Broad-spectrum bacteriostatic wet wipe composition and use thereof

Abstract

This invention discloses a broad-spectrum antibacterial wipe composition and its application. The composition comprises a non-woven fabric substrate and a liquid composition impregnated thereon. The liquid composition uses superprotein B derived from microbial fermentation as the core antibacterial ingredient, combined with 1,2-hexanediol and ethylhexylglycerin to form a synergistic preservative system, and adds skin-care and moisturizing ingredients such as squalane and polyols. This invention abandons traditional chemical preservatives, achieving highly efficient broad-spectrum antibacterial activity through multi-component synergy. It rapidly achieves an antibacterial rate of over 99.99% against Escherichia coli, Staphylococcus aureus, and Candida albicans, without skin irritation. It also has skin barrier repair and moisturizing functions. The core ingredients are derived from green and environmentally friendly sources, and microorganisms are less likely to develop drug resistance, making it especially suitable for infants and people with sensitive skin.

Description

Technical Field

[0001] This invention belongs to the field of personal hygiene products, specifically relating to a broad-spectrum antibacterial wipe composition and its application. Background Technology

[0002] As a convenient disposable cleaning and care product, wet wipes have become widely used in various scenarios such as daily life, infant care, medical protection, and outdoor travel due to their ease of use, high cleaning efficiency, and ability to be used without water. Consequently, the market demand for the antibacterial function of wet wipes is constantly increasing. Currently, the core antibacterial effect of antibacterial wipes on the market relies on the addition of chemically synthesized preservatives or disinfectants. The mainstream antibacterial ingredients are phenoxyethanol, parabens, alcohol, and quaternary ammonium salts. These traditional antibacterial systems have many inherent defects in practical applications, becoming a key technological bottleneck restricting the development of the antibacterial wipes industry towards safety, greenness, and multi-functionality. They also fail to meet the usage needs of special groups such as infants and people with sensitive skin.

[0003] Traditional antibacterial wipes pose significant safety concerns, with a high risk of skin irritation and sensitization. The aforementioned chemically synthesized antibacterial ingredients have poor biocompatibility with human skin. Long-term or frequent use can easily damage the skin's slightly acidic barrier, causing lipid loss and leading to discomfort such as dryness, tightness, and redness. Some ingredients can also easily induce allergic reactions. These irritation issues are even more pronounced for infants and young children whose skin barriers are not yet fully developed, as well as for individuals with thin stratum corneum and sensitive skin with low tolerance. This makes traditional antibacterial wipes unsuitable for the daily cleaning and antibacterial needs of these special populations.

[0004] Traditional antibacterial ingredients have very simple mechanisms of action, which can easily induce drug resistance in microorganisms, posing a public health risk. Existing chemical disinfectants mostly achieve their antibacterial effect by interfering with the metabolic processes of microorganisms through single targets, such as inhibiting microbial enzyme activity or blocking nucleic acid synthesis. Long-term use of these antibacterial wipes can cause microorganisms in the environment to gradually adapt and develop drug resistance, significantly reducing the long-term antibacterial effectiveness of the wipes and leading to the spread of drug-resistant strains, posing a potential threat to public health and safety.

[0005] Furthermore, the sources and uses of traditional antibacterial ingredients lack environmental friendliness and are inconsistent with the concept of green development. These antibacterial ingredients are mostly chemically synthesized products, and their production process is often accompanied by high energy consumption and high pollution. Moreover, some components are difficult to biodegrade in the natural environment. After the wipes are used, they enter the natural environment with solid waste, which can easily cause water and soil pollution. At the same time, the raw material sources of some synthetic ingredients are unsustainable, which contradicts the current green and low-carbon industrial development concept.

[0006] Finally, existing antibacterial wipes have limited functionality and lack synergy between cleansing, antibacterial properties, and skin care. Current product development focuses solely on achieving antibacterial effects, neglecting the damage to the skin barrier during cleansing and lacking targeted skin repair and moisturizing ingredients. This results in wipes further damaging the skin barrier while cleansing and inhibiting bacteria, leading to significant dryness after use and failing to meet the comprehensive needs of sensitive skin for integrated cleansing, antibacterial, and skin care.

[0007] To address the aforementioned issues, the industry has attempted to develop natural antibacterial ingredients, such as plant extracts, to replace chemical preservatives. However, existing natural antibacterial ingredients generally suffer from narrow antibacterial spectrum, low antibacterial efficiency, and poor product stability. Furthermore, they are difficult to combine with other formulation ingredients to achieve synergistic antibacterial effects, failing to meet the broad-spectrum and highly efficient antibacterial requirements of traditional chemical antibacterial systems. At the same time, some natural plant extracts still have certain skin irritation properties, and the high cost of raw material extraction makes it difficult to achieve large-scale industrial application.

[0008] Therefore, developing a novel antibacterial wipe system that is environmentally friendly, highly effective and broad-spectrum in antibacterial activity, safe and low-irritant, does not easily induce microbial resistance, and can also take into account the skin's repair and moisturizing functions has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0009] This invention aims to overcome the shortcomings of existing technologies where chemical antibacterial ingredients are highly irritating, easily cause allergies, may induce microbial resistance, and are not environmentally friendly. It provides a wet wipe formulation composition with bioactive proteins derived from microbial fermentation as the core, which is scientifically compounded with specific polyol preservative synergists to construct a wet wipe formula composition that is free of traditional chemical preservatives, highly effective, broad-spectrum, low-irritant, and highly safe, while also endowing the product with additional skin repair and moisturizing care functions.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] In one embodiment, the present invention provides a broad-spectrum antibacterial wipe composition comprising a nonwoven fabric substrate and a liquid composition impregnated on the nonwoven fabric substrate; the liquid composition comprises, by weight percentage, the following components: superprotein B 0.01%-5.0%, 1,2-hexanediol 0.1%-5.0%, ethylhexylglycerin 0.01%-2.0%, squalane 1%-5.0%, dipropylene glycol 1%-3.0%, glycerin 1%-3.0%, PEG-40 hydrogenated castor oil 0.3%-3.0%, with the balance being deionized water and a pH adjuster; the pH of the liquid composition is 4.5-6.5.

[0012] Specifically, the superprotein B is an active protein prepared by modification based on a polypeptide fusion protein with anti-Malaiseem activity. The polypeptide fusion protein with anti-Malaiseem activity is formed by fusing a polypeptide with a cell wall dissolving enzyme via GGGGS linker peptides. The polypeptide is either a P6 peptide with the amino acid sequence HELVHYGRKKRRQRRR (SEQ ID NO.1) or a P9 peptide with the amino acid sequence HELVHRRWRRWNRFNRRRCR (SEQ ID NO.2). The cell wall dissolving enzyme is selected from at least one of the following: chitosanase Csn (NCBI accession number WP_074867044.1), chitinase Chi (NCBI accession number WP_202492403.1), and β-1,6-glucanase Glu (NCBI accession number BAB91213.1). The superprotein B is prepared by bridging anchoring polypeptides targeting microbial cell membranes, high-density fermentation expression of Pichia pastoris, solvent-free extraction, separation and purification, and precise hydrolysis modification.

[0013] In another embodiment, the liquid composition of the present invention contains 0.5%-2.0% by weight of superprotein B and 1%-2.0% by weight of squalane.

[0014] In another embodiment, the pH adjuster of the present invention is one or a combination of citric acid and sodium citrate, and the amount of the pH adjuster added is an effective amount to stabilize the pH of the liquid composition at 4.5-6.5.

[0015] In another embodiment, the weight ratio of the nonwoven fabric substrate to the liquid composition of the present invention is 1:1-5 (e.g., 1:3), wherein the nonwoven fabric substrate is a spunlace nonwoven fabric that has been hydrophilically treated before being used to impregnate the liquid composition.

[0016] In another embodiment, the liquid composition of the present invention comprises, by weight percentage, the following components: 1% superprotein B, 1.5% 1,2-hexanediol, 0.5% ethylhexylglycerin, 2% squalane, 2% dipropylene glycol, 1% glycerin, 0.3% PEG-40 hydrogenated castor oil, 91.7% deionized water, and the pH is adjusted to 5.0 by a pH adjuster.

[0017] In one embodiment, the present invention provides a method for preparing a broad-spectrum antibacterial wipe composition, comprising the following steps: S1, weighing superprotein B, 1,2-hexanediol, ethylhexylglycerin, squalane, dipropylene glycol, glycerin, and PEG-40 hydrogenated castor oil according to the specified ratio, stirring and mixing to obtain a homogeneous mixture; S2, adding deionized water to the mixture from step S1 to a total weight of 100%, continuing to stir until all components are evenly dispersed, adding a pH adjuster to adjust the pH of the system to 4.5-6.5, and allowing it to stand until the liquid is stable to obtain a wipe liquid; S3, placing a non-woven fabric substrate in the wipe liquid from step S2, fully impregnating it at room temperature and pressure, draining off excess wipe liquid to obtain the broad-spectrum antibacterial wipe composition.

[0018] In another embodiment, the stirring speed in steps S1 and S2 of the present invention is 200-500 r / min, and the stirring time is 10-20 min; the settling time in step S2 is 5-10 min, and no stratification or sedimentation occurs during the settling process.

[0019] In another embodiment, the personal care product of the present invention is an antibacterial wipe without traditional chemical preservatives, wherein the traditional chemical preservatives are one or more of phenoxyethanol, parabens, and quaternary ammonium salts.

[0020] In another embodiment, the personal hygiene and care product of the present invention is suitable for infants, people with sensitive skin, and medical cleaning scenarios, specifically one or more of the following: sensitive skin hand and mouth wipes, infant body cleaning wipes, medical hand antibacterial wipes, and medical surface cleaning wipes.

[0021] The broad-spectrum antibacterial wipe composition disclosed in this invention abandons the use of traditional chemical antibacterial preservatives. Through the innovative selection of core antibacterial ingredients, the scientific construction of a synergistic antibacterial system, and the precise formulation of skin-care ingredients, it fundamentally solves the technical defects of existing antibacterial wipes, such as high irritation, easy induction of drug resistance, poor environmental friendliness, and single function. It achieves an organic unity of highly effective antibacterial properties, safety and gentleness, skin care and moisturizing, and green environmental protection. Compared with the prior art, it has the following significant beneficial effects:

[0022] (1) Highly efficient and broad-spectrum antibacterial activity with fast and stable antibacterial effect: This invention uses super protein B as the core antibacterial component, combined with 1,2-hexanediol and ethylhexylglycerin to construct a synergistic antibacterial system, which enhances the effect of the two. The polyol destroys the lipid bilayer of the microbial cell membrane and increases its permeability, which greatly improves the targeting efficiency of super protein B to the microbial cell wall. At the same time, it kills microorganisms through multiple pathways, so that the composition can achieve an antibacterial rate of more than 99.9% against Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Escherichia coli) and fungi (Candida albicans) within 2 minutes. It has a broad antibacterial spectrum, fast onset of action, and the antibacterial effect is not affected by environmental factors, and has excellent stability.

[0023] (2) Safe and low irritation: The core antibacterial ingredient of this invention, super protein B, is derived from biological fermentation and has good human compatibility. The entire system abandons high-risk chemical preservatives such as phenoxyethanol and MIT. In vitro skin model tests have confirmed that it has extremely low irritation and is especially suitable for infants and young children and people with sensitive skin.

[0024] (3) Less likely to induce drug resistance: The antibacterial mechanism of super protein B is a multi-target, multi-pathway physical action. It inhibits bacteria by destroying the cell wall and cell membrane of microorganisms and interfering with their lipid metabolism. This is completely different from the single metabolic interference mechanism of traditional chemical bactericides. Microorganisms cannot adapt to this antibacterial method through gene mutation, thus avoiding the development of drug resistance from the root. At the same time, the multi-mechanism antibacterial mode of the synergistic system further reduces the risk of cross-resistance, ensures the long-term antibacterial effectiveness of the product, and avoids the public health risks caused by the spread of drug-resistant strains.

[0025] (4) It combines skin repair and moisturizing functions, achieving integrated cleansing, antibacterial and care: The present invention precisely combines skin care ingredients such as squalane, dipropylene glycol and glycerin in the antibacterial system. Squalane can form a breathable protective film on the skin surface, lock in skin moisture and promote skin barrier repair, effectively offsetting the loss of skin lipids caused during the cleansing process. Dipropylene glycol and glycerin, as mild polyol moisturizers, work synergistically with squalane to enhance the long-lasting moisturizing effect, so that the wipes can relieve skin dryness and tightness while completing cleansing and antibacterial functions, achieving the unity of cleansing, antibacterial and skin care functions, and meeting the all-round care needs of sensitive skin.

[0026] (5) Green and environmentally friendly, in line with the concept of sustainable development: The core active ingredient of this invention, super protein B, is prepared by high-density fermentation expression of Pichia pastoris. The production process adopts solvent-free extraction, separation and purification process, with no harmful pollutant emissions. The raw material source is sustainable and environmentally friendly. All components in the formula are easily biodegradable. After the wet wipes are used, they will enter the natural environment with the waste and will not cause water and soil pollution. Moreover, the production process has low energy consumption. The entire life cycle from raw material preparation, production and processing to post-use degradation is in line with the concept of green and low-carbon industrial development.

[0027] (6) Wide range of applications and good industrialization prospects: The broad-spectrum antibacterial wipe composition of the present invention can be adjusted according to different usage scenarios to prepare a variety of products such as sensitive skin hand and mouth wipes, infant and toddler body cleaning wipes, medical hand antibacterial wipes, and outdoor cleaning wipes. It can be widely used in daily life, infant and toddler care, medical protection, outdoor travel and other fields. It can not only meet the cleaning and antibacterial needs of the general population, but also meet the special needs of special groups such as infants, sensitive skin, and medical practitioners. It has strong market adaptability and broad application prospects. Attached Figure Description

[0028] Figure 1 The results of the chicken embryo chorioallantoic membrane stimulation test are as follows: a) is the state where the test substance (the wet wipe liquid of the present invention) is in contact with the chicken embryo chorioallantoic membrane for 0 seconds, and b) is the state where the test substance (the wet wipe liquid of the present invention) is in contact with the chicken embryo chorioallantoic membrane for 5 minutes. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0030] The superprotein B used in this embodiment is based on a polypeptide fusion protein with anti-Malassezia activity (patent application number: 202211282912.3), which bridges and targets the cell membrane with anchoring polypeptides. It is prepared by high-density fermentation expression of Pichia pastoris, separation and purification by solvent-free extraction and precise hydrolysis process. This superprotein B contains natural substances such as proteins, polypeptides, amino acids and their derivatives, and lipids. It can inhibit bacteria by destroying the cell wall and cell membrane of microorganisms through multiple targets and interfering with lipid metabolism.

[0031] The specific preparation process is as follows:

[0032] Selection of basic polypeptide fusion protein: The polypeptide was selected as either the P6 peptide of SEQ ID NO.1 or the P9 peptide of SEQ ID NO.2. The cell wall lysing enzyme was selected as at least one of the following: chitosanase Csn (NCBI accession number WP_074867044.1), chitinase Chi (NCBI accession number WP_202492403.1), and β-1,6-glucanase Glu (NCBI accession number BAB91213.1). The anti-Malassezia active polypeptide fusion protein formed by fusing the two through GGGGS linker peptides was the basic protein.

[0033] Targeted modification: Anchoring peptides targeting microbial cell membranes are bridged to the peptide chain ends of the above-mentioned basic polypeptide fusion protein to complete the targeted modification of the protein; then, after fermentation expression, separation and purification, precise hydrolysis, enzyme inactivation and drying, the super protein B product is obtained.

[0034] The superprotein B contains proteins, peptides, amino acids and their derivatives, and lipid-based natural active substances. It can achieve antibacterial effects by disrupting the cell wall and cell membrane of microorganisms and interfering with microbial lipid metabolism through multiple targets. When combined with 1,2-hexanediol and ethylhexylglycerol, it can produce a significant synergistic antibacterial effect.

[0035] The pH adjuster used in this embodiment is citric acid and / or sodium citrate, which can be used alone or in combination as needed. The amount added is based on stabilizing the pH of the liquid composition at 4.5~6.5. The non-woven fabric substrate used is hydrophilic treated spunlace non-woven fabric, which has good liquid adsorption and skin adhesion.

[0036] Example 1: Broad-spectrum antibacterial wipes for sensitive skin's hands and mouth

[0037] The broad-spectrum antibacterial wipe composition of this embodiment comprises, by weight percentage, the following components in the liquid composition impregnated in the non-woven fabric substrate: superprotein B 1%, 1.5% 1,2-hexanediol, 0.5% ethylhexylglycerin, 2% squalane, 2% dipropylene glycol, 1% glycerin, 0.3% PEG-40 hydrogenated castor oil, and 91.7% deionized water; citric acid and sodium citrate are used as pH adjusters to adjust the pH of the liquid composition to 5.0.

[0038] The weight ratio of the nonwoven fabric substrate to the above liquid composition is 1:3.

[0039] Weigh out the following ingredients according to the above weight percentages: superprotein B, 1,2-hexanediol, ethylhexylglycerin, squalane, dipropylene glycol, glycerin, and PEG-40 hydrogenated castor oil. Place them in a stirring container and stir at 300 r / min for 15 min at 25°C to obtain a homogeneous mixture.

[0040] Add the prescribed amount of deionized water to the above homogeneous mixture and continue stirring at 300 r / min for 20 min to ensure uniform dispersion of each component. Then add the pH adjuster composed of citric acid and sodium citrate while stirring until the pH of the system stabilizes at 5.0. After stopping stirring, let stand for 8 min until the liquid has no layering or precipitation, and obtain a stable wet wipe solution.

[0041] The hydrophilically treated spunlace nonwoven fabric substrate is placed in the above-mentioned wet wipe liquid and soaked for 20 minutes at room temperature and pressure to fully saturate the nonwoven fabric with the wet wipe liquid. Then, excess wet wipe liquid is drained from the surface of the nonwoven fabric to obtain a broad-spectrum antibacterial wet wipe for sensitive skin hands and mouth.

[0042] Example 2: Broad-spectrum antibacterial wipes for infants and young children.

[0043] The broad-spectrum antibacterial wipe composition of this embodiment comprises, by weight percentage, the following components in the liquid composition impregnated in the non-woven fabric substrate: 0.5% superprotein B, 1.2% 1,2-hexanediol, 0.2% ethylhexylglycerin, 1% squalane, 0.6% dipropylene glycol, 1.5% glycerin, 1% PEG-40 hydrogenated castor oil, and 94% deionized water; citric acid is used as a pH adjuster to adjust the pH of the liquid composition to 5.5.

[0044] The weight ratio of the nonwoven fabric substrate to the above liquid composition is 1:3.

[0045] Weigh out the following ingredients according to the above weight percentages: superprotein B, 1,2-hexanediol, ethylhexylglycerin, squalane, dipropylene glycol, glycerin, and PEG-40 hydrogenated castor oil. Place them in a stirring container and stir at 200 r / min for 20 min at 28°C until fully mixed to obtain a homogeneous mixture.

[0046] Add the prescribed amount of deionized water to the above homogeneous mixture and continue stirring at 200 r / min for 15 min to ensure uniform dispersion of each component. Then add citric acid pH adjuster while stirring until the pH of the system stabilizes at 5.5. After stopping stirring, let stand for 5 min until the liquid is free of stratification and precipitation, thus obtaining a stable wet wipe solution.

[0047] The hydrophilically treated spunlace nonwoven fabric substrate is placed in the above-mentioned wet wipe liquid and soaked for 15 minutes at room temperature and pressure to fully saturate the nonwoven fabric with the wet wipe liquid. Then, excess wet wipe liquid is drained from the surface of the nonwoven fabric to obtain a broad-spectrum antibacterial wet wipe for infant and toddler body cleaning.

[0048] Example 3: Medical Cleaning and Antibacterial Wipes

[0049] The broad-spectrum antibacterial wipe composition of this embodiment comprises, by weight percentage, the following components in the liquid composition impregnated in the non-woven fabric substrate: superprotein B 2%, 1,2-hexanediol 1.2%, ethylhexylglycerin 0.8%, squalane 2%, dipropylene glycol 3%, glycerin 3%, PEG-40 hydrogenated castor oil 2%, and deionized water 86%; sodium citrate is used as a pH adjuster to adjust the pH of the liquid composition to 4.5.

[0050] The weight ratio of the nonwoven fabric substrate to the above liquid composition is 1:3.

[0051] Weigh out the following ingredients according to the above weight percentages: superprotein B, 1,2-hexanediol, ethylhexylglycerin, squalane, dipropylene glycol, glycerin, and PEG-40 hydrogenated castor oil. Place them in a stirring container and stir at 500 r / min for 10 min at 30°C to obtain a homogeneous mixture.

[0052] Add the prescribed amount of deionized water to the above homogeneous mixture and continue stirring at 500 r / min for 30 min to ensure uniform dispersion of each component. Then add sodium citrate pH adjuster while stirring until the pH of the system stabilizes at 4.5. After stopping stirring, let stand for 10 min until the liquid has no stratification and no precipitation, thus obtaining a stable wet wipe solution.

[0053] The hydrophilically treated spunlace nonwoven fabric substrate is placed in the above-mentioned wet wipe liquid and soaked for 30 minutes at room temperature and pressure to fully saturate the nonwoven fabric with the wet wipe liquid. Then, excess wet wipe liquid is drained from the surface of the nonwoven fabric to obtain medical cleaning and antibacterial wet wipes.

[0054] Example 4: Performance Testing

[0055] To verify the technical effect of the broad-spectrum antibacterial wipes composition of the present invention, the broad-spectrum antibacterial wipes for sensitive skin hands and mouth prepared in Example 1 were used as test samples. Antibacterial performance and skin irritation were tested. All tests were repeated 3 times and the average value was taken as the test result. At the same time, a blank control group without added super protein B was set up to conduct parallel tests.

[0056] 4.1 Antibacterial performance test

[0057] 4.1.1 Testing Basis

[0058] The antibacterial performance test was conducted in accordance with the method specified in GB 15979-2002 "Hygienic Standard for Disposable Sanitary Products".

[0059] 4.1.2 Test strains

[0060] Escherichia coli (ATCC 25922, Gram-negative bacteria), Staphylococcus aureus (ATCC 6538, Gram-positive bacteria), and Candida albicans (ATCC 10231, fungus).

[0061] 4.1.3 Test Procedure

[0062] 1) Neutralizer test grouping:

[0063] Group 1: 4.5 mL neutralizer + 0.4 mL hard water + 0.1 mL bacterial suspension → culture;

[0064] Group 2: (0.4 mL sample solution + 4.5 mL neutralizer) + 0.1 mL bacterial suspension → culture;

[0065] Group 3: 4.9 mL PBS + 0.1 mL bacterial suspension → culture;

[0066] Group 4: 0.5 mL PBS of the same batch + 0.5 mL neutralizer + culture medium → culture.

[0067] 2) Evaluation regulations for neutralizer test:

[0068] a) Similar amounts of test bacteria grow in Group 1, Group 2, and Group 3, and are between 1.0×10 4 CFU / mL and 9.0×10 4 CFU / mL. The error rate of the number of colonies between groups, calculated according to formula (E.2), should not exceed 15%;

[0069] The calculation of the error rate is shown in formula (E.2):

[0070]

[0071] In the formula:

[0072] E - Error rate of the number of colonies between groups;

[0073] X m -- Average number of colonies among the three groups, in colony forming units per piece (CFU / piece); [[ID=3⑧]]

[0074] X n -- Average number of colonies in each group, in colony forming units per piece (CFU / piece).

[0075] [[ID=④④]]b) No bacteria grow in Group 4;

[0076] c) If all three consecutive tests meet the above requirements, it is determined to be qualified.

[0077] 3) Test procedures

[0078] Add 0.5 mL of freshly prepared bacterial suspension to 4.5 mL of sample solution, mix well, and start timing for 2 min. Then, use a quantitative pipette to add 0.5 mL of the bacterial suspension to a 4.5 mL test tube containing neutralizing agent, mix thoroughly, and allow to neutralize for 10 min. Perform a 10-fold serial dilution, selecting an appropriate dilution. Inoculate 1.0 mL onto each of two Petri dishes. Pour 15 mL to 20 mL of nutrient agar (for bacteria) or Sabouraud dextrose agar (for yeast) cooled to 40-45 °C and melted into the Petri dishes containing the sample solution. Rotate the Petri dishes to mix thoroughly. After the agar solidifies, invert the Petri dishes and incubate at 36 °C ± 1 °C for 48 h (bacteria) or 72 h (yeast). Count the viable colonies. Simultaneously, use the diluted solution instead of the sample for parallel experiments as a positive control. The recovered bacterial count is 1.0 × 10⁻⁶. 4 CFU / mL ~9.0×10 4 CFU / mL.

[0079] Repeat the experiment 3 times and calculate the sterilization rate.

[0080] 4) Calculation

[0081] The sterilization rate can be calculated using the following formula:

[0082] K = [(Nc - Ns) / Nc] × 100%

[0083] In the formula:

[0084] K – Sterilization rate;

[0085] Nc — Average colony count of control samples, in units of colony forming units per milliliter (CFU / mL).

[0086] Ns – Average colony count of the test sample, expressed as colony forming units per milliliter (CFU / mL).

[0087] 4.1.4 Evaluation Criteria

[0088] If the inhibition rate is ≥90% in each test, it is determined to have antibacterial effect; if the inhibition rate is ≥99% in each test, it is determined to have strong antibacterial effect.

[0089] 4.1.5 Test Results

[0090] The antibacterial rate test results of the wet wipe liquid of the present invention and the blank control group against each test strain are shown in Table 1 below.

[0091] Table 1 Results of antibacterial performance test

[0092] ;

[0093] As shown in Table 1, the wet wipes of this invention exhibited an inhibition rate of 99.99% against Escherichia coli, Staphylococcus aureus, and Candida albicans after application, and the antibacterial effect was significantly improved compared to the blank control group. This demonstrates that the composition of this invention not only has a highly efficient, broad-spectrum, and rapid antibacterial effect against common pathogens, but also has excellent inhibitory effects on fungi, covering both bacteria and fungi, and is suitable for all scenarios requiring skin cleansing and antibacterial action.

[0094] 4.2 Skin irritation test

[0095] 4.2.1 Testing Basis

[0096] The HET-CAM test of chicken embryo chorioallantoic membrane was conducted in accordance with SN / T 2329--2009.

[0097] 4.2.2 Test Conditions

[0098] The chicken embryos were incubated at a temperature of 37.8℃ and a relative humidity of 60%, and healthy chicken embryos that had been incubated for 10 to 12 days were selected for the experiment.

[0099] 4.2.3 Test Procedure

[0100] The blunt end of the chicken embryo eggshell is removed to expose the chorioallantoic membrane (CAM), ensuring the membrane is intact and the blood vessels are clearly visible and undamaged. The chorioallantoic membrane (CAM) is a respiratory membrane surrounding the chicken embryo. Because the surface of the CAM is richly vascularized, it can be considered a complete organism. This experiment utilizes the intact, clear, and transparent nature of the mid-stage chorioallantoic membrane system in hatched chicken embryos. A certain amount of the test substance is directly contacted with the CAM, and after a specified exposure time, changes in chorioallantoic membrane toxicity indicators (such as hemorrhage, coagulation, and vascularization) are observed and scored. The stimulation score is calculated to assess the eye irritation of the test substance.

[0101] Take 0.1 mL of the wet wipe solution from Example 1 and drop it onto the exposed CAM surface for direct contact. Observe continuously for 300 s and record the initial time of bleeding, vascular lysis, and coagulation on the CAM (referred to as secH, secL, and secC, respectively). If there are no such irritation phenomena, the time is recorded as 301 s.

[0102] Calculate the stimulus score (IS) using the formula, and round the result to two decimal places:

[0103] ;

[0104] sec H (bleeding time) – The average time it takes for bleeding to begin to occur as observed on the CAM membrane, measured in seconds (s).

[0105] sec L (angiogenesis time) --- The average time at which angiogenesis begins to occur as observed on the CAM membrane, measured in seconds (s);

[0106] sec C (clotting time) ---- The average time, in seconds (s), to the onset of vascular lysis as observed on the CAM membrane;

[0107] Note: The value should be 301 when no stimulus occurs.

[0108] Use 0.1 mol / L sodium hydroxide solution as a positive control, and add deionized water to the blank control group, following the same procedure.

[0109] 4.2.4 Evaluation Criteria

[0110] An IS score < 1 indicates no irritation; 1 ≤ IS < 5 indicates mild irritation; 5 ≤ IS < 9 indicates moderate irritation; and IS ≥ 10 indicates strong irritation / corrosiveness. For the positive control 0.1 mol / L sodium hydroxide, an IS value between 10 and 19 is reliable.

[0111] 4.2.5 Test Results

[0112] The skin irritation test results of the wet wipe liquid of the present invention are shown in Table 2 below.

[0113] Table 2 Results of Skin Irritation Test

[0114] ;

[0115] From Table 2 and Figure 1 (Where a represents the state of the wet wipe solution of the present invention in contact with the chicken embryo chorioallantoic membrane for 0s, and b represents the state of the wet wipe solution of the present invention in contact with the chicken embryo chorioallantoic membrane for 5min.) It can be seen that after the wet wipe solution of the present invention comes into contact with the chicken embryo chorioallantoic membrane, no bleeding, angiolysis, coagulation, or other irritation phenomena occur, and the irritation score is 0, indicating non-irritation. This proves that the composition of the present invention uses microbially fermented modified superprotein B as the core antibacterial component, abandons traditional chemical preservatives, has excellent skin compatibility, no irritation risk, and can be safely applied to special populations such as infants and children with sensitive skin.

[0116] 4.3 Stability Testing

[0117] After sealing and packaging the wet wipes prepared in Examples 1-3, they were stored for 3 months under normal temperature (25℃±2℃), high temperature (40℃±2℃, RH75%±5%), and low temperature (-5℃±2℃) conditions, respectively. The appearance of the wet wipes, liquid separation, and changes in antibacterial rate were observed regularly.

[0118] The test results are as follows:

[0119] 1) Stability testing of samples stored at room temperature

[0120] Table 3. Results of stability tests on samples stored at room temperature

[0121] ;

[0122] 2) Stability testing of samples stored at high temperatures

[0123] Table 4. Stability test results of samples stored at high temperatures

[0124] ;

[0125] 3) Stability testing of samples stored at low temperatures

[0126] Table 5. Stability test results of samples stored at low temperatures

[0127] ;

[0128] The results in Tables 3 to 5 show that under the three storage conditions, the nonwoven fabric of the wet wipes showed no mold or damage, and the liquid showed no stratification, sedimentation, or odor. The antibacterial rate against each test strain remained above 99%, proving that the composition of the present invention has excellent storage stability and can meet the storage and use requirements of industrial products.

[0129] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A broad-spectrum antibacterial wipe composition, characterized in that, The invention comprises a nonwoven fabric substrate and a liquid composition impregnated on the nonwoven fabric substrate; the liquid composition comprises, by weight percentage, the following components: superprotein B 0.01%-5.0%, 1,2-hexanediol 0.1%-5.0%, ethylhexylglycerin 0.01%-2.0%, squalane 1%-5.0%, dipropylene glycol 1%-3.0%, glycerin 1%-3.0%, PEG-40 hydrogenated castor oil 0.3%-3.0%, with the balance being deionized water and a pH adjuster; the pH of the liquid composition is 4.5-6.

5.

2. The broad-spectrum antibacterial wipes composition according to claim 1, characterized in that, In the liquid composition, the weight percentage of superprotein B is 0.5%-2.0%, and the weight percentage of squalane is 1%-2.0%. The superprotein B is formed by fusing a polypeptide with a cell wall dissolving enzyme. The polypeptide is either the P6 peptide with the amino acid sequence SEQ ID NO.1 or the P9 peptide with the amino acid sequence SEQ ID NO.

2. The cell wall dissolving enzyme is selected from at least one of the following: chitosanase Csn with NCBI accession number WP_074867044.1, chitinase Chi with NCBI accession number WP_202492403.1, and β-1,6-glucanase Glu with NCBI accession number BAB91213.

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3. The broad-spectrum antibacterial wipes composition according to claim 1, characterized in that, The superprotein B was prepared by bridging anchoring peptides targeting microbial cell membranes, high-density fermentation expression of Pichia pastoris, solvent-free extraction, separation and purification, and modification by precise hydrolysis process. The polypeptide and cell wall lysin are fused via GGGGS linker peptides.

4. The broad-spectrum antibacterial wipes composition according to claim 1, characterized in that, The pH adjuster is one or a combination of citric acid and sodium citrate, and the amount of pH adjuster added is an effective amount to make the pH of the liquid composition between 4.5 and 6.

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5. The broad-spectrum antibacterial wipes composition according to claim 1, characterized in that, The weight ratio of the nonwoven fabric substrate to the liquid composition is 1:1-5. The nonwoven fabric substrate is a spunlace nonwoven fabric that has been hydrophilically treated before being used to impregnate the liquid composition.

6. The broad-spectrum antibacterial wipes composition according to any one of claims 1-4, characterized in that, The liquid composition comprises, by weight percentage, the following components: superprotein B 1%, 1,2-hexanediol 1.5%, ethylhexylglycerin 0.5%, squalane 2%, dipropylene glycol 2%, glycerin 1%, PEG-40 hydrogenated castor oil 0.3%, and deionized water 91.7%, with the pH adjusted to 5.0 by a pH adjuster.

7. A method for preparing a broad-spectrum antibacterial wipe composition as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Weigh out the following components according to the specified ratio: superprotein B, 1,2-hexanediol, ethylhexylglycerin, squalane, dipropylene glycol, glycerin, and PEG-40 hydrogenated castor oil. Stir and mix thoroughly to obtain a homogeneous mixture. S2. Add deionized water to the mixture from step S1 until the total weight is 100%. Continue stirring until all components are evenly dispersed. Add a pH adjuster to adjust the pH of the system to 4.5-6.

5. Let the mixture stand until it is stable to obtain a wet wipe liquid. S3. Place the nonwoven fabric substrate in the wet wipe liquid from step S2 and fully immerse it at room temperature and pressure. Drain excess wet wipe liquid to obtain the broad-spectrum antibacterial wet wipe composition.

8. The preparation method according to claim 7, characterized in that, The stirring speed in steps S1 and S2 is 200-500 r / min, and the stirring time is 20-30 min; the settling time in step S2 is 5-10 min, and no stratification or sedimentation occurs during the settling process.

9. The use of the broad-spectrum antibacterial wipe composition according to any one of claims 1-6 in the preparation of personal hygiene products, characterized in that, The personal hygiene product is an antibacterial wipe without traditional chemical preservatives, wherein the traditional chemical preservatives are one or more of phenoxyethanol, parabens, and quaternary ammonium salts.

10. The application according to claim 9, characterized in that, The personal care products mentioned are suitable for infants, people with sensitive skin, and medical cleaning scenarios, specifically one or more of the following: sensitive skin hand and mouth wipes, infant and toddler body cleaning wipes, medical hand antibacterial wipes, and medical surface cleaning wipes.

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