-temperature environments and not suitable for household use, but also solves the problem of long action time of conventional antibacterial factors.

[0033] The technical effects of the present invention include:

[0034] (1) The present invention selects quaternized lignin and PVA to prepare an electrospinning solution, introduces quaternary ammonium groups into it to improve antibacterial performance; selects PCL and quercetin, and fully utilizes the hydrophobic property of PCL and the antibacterial property of quercetin. The halogenamine precursor substance DMH is introduced into the PVA-QL spinning solution, and a composite nanofiber membrane is prepared by co-spinning, and the composite nanofiber membrane is chlorinated to achieve rapid killing of bacteria and viruses.

[0035] (2) The present invention selects PVA and PCL, and fully utilizes the inherent degradation characteristics of these polymers, and the addition of PVA can improve the problem of slow degradation rate of PCL. As a wipe, the composite nanofiber membrane can effectively kill bacteria and viruses on the surface of low-temperature-stored food packaging. Compared with commercially available wipeable antibacterial wet wipes, the nanofiber membrane is lighter in weight, only 0.5 g, reducing the transportation cost in actual application, and can be naturally degraded in the soil, reducing the burden on the ecosystem.

[0036] (3) The present invention improves the utilization of lignin with high added value and provides a good prospect for the future development of biodegradable wipes.

[0037] (4) The composite nanofiber membrane of the present invention can rapidly inactivate bacteria within 2 minutes. It can be applied to cartons (such as express packaging, whole-box fresh food packaging, etc.), plastic packaging (such as ice cream packaging, meat product packaging, etc.), and stainless steel equipment (such as trays, dining tables, door handles, etc.), and has good versatility in multiple scenarios. It has been developed into a wipeable disinfectant towel product. The evaluation cost is comparable to that of commercially available disinfectant wipes. In particular, the wipeable towel of the present invention has good degradation performance, is green and environmentally friendly, and can be promoted for household use.

[0038] Other features and advantages of the present invention will be described in detail in the following specific implementation section. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] By describing the exemplary embodiments of the present invention in more detail in conjunction with the drawings, the above and other objects, features, and advantages of the present invention will become more apparent.

[0040] Figure 1 Shows the characterization results of quaternized lignin: (a) Fourier transform infrared spectroscopy diagram of quaternized lignin; (b) Zeta potential diagram of quaternized lignin.

[0041] Figure 2 Shows the performance evaluation results of polyvinyl alcohol - quaternized lignin nanofiber membrane: (a) SEM image, (b) diameter distribution, (c) tensile strength, (d) elongation at break, (e) water contact angle.

[0042] Figure 3 Shows the performance evaluation results of PQL / PQ composite nanofiber membrane: (a) SEM image, (b) tensile strength, (c) elongation at break, (d) water contact angle.

[0043] Figure 4 Shows the performance evaluation results of PQL-Cl / PQ composite nanofiber membrane: (a), (b) SEM images, (c) elemental mapping diagram, (d) tensile strength, (e) elongation at break, (f) water contact angle, (g) Cl + content, (h) in vitro cytotoxicity test, (i) biodegradation experiment.

[0044] Figure 5 Shows the antibacterial performance evaluation results of PQL-Cl / PQ composite nanofiber membrane.

[0045] Figure 6 Shows the effect of PQL-Cl / PQ composite nanofiber membrane on bacterial cell membrane damage.

[0046] Figure 7 Shows the actual antibacterial application of PQL-Cl / PQ composite nanofiber membrane. DETAILED DESCRIPTION OF THE INVENTION

[0047] Preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein.

[0048] For those conditions not specified in the examples, they are all carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0049] Materials and Methods

[0050] Sodium lignosulfonate (BR, 96%), starch indicator (1%), polyethylene oxide were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (China). Polycaprolactone (average Mn 80000), quercetin (97%), 5,5-dimethylhydantoin (98%), sodium thiosulfate standard solution (0.1000 mol / L) were purchased from Shanghai Macklin Biochemical Co., Ltd. (China). (3-Chloro-2-hydroxypropyl) trimethyl ammonium chloride aqueous solution (65 wt%), iodine standard solution (0.05000 mol / L, 0.1 N), polyvinyl alcohol were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (China). Phosphate buffer solution (1×PBS) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (China). Sodium hypochlorite solution was purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd. Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 25922) were purchased from Beijing Na Biotechnology Co., Ltd. (China). Tryptic Soy Agar, Tryptic Soy Broth were purchased from BD Bacto (USA). Live&Dead Bacterial Staining Kit was purchased from Yeasen Biotech Co., Ltd. (Shanghai, China).

[0051] Electrospinning parameters for preparing the thin film in the examples: The spinning needle used was an 18-gauge blunt needle, the distance between the spinning needle and the drum collector was 15 - 17 cm, the rotation speed of the drum was 500 r / min, the syringe used was 10 mL, the flow rate was 0.8 mL / h, the high-voltage power supply was set to 18 - 20 KV, the temperature was 25 ± 2 °C, and the relative air humidity was 50 ± 1%.

[0052] Example 1 Preparation of Quaternized Lignin

[0053] Weigh the lignosulfonate and prepare a 20 wt% solution. Add sodium hydroxide to adjust the pH to 12 to create an alkaline environment. Heat it in a water bath to 80 °C, add a 65 wt% solution of (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride (CHPTAC), and then add a 20 wt% sodium hydroxide solution after 5 minutes to ensure the reaction proceeds under alkaline conditions. React at 85 °C for 4 hours. After the reaction is completed, dialyze using a dialysis bag with a molecular weight cut-off of 1000 kDa. Dialyze in a solution with a pH of 12 to remove unreacted CHPTAC and small molecule impurities, and then dialyze in deionized water. After dialysis is completed, collect by freeze-drying. Use Fourier transform infrared spectroscopy and Zeta potential to characterize the quaternized lignin.

[0054] Figure 1 Figure (a) shows the FT-IR spectra of quaternized lignin (QL) and sodium lignosulfonate (Li). A new peak characteristic of the quaternary ammonium group appears at approximately 1467 cm -1 for QL, indicating that the quaternary ammonium group has been successfully grafted onto the lignin. Figure 1 Figure (b) shows the Zeta potential diagrams of quaternized lignin and sodium lignosulfonate. In a deionized aqueous solution with a neutral pH, the potential of QL is much higher than that of lignin without grafted quaternary ammonium groups, indicating that the positively charged quaternary ammonium groups have been grafted onto the lignin surface, neutralizing some of the negative charges of sodium lignosulfonate. The above results show that the positively charged quaternary ammonium groups have been grafted onto the lignin.

[0055] Example 2 Preparation of Polyvinyl Alcohol - Quaternized Lignin Nanofiber Membrane

[0056] Dissolve quaternized lignin with different addition ratios (0 wt%, 0.2 wt%, 0.4 wt%, 0.6 wt%) in 10 mL of deionized water to make it fully dissolve. Add 8% polyvinyl alcohol and polyethylene oxide, and continuously stir at 90 °C for 2 hours to make it fully dissolve and mix evenly. Stir overnight at room temperature to form a stable PVA-QL electrospinning solution.

[0057] Electrospin each PVA-QL electrospinning solution to prepare polyvinyl alcohol - quaternized lignin nanofiber membranes, denoted as PVA, PQL-2, PQL-4, and PQL-6 in sequence.

[0058] Example 3 Preparation of Polycaprolactone - Quercetin Nanofiber Membrane

[0059] Weigh polycaprolactone and dissolve it in a solution of N,N-dimethylformamide and dichloromethane (the volume ratio of N,N-dimethylformamide to dichloromethane is 2:3), stir for 4 h to obtain a polycaprolactone solution with a final concentration of 15 wt%. Add quercetin to it, with the concentration of quercetin being 4 wt%, and stir overnight at room temperature to form a stable PCL-Que electrospinning solution.

[0060] Electrospin each PCL-Que electrospinning solution to prepare a polycaprolactone-quercetin nanofiber membrane, denoted as the PQ nanofiber membrane.

[0061] Example 4 Preparation of Blended Polyvinyl Alcohol - Quaternized Lignin / Polycaprolactone - Quercetin Composite Nanofiber Membrane

[0062] (1) Composite nanofiber membrane PQL / PQ

[0063] Prepare a polyvinyl alcohol-quaternized lignin electrospinning solution (according to the conditions of PQL-6) and a polycaprolactone-quercetin electrospinning solution (according to the conditions of PQ) respectively. After stirring evenly at room temperature, perform electrospinning to obtain a composite nanofiber membrane. After the electrospinning is completed, the composite nanofiber membrane is dried in a vacuum drying oven at 25 °C, denoted as PQL / PQ.

[0064] (2) Composite nanofiber membrane PQL-DMH / PQ

[0065] Prepare a polyvinyl alcohol-quaternized lignin electrospinning solution (according to the conditions of PQL-6) and a polycaprolactone-quercetin electrospinning solution (according to the conditions of PQ) respectively. Add 0.2 wt% of 5,5-dimethylhydantoin to the polyvinyl alcohol-quaternized lignin electrospinning solution. After stirring evenly at room temperature, perform electrospinning to obtain a composite nanofiber membrane. After the electrospinning is completed, the composite nanofiber membrane is dried in a vacuum drying oven at 25 °C, denoted as the PQL-DMH / PQ nanofiber membrane.

[0066] Example 5 Chlorination of Composite Nanofiber Membrane

[0067] Prepare a sodium hypochlorite-hydrochloric acid mixed solution with a pH of 5 to enable uniform gas generation. Place the dried composite nanofiber membrane (PQL-DMH / PQ) prepared in Example 4 in a beaker, separate it from the solution using a polytetrafluoroethylene partition, and chlorinate it for 2 h under the condition of uniform gas generation in the sodium hypochlorite-hydrochloric acid mixed solution to obtain a chlorinated composite nanofiber membrane, denoted as PQL-Cl / PQ.

[0068] Characterization of Material Properties

[0069] 1. Structural characterization

[0070] The field emission scanning electron microscope (SEM) was used to test polyvinyl alcohol-quaternized lignin nanofiber membranes and blended PQL / PQ, PQL-DMH / PQ, and PQL-Cl / PQ composite nanofiber membranes in different proportions to examine the microstructure of the film surface.

[0071] 2. Mechanical Property Characterization

[0072] PQL nanofiber membranes with different contents and blended PQL / PQ, PQL-DMH / PQ, and PQL-Cl / PQ composite nanofiber membranes were all cut into rectangles of 1×8 cm to measure the thickness of the nanofiber membranes. The tensile properties of the nanofiber membranes were measured using a tensile testing machine. The measurement was carried out at a speed of 50 mm / min and an initial distance between the clamps of 50 mm. Each group of samples was measured three times and the average value was taken.

[0073] 3. Water Contact Angle Characterization

[0074] The water contact angles of PQL nanofiber membranes with different contents and blended PQL / PQ, PQL-DMH / PQ, and PQL-Cl / PQ composite nanofiber membranes were measured using WCA analysis to evaluate the surface wettability of the materials.

[0075] 4. Determination of Available Chlorine Content and Chlorine Stability during Storage

[0076] The available chlorine content of the samples was determined using the iodometric method. The samples were weighed and cut into pieces, then placed in a 0.001N sodium thiosulfate solution and stirred for 20 min. 1 mL of starch indicator was added to it. To simulate the dechlorination process of the composite nanofiber membranes, a 0.001N iodine standard solution was used for titration. The volume of the iodine standard solution consumed was recorded for the calculation of the available chlorine content. The formula is as follows:

[0077]

[0078] In the formula:

[0079] V 0 : The volume of the iodine standard solution consumed without adding the composite nanofiber membrane, mL.

[0080] V i : The volume of the iodine standard solution consumed after adding the composite nanofiber membrane, mL.

[0081] m: The mass of the added composite nanofiber membrane, g.

[0082] To simulate the storage process of the samples, the samples were placed in a sealed, dry, and dark environment at room temperature and their available chlorine contents were measured at different times (1w, 2w, 3w, 4w).

[0083] 5. Antibacterial Performance

[0084] (1) Determination of Antibacterial Rate by Agar Plate Counting Method

[0085] The antibacterial performance of the composite nanofiber membrane was evaluated by the agar plate counting method. Gram-positive bacteria represented by Staphylococcus aureus (ATCC 6538) and Gram-negative bacteria represented by Escherichia coli (ATCC 25922) were selected to evaluate the antibacterial performance of the composite nanofiber membrane. Escherichia coli and Staphylococcus aureus were separately inoculated into 10 mL of liquid medium and cultured at 200 r / min and 37 °C until 1×10 8 CFU / mL, and then the bacterial solution was diluted to 1×10 5 CFU / mL for standby. 100 μL of the bacterial solution was dropped onto different types of membranes (2 cm × 3 cm). After culturing at 37 °C for 2 min, 5 min, 10 min, and 20 min, 900 μL of phosphate buffer solution was added thereto. After mixing evenly, 100 μL of the bacterial solution was taken and inoculated onto a solid agar plate, spread evenly, and cultured overnight in a constant temperature and humidity incubator. The antibacterial performance of the material was evaluated by the plate counting method. The calculation formula for the antibacterial rate is as follows:

[0086]

[0087] (2) Determination of Bacterial Dead / Live Viability

[0088] A bacterial suspension of 1×10 8 CFU / mL was incubated with the composite nanofiber membrane overnight at 37 °C, and untreated bacteria were used as a control. After incubation, it was centrifuged at 10000 rpm for 1 min, the precipitate was taken, washed with physiological saline, and finally resuspended in 100 μL of physiological saline. It was stained with a live / dead bacteria staining kit at room temperature for 15 min. The two fluorescent dyes were DMAO and EthD-Ⅲ, with a ratio of 1:2. 1 μL of the mixed and uniform dye was added to the bacterial solution. After staining, 5 μL of the bacterial suspension was dropped onto a glass slide, and the viability of the bacteria was observed under a confocal microscope at excitation / emission wavelengths of 496 / 528 nm (DMAO, green) and 532 / 625 nm (EthD-Ⅲ, red).

[0089] (3) SEM Characterization of Bacterial Morphology

[0090] An overnight cultured bacterial suspension of 1×10 9CFU / mL. Take 1000 μL of the Escherichia coli suspension and the Staphylococcus aureus suspension and incubate them with the PQL-Cl / PQ composite nanofiber membrane at 37 °C for 2 h. The one without adding the material serves as the control group. The treated bacterial suspension is centrifuged at 5000 rpm for 5 min, washed three times with 0.01 M PBS. Add 1 mL of 2.5% glutaraldehyde solution and fix it at 4 °C for 4 h. Centrifuge at 5000 rpm for 5 min and discard the supernatant. Wash it three times with 0.01 M PBS. Then add 1 mL of 30% ethanol, 50% ethanol, 70% ethanol, 90% ethanol, and 100% ethanol in sequence for dehydration, 10 min each time. After that, drop it on a silicon wafer and dry it overnight in a freeze dryer. After completion, mount the sample on the short board of a scanning electron microscope and spray a conductive layer, and then observe it under the scanning electron microscope.

[0091] 6. Applications of the composite nanofiber membrane

[0092] (1) Applications of the composite nanofiber membrane on the surface of cold-chain food packaging

[0093] Select Gram-positive bacteria represented by Staphylococcus aureus and Gram-negative bacteria represented by Escherichia coli to evaluate the applications of the composite nanofiber membrane on the surface of cold-chain food packaging. For cold-chain food packaging, choose two different types of ice cream packaging (box packaging and bag packaging). Dilute the bacterial liquid to 1×10 4 CFU / mL for standby. Take 200 μL of the bacterial liquid and inoculate it on the packaging surface. After inoculation, place the packaging at different storage temperatures (4 °C, -20 °C, -80 °C) to simulate the temperatures during the transportation and storage of cold-chain food. The next day, take out the packaging and wipe it with the composite nanofiber membrane. The control group is not wiped. Cut out a 1×1 cm square on the packaging surface and put it into 1 mL of liquid LB medium, culture it at 200 r / min and 37 °C for 2 h. Take 100 μL of the bacterial liquid, drop it on a solid agar plate, spread it evenly, and culture it overnight in a constant temperature and humidity incubator. Evaluate the wipe sterilization effect of the composite nanofiber membrane on cold-chain food packaging through the number of colonies on the agar plate.

[0094] (2) Applications of the composite nanofiber membrane on the surface of stainless steel materials

[0095] Take 100 μL of the above-mentioned bacterial liquid and inoculate it on the surface of stainless steel materials. After inoculation, simulate the wiping of the surface of stainless steel materials. The control group is not wiped. Use a sterile cotton swab to take samples on the surface of stainless steel materials. After sampling, culture it in a liquid medium for 2 h and then spread it on an agar plate. Evaluate the wipe sterilization effect of the composite nanofiber membrane on cold-chain food packaging through the number of colonies on the agar plate.

[0096] 7. Safety Evaluation of Composite Nanofiber Membrane

[0097] The CCK-8 kit was used to evaluate the in vitro cytotoxicity of the composite nanofiber membrane. The 6-cm 2 composite nanofiber membrane was immersed in DMEM medium for 24 h, and the sample extract was obtained by filtration. Mouse fibroblast L929 cells were selected and inoculated into 96-well plates at a concentration of 5×10 3 / mL. The temperature of the cell incubator was 37 °C, and the CO 2 content was 5%. After culturing for 24 h, 100 μL of the sample extract was added to the 96-well plates. After incubation for 24 h, 10 μL of CCK-8 solution was added to each well and incubated at 37 °C for 1 h. A commercially available polyethylene plastic film (PE film) was used as the control group. The OD value was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0098] Cell survival rate = (As - Ab) × 100 / (Ac - Ab)

[0099] As: Absorbance of the experimental group (containing cells, medium, CCK-8 solution, and drug solution);

[0100] Ac: Absorbance of the negative control group (containing cells, medium, CCK-8 solution, without drug);

[0101] Ab: Absorbance of the blank group (containing medium, CCK-8 solution, without cells, drug).

[0102] 8. Degradability of Composite Nanofiber Membrane

[0103] The soil burial natural degradation method was used for the biodegradation experiment of the composite nanofiber membrane. The soil selected was natural soil in the outdoor environment. The size of the composite nanofiber membrane was selected as 3 cm × 3 cm, and it was placed in the same soil environment. On the 1st, 3rd, 5th, 7th, 14th, and 28th days, the degradation of the nanofiber membrane was observed and recorded by photography.

[0104] Performance Evaluation and Application of Nanofiber Membrane

[0105] 1. Performance Evaluation of Polyvinyl Alcohol - Quaternized Lignin Nanofiber Membrane

[0106] As Figure 2 shown in (a) of , the surface morphology and diameter distribution of PVA nanofiber membrane, PQL-2 nanofiber membrane, PQL-4 nanofiber membrane, and PQL-6 nanofiber membrane were observed using a field emission scanning electron microscope (SEM). With the increase in the addition amount of quaternized lignin, their apparent morphology did not change much.

[0107] As Figure 2As shown in (b), with the increase in the addition amount of quaternized lignin, the fiber diameter of the nanofibers increased slightly. Specifically, the fiber diameter of the nanofibers added with 8% PVA solution was mainly distributed around 205 nm. With the addition of quaternized lignin, the fiber diameter showed an increasing trend (from around 205 nm to around 307 nm). This might be because after the addition of quaternized lignin, hydrogen bonds were formed between the hydroxyl groups of quaternized lignin and the hydroxyl groups of polyvinyl alcohol. Due to the interaction of hydrogen bonds, the viscosity of the spinning solution increased, and at the same voltage, the diameter increased slightly.

[0108] The tensile properties of the nanofiber membrane are very important for dynamic wiping. A tensile testing machine was used to evaluate the mechanical properties of the nanofiber membrane. The elongation at break is the ratio of the elongation of the sample until fracture to its original length. The tensile strength is the stress at which the material produces the maximum uniform plastic deformation. As Figure 2 As shown in (c) and (d), after adding quaternized lignin, the tensile strength increased to twice that of the PVA nanofiber membrane, greatly optimizing the mechanical properties of the nanofiber membrane, but the elongation at break gradually decreased. The reason is that after adding quaternized lignin, hydrogen bonds were formed between the hydroxyl groups of quaternized lignin and the hydroxyl groups of polyvinyl alcohol. The interaction between hydrogen bonds increased the binding force between molecules, resulting in an increase in the tensile strength of the nanofibers and a decrease in the elongation at break.

[0109] Figure 2 (e) shows the water contact angle of the quaternized lignin nanofiber membrane. It can be seen that after adding quaternized lignin, the water contact angle decreased from 48.57° to 34.21°, and with the addition of quaternized lignin, the water contact angle gradually decreased. This is because quaternized lignin is a water-soluble substance, so the addition of quaternized lignin led to better solubility of the nanofiber membrane in water. Considering that the increase in the addition amount of quaternized lignin did not have a great impact on the mechanical properties, apparent morphology, and water contact angle of the PVA-QL nanofiber membrane, the present invention selected PQL-6 (denoted as PQL) for subsequent research.

[0110] 2. Performance Evaluation of PQL / PQ Composite Nanofiber Membrane

[0111] The surface morphologies of polycaprolactone - quercetin (PQ) nanofiber membrane, PQL nanofiber membrane, and PQL / PQ composite nanofiber membrane were observed using a field emission scanning electron microscope (SEM). As Figure 3As shown in (a), the fiber structure of the PQ nanofiber membrane shows adhesion, which may be due to too high solution concentration and incomplete evaporation of the organic reagent. From the scanning electron microscope images of the PQL / PQ composite nanofiber membrane, it can be seen that distinct fiber morphology and adhered fiber morphology appear simultaneously, indicating that the composite nanofiber membrane obtained by blending has two morphologies, namely the PQL nanofiber membrane and the PQ nanofiber membrane, meaning the successful synthesis of the composite nanofiber membrane.

[0112] The mechanical properties of the nanofiber membrane were evaluated using a tensile testing machine. As Figure 3 shown in (b) and (c), the tensile strength of the PQL / PQ composite nanofiber membrane is 8.73 MPa, which is much higher than that of the single nanofiber membrane; the elongation at break of the PQL / PQ composite nanofiber membrane is slightly lower compared to the single nanofiber membrane.

[0113] The surface wettability of the composite nanofiber membrane and the single nanofiber membrane was evaluated using a WCA analyzer. As Figure 3 shown in (d), the PQL nanofiber membrane has good water solubility, so it cannot be used for wiping disinfection and sterilization. While the PQ nanofiber membrane has excellent hydrophobic properties, with a water contact angle as high as 102°. The water contact angle of the PQL / PQ composite nanofiber membrane is 75.09°, which is much higher than that of the single PQL nanofiber membrane, proving that the PQ nanofiber membrane increases the water contact angle of the composite nanofiber membrane and provides a hydrophobic substrate for the subsequent application of the composite nanofiber membrane.

[0114] 3. Performance Evaluation of PQL - Cl / PQ Composite Nanofiber Membrane

[0115] The apparent morphology of the nanofiber membrane before and after chlorination was observed using a field emission scanning electron microscope. Figure 4 (a) is the composite nanofiber membrane PQL-DMH / PQ obtained by adding the haloamine precursor 5,5-dimethylhydantoin (DMH) during the blending process, and there is not much change in its morphology compared to the original PQL / PQ composite nanofiber membrane. Figure 4 (b) is the composite nanofiber membrane PQL-Cl / PQ obtained by chlorinating the PQL-DMH / PQ composite nanofiber membrane. Obviously, the morphology of the PQL-Cl / PQ composite nanofiber membrane has changed, and the fibers have changed from the original circular cross-section to flat oval or ribbon-shaped, which may be due to the pressure applied to the nanofibers during the operation.

[0116] Figure 4 The element mapping diagram in (c) reflects the uniform distribution of N and Cl elements in the PQL-Cl / PQ composite nanofiber membrane, and the large amount of Cl element indicates the smooth progress of the chlorination reaction.

[0117] The mechanical properties of the PQL-Cl / PQ composite nanofiber membrane have decreased, as shown in Figure 4 (d) and (e) of

[0118] A water contact angle measuring instrument was used to evaluate the surface wettability of the nanofiber membrane after the introduction of DMH and the chlorination reaction. As shown in Figure 4 (f) of, the water contact angle of the PQL-DMH / PQ composite nanofiber membrane with the addition of the haloamine precursor decreased from 75.09° to 62.44°. This may be because DMH contains N-H bonds that can form hydrogen bonds with the hydroxyl groups of water molecules, increasing hydrophilicity and thus decreasing the water contact angle. After the chlorination reaction, the N-H bonds are transformed into N-Cl bonds, and the hydrogen bonds disappear, and the water contact angle of the PQL-Cl / PQ composite nanofiber membrane is restored.

[0119] Considering that the disposable wipes will not be put into use immediately after preparation, the present invention simulates the storage process of the disposable wipes and places them in a sealed environment at room temperature to test the Cl + content of the PQL-Cl / PQ composite nanofiber membrane at different storage times, so as to evaluate the storage stability of the PQL-Cl / PQ composite nanofiber membrane. The iodometric method was used to evaluate the available chlorine content of the composite nanofiber membrane after chlorination. As shown in Figure 4 (g) of, as the storage time increases, from 1 d to 7 d, the Cl + content decreases rapidly, but stabilizes after 7 d, and the Cl + content remains between 200 p.p.m - 300 p.p.m. This means that the PQL-Cl / PQ composite nanofiber membrane has good storage stability.

[0120] The present invention uses the CCK-8 method to evaluate the in vitro cytotoxicity of the nanofiber membrane. The in vitro cytotoxicity of the PQL nanofiber membrane, PQ nanofiber membrane, PQL / PQ composite nanofiber membrane, PQL-DMH / PQ composite nanofiber membrane, and PQL-Cl / PQ composite nanofiber membrane was studied using mouse fibroblast L929 cells. The results of analyzing cell viability by the CCK-8 method are shown in Figure 4 (h) of. Whether it is a single nanofiber membrane or a composite nanofiber membrane, the cell survival rate is above 70%, and there is no significant difference from the negative control PE membrane. It can be seen that the PQL-Cl / PQ composite nanofiber membrane has good cell biocompatibility and no cytotoxicity. Therefore, it can be applied to the surface of low-temperature food packaging and processing and transportation equipment.

[0121] To evaluate the burden on the ecosystem brought by the nanofiber membrane, the present invention simulated the biodegradation process of the nanofiber membrane, as shown in Figure 4As shown in (i) of [Figure 0]. The PE film was selected as the control group. It can be seen that except for the PE film, the nanofiber membranes of other groups showed obvious shape changes at 14 days, and the surfaces of the PQL-DMH / PQ and PQL-Cl / PQ composite nanofiber membranes shrank. The PQL nanofiber membrane had been completely degraded on the 3rd day, probably because PVA is water-soluble and the nanofiber membrane absorbed water in the soil to complete the degradation. Due to the presence of PQL nanofiber membranes in the PQL-DMH / PQ and PQL-Cl / PQ composite nanofiber membranes, on the 3rd day, both showed characteristics such as surface shrinkage and area reduction. All the membranes showed obvious biodegradation after 2 weeks. The degradation characteristics of the PQL-DMH / PQ and PQL-Cl / PQ composite nanofiber membranes were more obvious, indicating that the blended composite nanofiber membranes optimized the problem of the slow degradation rate of the PQ single nanofiber membrane, showing that both single nanofiber membranes and composite nanofiber membranes had good biodegradability under natural soil conditions.

[0122] 4. Evaluation of Antibacterial Performance of Composite Nanofiber Membrane

[0123] Based on the resistance of the novel coronavirus to disinfection factors, indicator microorganisms, Staphylococcus aureus (S. aureus ATCC 6538) and Escherichia coli (E. coli ATCC25922), were selected as indicator microorganisms for chemical disinfection. Therefore, in the present invention, S. aureus ATCC 6538 and E. coli ATCC25922 were selected as model bacteria to evaluate the antibacterial ability of the PQL-Cl / PQ composite nanofiber membrane. The PQL-DMH / PQ composite nanofiber membrane, commercially available germicidal wipes, and the PQL-Cl / PQ composite nanofiber membrane were used for antibacterial experiments. As Figure 5 As shown in (a) and (c) of [Figure 8], after Staphylococcus aureus and Escherichia coli contacted different membranes for 2 minutes, the colony-forming units (CFUs) on the surfaces of the commercially available germicidal wipes and the PQL-Cl / PQ composite nanofiber membrane were significantly less than those on the PQL-DMH / PQ composite nanofiber membrane. According to the agar plate counting method, the contact killing antibacterial rate of the PQL-Cl / PQ composite nanofiber membrane against Escherichia coli and Staphylococcus aureus within 2 minutes was 99.9% ( Figure 5 as shown in (b) and (d) of [Figure 10]).

[0124] This invention studied the effect of PQL-Cl / PQ composite nanofiber membrane on the damage of bacterial cell membranes, and selected two dyes, DMAO and EthD-Ⅲ. DMAO is a green nucleic acid fluorescent dye that can stain both live and dead bacteria. EthD-III is a red nucleic acid fluorescent dye that only stains dead bacteria with damaged cell membranes. When DMAO and EthD-III are used in combination for staining, bacteria with intact cell membranes appear green, while bacteria with damaged cell membranes appear green and red. The PQL-Cl / PQ composite nanofiber membrane was incubated with bacteria overnight at 37 °C, and the survival state of the bacteria could be visually observed by the colors red and green. As Figure 6 shown in (a) and (b) of

[0125] 5. Practical Application of Composite Nanofiber Membrane

[0126] This invention simulated the household use scenario of the PQL-Cl / PQ composite nanofiber membrane. Common packages for storing food at low temperatures, such as bag packaging and carton packaging, were selected, and 4 °C, -20 °C, and -80 °C were used to simulate the storage temperature conditions for low-temperature stored food. As Figure 7 shown in (a) and (b) of 4 CFU of pathogenic bacteria were inoculated on the outer surface of the food package, and it was stored overnight under different temperature conditions to simulate the real scenario of bacteria infecting the food package. In the experimental group, a 6×6 cm PQL-Cl / PQ composite nanofiber membrane was used to wipe for 2 minutes, and the control group was not treated at all. Sterile cotton swabs were used to sample and culture on the package surface. As Figure 7 shown in (e) and (f) of

[0127] During the storage and transportation of low-temperature foods, stainless steel materials are often used. Such as the stainless steel plates and stainless steel drawers used in cold storage construction; and in the daily catering production rooms and food factory production lines, stainless steel materials are also often used, such as stainless steel tabletops and stainless steel casings of operating equipment. This invention selected stainless steel trays to replace stainless steel materials ( Figure 7 shown in (c) of 4CFU bacteria. In the experimental group, a stainless - steel tray was wiped with a PQL - Cl / PQ composite nanofiber membrane of 6×6 cm for 2 minutes, while the control group was not treated at all. After treatment, a sterile cotton swab was used to wipe and sample the tray. After culturing with a liquid medium at 37°C for 2 hours, the bacterial liquid was spread on an agar plate. After being wiped with the PQL - Cl / PQ composite nanofiber membrane for 2 minutes, the colony - forming units on the agar plate were far less than those of the non - wiped control group, and the antibacterial rate was as high as 99.9% ( Figure 7 as shown in (d)). The above experiments prove that the PQL - Cl / PQ composite nanofiber membrane can be used to wipe equipment, desktops, etc. made of stainless steel to prevent bacteria from adhering to the surface of stainless - steel materials, thus affecting the quality of food during the food - processing process.

[0128] The present invention prepares a multi - scenario - applicable low - temperature - resistant wipe for rapid inactivation of novel viruses, which has low - temperature adaptability and the functions of rapidly killing bacteria and viruses. The present invention selects two spinning solutions of PVA - QL and PCL - Que for blended spinning to prepare a nanofiber membrane. The addition of quaternary ammonium groups can attract negatively charged bacterial particles and virus particles, so that the PQL - Cl / PQ composite nanofiber membrane can quickly play a role when contacting bacterial and virus particles. The introduction of PVA optimizes the problem of the slow degradation rate of the single PCL composite nanofiber membrane. The addition of PCL and quercetin endows the composite nanofiber membrane with hydrophobic properties, making the nanofiber membrane have wipe - able properties. The PQL - Cl / PQ composite nanofiber membrane can rapidly kill Escherichia coli and Staphylococcus aureus within 2 minutes. It has good versatility for cardboard boxes, plastic packages, and stainless - steel equipment, and has been developed into a disinfection wipe product for coronaviruses and food - borne pathogenic bacteria. In particular, the PQL - Cl / PQ composite nanofiber membrane is extremely light in weight, greatly reducing the transportation cost. More prominently, the wipe has excellent degradation performance and is environmentally friendly. In summary, the PQL - Cl / PQ composite nanofiber membrane can be used for wiping, disinfecting, and antibacterial of low - temperature food packaging, which is of great significance for effectively blocking the spread of viruses through cold - chain logistics and is suitable for household use.

[0129] The embodiments of the present invention have been described above. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A composite nanofiber membrane, characterized in that: The composite nanofiber membrane is a blended polyvinyl alcohol-quaternized lignin nanofiber membrane and a polycaprolactone-quercetin nanofiber membrane; the composite nanofiber membrane contains an N-halamine precursor.

2. The method for preparing the composite nanofiber membrane according to claim 1, characterized in that: The following steps are involved: (1) mixing an aqueous solution of quaternized lignin with polyvinyl alcohol and polyethylene oxide, and heating to fully dissolve the mixture to obtain a stable polyvinyl alcohol-quaternized lignin electrospinning solution; (2) uniformly mixing the polycaprolactone solution and quercetin to obtain a stable polycaprolactone-quercetin electrospinning solution; (3) The N-halamine precursor is uniformly mixed with the polyvinyl alcohol-quaternized lignin electrospinning solution, and then electrospun together with the polycaprolactone-quercetin electrospinning solution to obtain the composite nanofiber membrane.

3. The method for preparing the composite nanofiber membrane according to claim 2, characterized in that: In step (1), the concentration of quaternary ammonium lignin in the polyvinyl alcohol-quaternary ammonium lignin electrospinning solution is 0.2-0.8 wt %, and the concentration of polyvinyl alcohol is 6-10 wt %; The conditions for heating to fully dissolve include: first stirring continuously at 85-95° C. for 1-3 hours, and then stirring at room temperature overnight.

4. The method for preparing the composite nanofiber membrane according to claim 2, characterized in that: In step (1), the quaternized lignin is prepared by a method comprising the following steps: Under alkaline conditions, a lignin sulfonate solution is mixed with a (3-chloro-2-hydroxypropyl)trimethylammonium chloride solution, and heated to react. After the reaction is completed, the quaternized lignin is purified by dialysis to obtain the quaternized lignin; Preferably, the alkaline condition is a pH of 11.5 to 12.5; Preferably, the heating reaction conditions include: temperature of 80-90°C and time of 3-6h; Preferably, the dialysis purification uses a dialysis bag with a molecular weight cutoff of 800 to 1200 KDa and is carried out in a solution with a pH of 11.5 to 12.

5.

5. The method for preparing the composite nanofiber membrane according to claim 2, characterized in that: In step (2), the solvent of the polycaprolactone solution is N,N-dimethylformamide and dichloromethane, and the volume ratio of the two is 1:1-2; The concentration of the polycaprolactone solution is 12-18 wt %, and the concentration of the quercetin is 2-8 wt %.

6. The method for preparing the composite nanofiber membrane according to claim 2, characterized in that: In step (3), the amount of the N-halamine precursor added is 0.1 to 1 wt % of the polyvinyl alcohol-quaternized lignin electrospinning solution; The N-halamine precursor compound is one or more of 5,5-dimethylhydantoin, 2,2,6,6-tetramethylpiperidinol and cyanuric acid; The electrospinning parameters include: a No. 18 blunt needle is selected as the spinning needle, the distance between the spinning needle and the drum collector is 15 to 17 cm, the rotation speed of the drum is 400 to 600 r / min, the syringe is 10 mL, the flow rate is 0.6 to 1.0 mL / h, the high voltage power supply is set to 18 to 20 KV, the temperature is 25±2° C., and the relative air humidity is 50±1%.

7. A chlorine-containing composite nanofiber membrane, characterized in that: The chlorine-containing composite nanofiber membrane is obtained by subjecting the composite nanofiber membrane according to claim 1 to chlorination treatment.

8. The chlorine-containing composite nanofiber membrane according to claim 7, characterized in that: The chlorination treatment is to contact the composite nanofiber membrane with chlorine gas; Preferably, the chlorination treatment method comprises: placing the composite nanofiber membrane close to a sodium hypochlorite-hydrochloric acid mixed solution but not in direct contact with the mixed solution, while allowing the gas generated by the sodium hypochlorite-hydrochloric acid mixed solution to contact the composite nanofiber membrane and chlorinate it; further preferably, the pH of the sodium hypochlorite-hydrochloric acid mixed solution is 4.5-5.

5.

9. Use of the composite nanofiber membrane according to claim 1 or the chlorine-containing composite nanofiber membrane according to claim 7 or 8 in the preparation of disinfection products.

10. A low-temperature resistant wipe suitable for multiple scenarios, characterized in that: The low-temperature resistant wipeable towel is made from the composite nanofiber membrane according to claim 1, or the chlorine-containing composite nanofiber membrane according to claim 7 or 8.

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