Small molecule peptide composition for sanitary disinfection and application thereof

Abstract

The invention focuses on the fields of biotechnology and sanitary disinfection, and successfully separates five brand-new small molecule peptides (small molecule peptides K, L, M, N and O) from herba violae and spica prunellae. According to the innovative extraction method from raw material pretreatment, mixed extraction to multi-step purification, a disinfection action mechanism of destroying pathogen cell membranes, interfering energy metabolism and inhibiting biosynthesis by small molecular peptides is defined. The small molecule peptides show the advantages of efficient disinfection activity, high safety, good stability and difficult generation of drug resistance. On the basis, a series of products such as hygienic disinfection spray, wet tissues and hand sanitizer are developed, the market application prospect is wide, remarkable economic and social benefits can be brought, and a brand-new and high-quality solution is provided for the field of hygienic disinfection.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and sanitary disinfection, and in particular relates to the extraction of novel small molecule peptides from specific plant resources, and the innovative application of these small molecule peptides in sanitary disinfection. Background Art

[0002] In the field of sanitation and disinfection, traditional disinfection methods and products have many drawbacks. Chemical disinfectants such as chlorine-containing disinfectants have strong bactericidal properties, but they are highly irritating and can damage the human respiratory tract and skin. They are also prone to leaving harmful residues and polluting the environment. Alcohol disinfectants are flammable and have poor disinfection effects on some viruses and spores. Although natural plant extracts have safety advantages, their active ingredients are complex, their disinfection effects are unstable, and their mechanisms of action are unclear. Small molecule peptides have emerged in bioactivity research. However, efficient, stable, and naturally derived small molecule peptide products developed for sanitation and disinfection are still extremely scarce. Therefore, the development of new, high-performance small molecule peptides for sanitation and disinfection is of great significance and market demand. Summary of the Invention

[0003] A small molecule peptide combination for hygienic disinfection, comprising the following five small molecule peptides: Small molecule peptide K: Asn - Ser - Glu - Pro - His - Cys - Lys - Trp - Ile - Gly- Tyr - Asp - Leu - Val - Thr (NS EPHCKWIGYD LVT); Small molecule peptide L: His - Gln - Arg - Cys - Asp - Trp - Ser - Pro - Gly - Lys- Val - Tyr - Cys - Leu - Met (HQRCDWS PGKVYCLM); Small molecule peptide M: Gly - Cys - His - Lys - Asp - Pro - Ile - Leu - Ser - Tyr- Asn - Glu - Val - Met - Thr (GC KHDPI LSYNEVMT); Small molecule peptide N: Thr - Glu - Asp - Cys - His - Pro - Arg - Gly - Ile - Val- Leu - Ser - Phe - Tyr - Lys (TEDCHPR GIVLSFYK); Small molecule peptide O: Val - Pro - Cys - Lys - Trp - Asp - His - Ile - Gly - Leu - Met - Thr - Phe - Asn - Glu (VPCKW DHI GLMTFNE).

[0004] Furthermore, the small molecule peptide combination is derived from extracts of Viola philippica and Prunella vulgaris.

[0005] Furthermore, the method for extracting the small molecule peptide combination comprises the following steps: Raw material pretreatment: Select high-quality Viola yedoensis whole herb and Prunella vulgaris fruit spikes, remove impurities, wash, and dry in a 40°C forced air drying oven to constant weight. Use a grinder to grind each into a powder with a particle size of approximately 0.35 mm. Mixed extraction: Viola yedoensis and Prunella vulgaris powder were mixed in a mass ratio of 2:3, and 10 volumes of a 70% ethanol-water mixture were added. Ultrasonic-assisted reflux extraction was performed at 60°C for 3 times, each for 2 hours, at an ultrasonic frequency of 50 kHz and a power of 250 W. The three extracts were combined and concentrated under reduced pressure to 1 / 5 of the original volume. Macroporous resin adsorption separation: The concentrate was passed through a pretreated D101 macroporous adsorption resin column. The column was first rinsed with deionized water to remove impurities, and then eluted with a 45% by volume ethanol solution, and the eluate was collected. Enzymatic hydrolysis: Adjust the pH of the eluate to 7.3, add a mixture of trypsin and pepsin at a mass ratio of 4:1, and add 3.5% of the total mass of the eluate. Incubate in a 43°C water bath with shaking at 200 rpm for 6 hours. Ultrafiltration purification: The enzymatic hydrolysate is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 2500Da to remove undigested macromolecular impurities and collect the permeate; Ion exchange chromatography: Load the permeate onto a CM-Sepharose Fast Flow strong cation exchange resin column. First, rinse the column with 0.03 M phosphate buffer (pH 7.0) to remove unbound impurities. Then, elute with a gradient of 0-0.6 M sodium chloride solution at a flow rate of 1.2 mL / min. Collect the elution peak with disinfectant activity. Gel filtration chromatography: The collected elution peak was loaded onto a Sephadex G-30 gel column and eluted with 0.12 M phosphate buffer (pH 7.2) to collect the target small molecule peptide component; Freeze drying: The collected small molecule peptide components are freeze-dried to obtain small molecule peptide dry powder.

[0006] Furthermore, in the macroporous resin adsorption separation step, the flow rate of the concentrated solution through the D101 macroporous adsorption resin column is 2.5 mL / min.

[0007] Furthermore, in the ultrafiltration purification step, the ultrafiltration pressure is controlled at 0.12-0.25 MPa.

[0008] Furthermore, in the ion exchange chromatography step, when gradient elution with sodium chloride solution is performed, the eluent flow rate is 1.2 mL / min.

[0009] Furthermore, in the gel filtration chromatography step, the eluent flow rate is 0.6 mL / min.

[0010] Furthermore, in the method for extracting the small molecule peptide combination described in 3, in the freeze-drying step, the freezing temperature is controlled below -55°C and the vacuum degree is below 15Pa.

[0011] In another aspect, the present invention provides a sanitary disinfection product comprising the small molecule peptide combination.

[0012] Furthermore, the sanitary disinfection product is a sanitary disinfection spray, sanitary disinfection wipes or sanitary disinfection hand soap.

[0013] Beneficial effects of the invention: Highly effective disinfectant: The small-molecule peptide of this invention exhibits significant inhibitory and killing effects against a variety of common pathogenic microorganisms, including Staphylococcus aureus, Escherichia coli, Candida albicans, and influenza virus. Effective disinfection is achieved at low concentrations (5-30 μg / mL), with activity surpassing that of many traditional disinfectants and existing botanical disinfectants. Experimental data show that at a concentration of 15 μg / mL, the small-molecule peptide K exhibits an inhibition zone diameter of 20 mm against Staphylococcus aureus, while the inhibition zone of a traditional plant extract disinfectant at the same concentration is only 12 mm. Highly safe: The small molecule peptide is derived from natural Viola yedoensis and Prunella vulgaris and undergoes multi-step purification, free of harmful chemicals. Animal studies have shown that high doses have no significant effect on the physiological parameters of experimental animals, are non-irritating to human skin and mucous membranes, and pose no allergic risk, making it safe for use in sanitary disinfection. Good stability: Small molecule peptides can maintain high disinfection activity under different pH values ​​(4-9) and temperature conditions (20-60°C). In an environment with a pH value of 5-8 and a temperature of 25-45°C, the activity of small molecule peptides is basically unchanged after long-term storage, making the product easy to store and transport. Not easy to develop drug resistance: Small molecule peptides have diverse mechanisms of action and act on multiple targets of pathogens. It is difficult for pathogens to develop drug resistance through a single gene mutation. They can effectively exert a long-term disinfection effect and reduce the possibility of pathogens developing drug resistance. DETAILED DESCRIPTION

[0014] Example 1

[0015] (1) Extraction of small molecule peptides Prepare the raw materials: Accurately weigh 120g of Viola yedoensis whole herb and 180g of Prunella vulgaris fruit spikes and process according to the pretreatment method described above to obtain plant powder. Ensuring raw material quality and pretreatment specifications are crucial for subsequent extraction of active ingredients. Mixed Extraction: Combine the two plant powders and add 3000 mL of a 70% ethanol-water mixture. Reflux extraction was performed three times, two hours each, at 60°C, 50 kHz ultrasonic frequency, and 250 W power. Combine the extracts and concentrate under reduced pressure to 600 mL. During reflux extraction, strictly control the temperature, ultrasonic parameters, and time to ensure adequate extraction. During reduced pressure concentration, pay attention to the temperature and vacuum level to avoid loss of active ingredients. Macroporous Resin Adsorption Separation: Slowly pass the concentrate through a pretreated D101 macroporous adsorption resin column at a flow rate of 2.5 mL / min. First, rinse the column with deionized water until the effluent is clear and impurities are removed. Then, elute with 1200 mL of 45% ethanol solution and collect the eluate. Closely monitor the effluent color and composition to ensure adequate adsorption and elution of the active ingredients. Enzymatic hydrolysis: Adjust the pH of the eluent to 7.3, add 6g of a mixed enzyme solution (trypsin: pepsin ratio 4:1 by mass), and incubate in a 43°C water bath with shaking at 200 rpm for 6 hours. Maintain a stable temperature and pH during hydrolysis. Regularly monitor and adjust the pH to ensure adequate contact between the enzyme and substrate. Ultrafiltration: The enzymatic hydrolysate is filtered through an ultrafiltration membrane with a molecular weight cutoff of 2500 Da, and the permeate is collected. During ultrafiltration, pay attention to pressure control, maintaining a pressure of 0.12-0.25 MPa to prevent membrane clogging and rupture. Ion Exchange Chromatography: The permeate was loaded onto a CM-Sepharose Fast Flow strong cation exchange resin column. The column was first rinsed with 600 mL of 0.03 M phosphate buffer (pH 7.0) to remove unbound impurities. Elution was then performed using a gradient of 0-0.6 M sodium chloride solution at a flow rate of 1.2 mL / min, and the peak exhibiting disinfectant activity was collected. Ion exchange chromatography ensures effective separation of the target small molecule peptide by precisely controlling the eluent concentration gradient and flow rate. Gel filtration chromatography: Load the collected elution peak onto a Sephadex G-30 gel column and elute with 0.12M phosphate buffer (pH 7.2) to collect the target small molecule peptide fraction. During gel filtration chromatography, maintain a stable eluent flow rate of 0.6 mL / min to ensure timely collection of the target fraction. Lyophilization: The collected small molecule peptide components are freeze-dried to obtain small molecule peptide dry powder. The freeze-drying process strictly controls the temperature below -55°C and the vacuum degree below 15Pa to ensure that the activity of the small molecule peptide is not affected. (II) Screening and sequencing process of small molecule peptides Construction of a crude small-molecule peptide library: After mixed extraction of Viola yedoensis and Prunella vulgaris, macroporous resin adsorption separation, and preliminary enzymatic hydrolysis, a mixture containing various small-molecule peptides and impurities was obtained. Initial fractionation by molecular size was performed using centrifugal ultrafiltration, followed by isoelectric focusing electrophoresis to subdivide the molecules based on their isoelectric points, thus constructing a crude small-molecule peptide library. The components of the library exhibited similar physicochemical properties, providing a rich sample for subsequent screening. Initial screening of sanitation and disinfection activity: A high-throughput screening model was used to screen the crude extract library of small molecule peptides. A large number of 96-well plates were prepared. When screening for antibacterial activity, Staphylococcus aureus, Escherichia coli, Candida albicans and other common pathogenic microorganisms were inoculated in different wells. The bacterial solution was adjusted to the logarithmic growth phase concentration, such as 1×10 5 CFU / mL. For antiviral activity screening, sensitive cell lines such as MDCK cells (for influenza virus) and Vero cells (for other common viruses) are pre-cultured in 96-well plates to form monolayers. Each component of the crude small peptide extract library is diluted to a specific concentration gradient and added to the 96-well plate inoculated with microorganisms or cells. A positive control group (containing known effective antibiotics, antiviral drugs, etc.) and a negative control group (containing only culture medium, bacterial suspension, or cells, without the crude small peptide extract) are also established. The 96-well plates are placed under appropriate culture conditions: bacteria are cultured at 37°C for 24 hours, fungi are cultured at 28°C for 48 hours, and virus-infected cells are incubated for an appropriate time based on the characteristics of the virus (e.g., 48 hours for MDCK cells infected with influenza virus). At the end of the incubation period, antibacterial screening is performed by observing microbial growth in the wells, such as changes in turbidity (measured by absorbance at a specific wavelength on a microplate reader). For antiviral screening, the MTT assay is used to assess cell viability and evaluate the protective effect of the crude small peptide extract against virus-infected cells. Small peptide crude extracts that significantly inhibited microbial growth or increased the survival rate of virus-infected cells at a certain concentration were labeled as potential active components and entered the next step of screening. The initial screening identified approximately 70 potential sanitary disinfectant active components from the numerous small peptide crude extracts. Subdivision and Purification of Active Components: The crude extracts of 70 potentially active small peptides identified in the initial screening were further subdivided and purified. High-performance liquid chromatography (HPLC) was first used in conjunction with different types of chromatographic columns (e.g., reverse-phase C18 columns, ion-exchange columns, etc.), selecting a separation mode based on the characteristics of each component to isolate the small peptides in each crude extract as relatively single peaks. The corresponding solutions were collected, theoretically containing a single primary small peptide but potentially containing small amounts of impurities. Preparative HPLC was then used to prepare and purify the initially isolated small peptides on a large scale, optimizing elution conditions to increase the purity of the target small peptides. Finally, mass spectrometry (MS) was used to determine the initial molecular weights of the purified small peptides. Comparisons were made with a database of known small peptides to exclude small peptides with known sequences and retain those with potentially novel sequences. This step yielded approximately 180 suspected novel small peptide samples. Amino acid sequencing: Amino acid sequencing was performed on the 180 retained suspected new small peptide samples. Tandem mass spectrometry (MS / MS)-based sequencing was primarily used. The small peptide samples were ionized and subjected to primary mass spectrometry analysis on a mass spectrometer to determine the precise molecular weight. Specific peptide ions were selected for secondary mass spectrometry analysis, where high-energy collisions induce peptide fragmentation, producing a series of fragment ions with specific mass differences. Professional bioinformatics software (such as Mascot and SEQUEST) was used to analyze the fragment ion data based on their mass and fragmentation patterns, and to infer the amino acid sequence of the small peptide. For some complex sequences or difficult-to-determine sites, traditional Edman degradation sequencing was used for verification. Edman degradation progressively cleaves and identifies amino acids from the N-terminus of the peptide chain to determine the peptide sequence. Amino acid sequencing successfully obtained the accurate amino acid sequences of 180 small peptides. Rescreening and Optimization of Disinfection Activity: 180 sequenced small peptides were comprehensively rescreened for disinfection activity. This antimicrobial rescreening employed more precise minimum inhibitory concentration (MIC) assays, such as the broth dilution method combined with a checkerboard titration. This not only determined the MIC values ​​of the small peptides against common pathogens, but also investigated their combined antimicrobial effects with other antimicrobial agents and evaluated their antimicrobial activity in diverse microbial communities. In addition to assessing cell viability using the MTT assay, the antiviral rescreening also employed real-time quantitative PCR to precisely determine the inhibition rate of viral nucleic acid replication, and immunofluorescence assays to observe changes in protein expression following viral infection. Based on the rescreening results, small peptides were selected that exhibited high inhibitory activity against a variety of common pathogens (such as Staphylococcus aureus, Escherichia coli, Candida albicans, and influenza virus). The amino acid sequences of the selected active small peptides were optimized using site-directed mutagenesis. Without altering key active sites, amino acid residues that could affect stability, solubility, or activity were substituted, and the optimized peptides were retested for disinfection activity. After multiple rounds of optimization and screening, five small molecule peptides with optimal sanitary disinfection activity, good stability and suitable solubility were finally identified, namely small molecule peptides K, L, M, N and O. (III) Small molecule peptide disinfection activity test Antibacterial activity test Experimental strains: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Candida albicans (ATCC 10231) were selected as test strains. These strains are common pathogens and are representative. Culture medium preparation: Use nutrient broth and nutrient agar for Staphylococcus aureus and Escherichia coli; use Sabouraud medium and Sabouraud agar for Candida albicans. Prepare the culture medium strictly according to the standard formula and autoclave to ensure sterility. Experimental Methods: The minimum inhibitory concentration (MIC) of small peptides was determined using the broth dilution method. Small peptide K-O was prepared in sterile water to a concentration gradient of 5-100 μg / mL. In a 96-well microplate, 100 μL of the corresponding culture medium was added to each well, followed by 100 μL of the small peptide solution at different concentrations. Bacterial suspensions in logarithmic growth phase or fungal spore suspensions were diluted with 1×10 5 CFU / mL (bacteria) or 1×10 4An inoculum of 100 CFU / mL (for fungi) was added to each well. A positive control (containing a known effective antibacterial or antifungal drug) and a negative control (containing only culture medium and bacterial suspension, without the small molecule peptide) were also established. The 96-well plate was incubated at 37°C for 24 hours (for bacteria) or 48 hours (for fungi). At the end of the incubation period, each well was observed for bacterial or fungal growth. The minimum small molecule peptide concentration that resulted in no bacterial or fungal growth was defined as the MIC. Experimental results: The small molecule peptide K-O has good antibacterial activity against Staphylococcus aureus, Escherichia coli and Candida albicans. The MIC of small molecule peptide K against Staphylococcus aureus was 7 μg / mL, the MIC against Escherichia coli was 10 μg / mL, and the MIC against Candida albicans was 13 μg / mL; the MIC of small molecule peptide L against Staphylococcus aureus was 9 μg / mL, the MIC against Escherichia coli was 12 μg / mL, and the MIC against Candida albicans was 15 μg / mL; the MIC of small molecule peptide M against Staphylococcus aureus was 11 μg / mL, the MIC against Escherichia coli was 14 μg / mL, and the MIC against Candida albicans was 17 μg / mL; the MIC of small molecule peptide N against Staphylococcus aureus was 8 μg / mL, the MIC against Escherichia coli was 11 μg / mL, and the MIC against Candida albicans was 14 μg / mL; the MIC of small molecule peptide O against Staphylococcus aureus was 10 μg / mL, the MIC against Escherichia coli was 13 μg / mL, and the MIC against Candida albicans was 1 (III) Small molecule peptide disinfection activity test Antibacterial activity test Killing Curve Experiment: To further explore the dynamic killing process of small molecule peptides against bacteria, a killing curve experiment was conducted using Escherichia coli as an example. E. coli were inoculated into nutrient broth containing different concentrations of small molecule peptide K (0, 5, 10, and 15 μg / mL) and cultured at 37°C with shaking at 180 rpm. The bacterial suspension was collected at 0, 1, 2, 3, 4, 5, and 6 hours, and a 10-fold serial dilution was performed. 100 μL of the dilution was plated on a nutrient agar plate. After incubation at 37°C for 24 hours, the colonies were counted and a killing curve was plotted. The results showed that the number of E. coli colonies in the experimental group containing small molecule peptide K gradually decreased over time. At a concentration of 10 μg / mL, the bacterial count decreased by an order of magnitude after 3 hours, and almost no viable bacteria were detected after 6 hours, indicating that small molecule peptide K can continuously and efficiently kill E. coli, rather than simply inhibiting its growth. Similar experiments on Staphylococcus aureus and Candida albicans with other small molecule peptides L-O also showed similar bactericidal trends, further confirming the potent bactericidal properties of small molecule peptides. Antimicrobial synergy studies: Considering the potential for synergistic effects when multiple antimicrobial agents are used in combination, synergistic antimicrobial activity between small peptides and with traditional antimicrobial agents was investigated. Using Staphylococcus aureus as the study target, groups were divided into groups using small peptides alone, groups using small peptide combinations (e.g., a 1:1 mixture of small peptide K and small peptide L), groups using small peptides in combination with traditional antimicrobial agents (e.g., small peptide K combined with penicillin), and a control group (containing only bacteria and culture medium). The minimum inhibitory concentration (FIC) index of the combined drugs was determined using the checkerboard dilution method. The FIC index is calculated as: the MIC of drug A in combination / the MIC of drug A alone + the MIC of drug B in combination / the MIC of drug B alone. An FIC index ≤ 0.5 indicates synergism between the two drugs; an FIC index of 0.5 < 4 indicates additive activity; and an FIC index > 4 indicates antagonism. Experimental results showed that when small molecule peptides K and L were used together, the FIC index was 0.4, demonstrating a significant synergistic antimicrobial effect and significantly enhancing the inhibitory effect against Staphylococcus aureus. When small molecule peptide K was used in combination with penicillin, the FIC index was 0.6, indicating an additive effect, also enhancing the antimicrobial activity against Staphylococcus aureus to a certain extent. This provides a theoretical basis for the combined use of small molecule peptides in practical sanitary disinfection products. Antiviral activity test Experimental Viruses and Cells: Influenza virus type A (H1N1) and human embryonic kidney cells (HEK293) were used as experimental models. Respiratory syncytial virus (RSV) and human lung adenocarcinoma cells (A549) were also introduced for extended studies. HEK293 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, while A549 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Both cells were incubated at 37°C in a 5% CO2 incubator until cell confluence reached 80%-90% before use in experiments. Influenza virus and respiratory syncytial virus are important pathogens that cause respiratory infections, and the selection of these two viruses and corresponding cell models is of great clinical significance. Experimental method: For influenza virus type A (H1N1), HEK293 cells were cultured at a density of 1×10 5The cells were seeded into 96-well plates at a density of 1000 μg / well. After 24 hours of culture, the culture medium was discarded and the cells were washed twice with PBS. The small molecule peptide K-O was prepared into solutions of different concentrations in serum-free DMEM medium, with 5 replicates for each concentration. A virus control group (influenza virus type A (H1N1) only) and a cell control group (cells only) were also set up. After adding the small molecule peptide solutions of different concentrations, 100 TCID 50 Influenza A virus (H1N1) was incubated at 37°C, 5% CO2 for 48 hours. Real-time fluorescence quantitative PCR was used to detect the relative content of influenza virus nucleic acid in cells to evaluate the inhibitory effect of small molecule peptides on viral replication. MTT assay was also used to detect cell viability to evaluate the protective effect of small molecule peptides on virus-infected cells. For respiratory syncytial virus (RSV), A549 cells were plated at 1×10 5 The cells were inoculated at a density of 100 cells / well in a 96-well plate and cultured for 24 hours, and then treated similarly to the above method. The enzyme-linked immunosorbent assay (ELISA) method was used to detect the content of RSV F protein in the cell culture supernatant. This protein is a key protein for RSV infection and pathogenicity, and its content can reflect the replication and infection of the virus. Experimental Results: For influenza A (H1N1) virus, increasing small peptide concentration significantly reduced the relative content of influenza viral nucleic acid in cells, while cell viability gradually increased. For example, at a concentration of 20 μg / mL, small peptide K reduced the relative content of viral nucleic acid by 80% compared to the virus control group, while cell viability increased to 85%. Small peptide K-O also demonstrated significant inhibitory activity against respiratory syncytial virus (RSV). At a concentration of 25 μg / mL, small peptide L reduced the content of RSV F protein in the cell culture supernatant by 75% compared to the virus control group. These results demonstrate that small peptides exhibit significant antiviral activity against a variety of common respiratory viruses, broadening their application in antiviral hygiene and disinfection. Security Testing Hemolysis test: Fresh healthy rabbit blood was collected, washed three times with normal saline, and prepared into a 2% red blood cell suspension. In a 96-well plate, 100 μL of the red blood cell suspension was added to each well, followed by 100 μL of a small peptide K-O solution at varying concentrations (10, 20, 50, and 100 μg / mL). A positive control group (distilled water was added to completely hemolyze the red blood cells) and a negative control group (normal saline were added) were also set up. After incubation at 37°C for 2 hours, the cells were centrifuged at 3000 rpm for 5 minutes, and the supernatant was measured for absorbance at 540 nm on a microplate reader. Higher absorbance values ​​indicate a greater degree of hemolysis. The results showed that the absorbance values ​​of the various small peptide experimental groups at different concentrations were similar to those of the negative control group and significantly lower than those of the positive control group. When the concentration of the small molecule peptide reached 100 μg / mL, the absorbance value of the small molecule peptide K-O group showed no statistically significant difference compared with that of the negative control group (P>0.05), indicating that the small molecule peptide K-O had no obvious hemolytic effect on red blood cells within the experimental concentration range and had potential application safety in blood contact-related scenarios (such as wound disinfection). Genotoxicity test (comet assay): Human peripheral blood lymphocytes were used for the comet assay to assess the genotoxicity of small peptides. Human peripheral blood lymphocytes were incubated with various concentrations (10, 50, 100, 500, and 1000 μg / mL) of the small peptide in K-O solution at 37°C for 4 hours. A positive control group (using the known mutagen methyl methanesulfonate) and a negative control group (containing only cell culture medium) were also set up. After incubation, a comet assay kit was used. The cell suspension was mixed with low-melting-point agarose and plated on a glass slide. After lysis, electrophoresis, and staining, DNA damage was observed under a fluorescence microscope. The genotoxicity of the small peptides was determined by analyzing parameters such as comet tail length and tail moment. The results showed that the comet tail length and tail moment of each small peptide experimental group were not significantly different from those of the negative control group (P>0.05), and were significantly lower than those of the positive control group. Even at the highest concentration of 1000 μg / mL, the small molecule peptide K-O did not induce obvious DNA damage, indicating that the small molecule peptide K-O is non-genotoxic and will not cause damage to the genetic material of organisms during long-term use, further ensuring its safety in the field of sanitation and disinfection. Preparation of sanitary disinfection products Sanitary disinfectant spray Formula: Taking small molecule peptide K as an example, each 100mL spray solution contains 20mg of small molecule peptide K, 5mL of propylene glycol, 1.2mL of polysorbate 80, and 0.1g of ethyl paraben. Purified water is added to 100mL. Propylene glycol keeps the spray solution moist, preventing the small molecule peptide from drying out and becoming inactive during the spray process. Polysorbate 80 acts as a surfactant, enhancing the dispersion of the small molecule peptide and ensuring uniform spraying. Ethyl paraben acts as a preservative, inhibiting microbial growth and extending the product's shelf life. Preparation process: First, dissolve the small molecule peptide K in a small amount of purified water. While stirring with a magnetic stirrer, slowly add propylene glycol, polysorbate 80, and ethyl paraben in sequence. Stir thoroughly for 30 minutes to completely dissolve and mix the ingredients. Then, dilute the volume to 100 mL with purified water and continue stirring for 20 minutes. Filter the prepared solution through a 0.22μm microporous filter membrane to sterilize the product to ensure sterility. Finally, the filtered solution is dispensed into spray bottles and tested for leaks before packaging to ensure no leakage during storage and use. Quality Control: The spray solution should be clear and transparent, free of precipitation, turbidity, or foreign matter. The content of small-molecule peptide K in the spray solution should be determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), and the content should be within 90%-110% of the labeled amount. This method accurately measures small-molecule peptides with high sensitivity and selectivity. The pH of the spray solution should be between 6.2 and 7.0. Within this pH range, small-molecule peptides exhibit excellent stability and minimal irritation to the skin and mucous membranes. Microbial limit tests must meet relevant standards. The total bacterial count per milliliter of spray solution must not exceed 100 CFU, and the total mold and yeast count must not exceed 10 CFU. Pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli must not be detected. Furthermore, the spray bottle's spray performance, including spray angle and spray particle size distribution, should be tested to ensure uniform coverage of the target area and that the spray particle size is between 50 and 150 μm to ensure effective disinfection. Hygienic disinfectant wipes Formula: Each 100g of wipes contains 30mg of small-molecule peptide L, 7g of glycerin, 2mL of polyoxyethylene sorbitan monolaurate (Tween-20), and 0.15g of methylparaben. Purified water is added to 100g. Glycerin maintains the wet wipes' moisture, allowing the small-molecule peptide to continue functioning; Tween-20 enhances the dispersion of the small-molecule peptide in the wipes' liquid; and methylparaben acts as a preservative to prevent microbial growth during storage. Preparation: Dissolve small molecule peptide L in an appropriate amount of purified water. Add glycerin, Tween-20, and methylparaben while stirring and mix thoroughly. Heat to 40-45°C to promote dissolution of the ingredients, but control the temperature to avoid denaturation of the small molecule peptide. Cut non-woven fabric into 15cm x 20cm squares and soak them in the prepared wipe solution for 30 minutes to ensure full absorption of the solution and uniform distribution of the small molecule peptide L. After soaking, remove the non-woven fabric, drain any excess liquid, and heat-seal the package at a temperature of 120-130°C to ensure a tight seal and prevent moisture evaporation and microbial contamination. Quality Control: Wet wipes should appear smooth, wrinkle-free, and free of damage, with moderate wetness. Ultraviolet spectrophotometry is used to determine the content of small-molecule peptide L in the wipes, which should be between 93% and 107% of the labeled amount. This method is simple, rapid, and suitable for routine testing of small-molecule peptide content. The pH of the wipes should be between 5.8 and 6.5 to ensure comfort and safety. Microbial limit testing requires that the total bacterial count per wipe must not exceed 20 CFU, and the total mold and yeast count must not exceed 10 CFU. Pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli must not be detected. Furthermore, the moisture content of the wipes should be maintained between 70% and 80% to ensure moisture retention while preventing microbial growth or reduced activity of the small-molecule peptides due to excessive moisture. Hygienic disinfectant hand soap Formula: Each 100g of hand sanitizer contains 25mg of small molecule peptide M, 13g of sodium laureth sulfate, 5g of cocamidopropyl betaine, 9g of glycerin, an appropriate amount of citric acid (adjust the pH to 7.0-7.8), and an appropriate amount of fragrance. Purified water is added to 100g. Sodium laureth sulfate and cocamidopropyl betaine act as surfactants, providing excellent cleaning and foaming properties. Glycerin moisturizes and prevents dryness after washing. Citric acid adjusts the pH of the hand sanitizer to a level close to the skin's physiological pH. The fragrance improves the product's odor and enhances the user experience. Preparation: Dissolve sodium laureth sulfate and cocamidopropyl betaine in an appropriate amount of purified water. Heat to 65-70°C and stir thoroughly to fully dissolve the surfactant. Dissolve small molecule peptide M in a small amount of purified water and slowly add to the above solution. Continue stirring for 25 minutes to ensure uniform dispersion of the small molecule peptide M. Adjust the pH by adding glycerin and citric acid, then add an appropriate amount of flavor and stir thoroughly. Finally, add purified water to 100g, stir thoroughly, and cool to room temperature. During the preparation process, strictly control the temperature and stirring speed to avoid excessive foaming that may affect product quality. Maintain a clean and hygienic production environment to prevent microbial contamination. Quality Control: Hand sanitizer should appear as a uniform emulsion, free of stratification or precipitation, and possess a normal color. Ultra-high performance liquid chromatography (UPLC) is used to determine the content of small-molecule peptide M in hand sanitizer. The content should be between 95% and 105% of the labeled amount. UPLC offers higher separation efficiency and analysis speed, enabling more accurate determination of small-molecule peptide content. The pH of the hand sanitizer should be between 7.0 and 7.8, which is consistent with the pH range for skin-friendly products. Foam performance testing is conducted using a Roche foam analyzer. The initial foam height of the hand sanitizer must be no less than 150 mm, and after 5 minutes, the foam height must be no less than 100 mm to ensure effective cleaning. Stability testing is conducted by placing the hand sanitizer at different temperatures (4°C, 25°C, and 40°C) for 3 months to observe for stratification, discoloration, or odor change, thereby assessing the product's physical and chemical stability. Microbial limit testing requires that the total number of bacteria in each gram of hand sanitizer must not exceed 1000 CFU, the total number of molds and yeasts must not exceed 100 CFU, and pathogenic bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa must not be detected to ensure the safety of the product during storage and use.

Claims

1. A small molecule peptide combination for sanitary disinfection, characterized in that: Contains the following 5 small molecule peptides: Small molecule peptide K: Asn - Ser - Glu - Pro - His - Cys - Lys - Trp - Ile - Gly -Tyr - Asp - Leu - Val - Thr (NS EPHCKWIGYD LVT); Small molecule peptide L: His - Gln - Arg - Cys - Asp - Trp - Ser - Pro - Gly - Lys -Val - Tyr - Cys - Leu - Met (HQRCDWS PGKVYCLM); Small molecule peptide M: Gly - Cys - His - Lys - Asp - Pro - Ile - Leu - Ser - Tyr -Asn - Glu - Val - Met - Thr (GC KHDPI LSYNEVMT); Small peptide N: Thr - Glu - Asp - Cys - His - Pro - Arg - Gly - Ile - Val -Leu - Ser - Phe - Tyr - Lys (TEDCHPR GIVLSFYK); Small molecule peptide O: Val - Pro - Cys - Lys - Trp - Asp - His - Ile - Gly - Leu -Met - Thr - Phe - Asn - Glu (VPCKW DHI GLMTFNE).

2. The small molecule peptide combination for sanitary disinfection according to claim 1, characterized in that: The small molecule peptide combination is derived from extracts of Viola philippica and Prunella vulgaris.

3. A method for extracting a small molecule peptide combination as claimed in claim 1, characterized in that: The following steps are involved: Raw material pretreatment: Select high-quality Viola yedoensis whole herb and Prunella vulgaris fruit spikes, remove impurities, wash, and dry in a 40°C forced air drying oven to constant weight. Use a grinder to grind each into a powder with a particle size of approximately 0.35 mm. Mixed extraction: Viola yedoensis and Prunella vulgaris powder were mixed in a mass ratio of 2:3, and 10 volumes of a 70% ethanol-water mixture were added. Ultrasonic-assisted reflux extraction was performed at 60°C for 3 times, each for 2 hours, at an ultrasonic frequency of 50 kHz and a power of 250 W. The three extracts were combined and concentrated under reduced pressure to 1 / 5 of the original volume. Macroporous resin adsorption separation: The concentrate was passed through a pretreated D101 macroporous adsorption resin column. The column was first rinsed with deionized water to remove impurities, and then eluted with a 45% by volume ethanol solution, and the eluate was collected. Enzymatic hydrolysis: Adjust the pH of the eluate to 7.3, add a mixture of trypsin and pepsin at a mass ratio of 4:1, and add 3.5% of the total mass of the eluate. Incubate in a 43°C water bath with shaking at 200 rpm for 6 hours. Ultrafiltration purification: The enzymatic hydrolysate is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 2500Da to remove undigested macromolecular impurities and collect the permeate; Ion exchange chromatography: Load the permeate onto a CM-Sepharose Fast Flow strong cation exchange resin column. First, rinse the column with 0.03 M phosphate buffer (pH 7.0) to remove unbound impurities. Then, elute with a gradient of 0-0.6 M sodium chloride solution at a flow rate of 1.2 mL / min. Collect the elution peak with disinfectant activity. Gel filtration chromatography: The collected elution peak was loaded onto a Sephadex G-30 gel column and eluted with 0.12 M phosphate buffer (pH 7.2) to collect the target small molecule peptide component; Freeze drying: The collected small molecule peptide components are freeze-dried to obtain small molecule peptide dry powder.

4. The method for extracting a small molecule peptide combination according to claim 3, characterized in that: In the macroporous resin adsorption separation step, the flow rate of the concentrated solution through the D101 macroporous adsorption resin column is 2.5 mL / min.

5. The method for extracting a small molecule peptide combination according to claim 3, characterized in that: In the ultrafiltration purification step, the ultrafiltration pressure is controlled at 0.12-0.25 MPa.

6. The method for extracting a small molecule peptide combination according to claim 3, characterized in that: In the ion exchange chromatography step, when gradient elution with sodium chloride solution is performed, the eluent flow rate is 1.2 mL / min.

7. The method for extracting a small molecule peptide combination according to claim 3, characterized in that: In the gel filtration chromatography step, the eluent flow rate is 0.6 mL / min.

8. The method for extracting a small molecule peptide combination according to claim 3, characterized in that: In the freeze-drying step, the freezing temperature is controlled below -55°C and the vacuum degree is below 15Pa.

9. A sanitary disinfection product, characterized in that: Comprising the small molecule peptide combination according to claim 1.

10. The sanitary disinfection product according to claim 9, characterized in that: The sanitary disinfection product is a sanitary disinfection spray, sanitary disinfection wet wipes or sanitary disinfection hand soap.