A polypeptide composition and its use

Abstract

The present invention relates to the field of biotechnology, and in particular to a polypeptide composition and its use, wherein the composition is composed of polypeptide and low-concentration ethanol as main components, and adjuvants or auxiliary components. The present invention also discloses the use of the composition in microorganisms, and the composition has significant antibacterial and antibacterial effects. Compared with the prior art, the polypeptide and low-concentration ethanol can act synergistically after compounding, and while reducing the use concentration of each, the antibacterial effect thereof is not reduced, and the composition has high safety, no hormones, and no antibiotics. At the same time, the composition of the polypeptide and low-concentration ethanol of the present invention can effectively reduce the use concentration of the polypeptide, save costs, and the low-concentration ethanol is also easy to transport, and has good market application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polypeptide composition and use thereof. Background Art

[0002] The demand for disinfection products is growing day by day. Most of the common disinfection products on the market are chemical products. While disinfecting and sterilizing, they also bring some adverse effects such as irritation to the respiratory system, irritation to the skin, mixed production of harmful substances, production of resistant bacteria, etc. Among them, ethanol is the most common, widely used, efficient and fast broad-spectrum bactericide.

[0003] The antibacterial effect of ethanol is non-specific. It mainly denatures and coagulates proteins, dissolves cells, and thus destroys cell metabolism, making it impossible for microorganisms to survive. Disinfectants with ethanol as the main ingredient on the market include Dr. Xiao skin disinfectant, Purell hand disinfectant, Dettol wash-free antibacterial hand sanitizer, etc. Although ethanol is relatively less irritating, since ethanol evaporates and takes away moisture from the skin surface, frequent use of ethanol disinfectants can cause dry skin (Zhang Qiuping. Preparation and Properties of Chitosan / Ethanol Disinfection Gel [D]. Jilin University, 2018.), cracks, decreased skin resistance, epidermal keratinization, etc. Although the product experience can be improved by adding skin care ingredients, long-term use still has a strong negative effect on skin cells.

[0004] Ethanol is widely used as a disinfectant for the disinfection of skin and mucous membranes, as well as the surfaces of medical devices and precision instruments. It is also widely used as a compound component of various skin and mucous membrane disinfectants. The Chinese Pharmacopoeia recommends the use of ethanol as a disinfectant at a concentration of 70% to 75% by volume. The quantitative bactericidal effect of ethanol solutions with a concentration of 40% or less does not meet the standard requirements (Lei Yi, Jiang Lijuan, Chen Baobao, Zhang Zhicheng, Kou Jingyuan, Zhang Xiaoling. Observation on the bactericidal effect of ethanol solutions of different concentrations [J]. Chinese Journal of Disinfection, 2020, 37(09): 656-658.). Studies have shown that when the ethanol concentration is between 60% and 95%, the antibacterial ability is optimal (Foddai ACG, Grant IR, Dean M. Efficacy of Instant Hand Sanitizers against Foodborne Pathogens Compared with Hand Washing with Soap and Water in Food Preparation Settings: A Systematic Review [J]. Journal of food If the concentration is higher or lower than this range, the disinfection effect cannot be achieved. If the ethanol concentration is too high, the bacterial surface protein will coagulate quickly, which will affect the continued penetration of ethanol into the bacteria, thus affecting the disinfection effect. If the ethanol concentration is too low, although ethanol can enter the bacteria, it cannot coagulate the bacterial protein and affect the disinfection effect, and may even only play an antibacterial role (Wang Hong, Liao Xinjuan, Yan Dongzhen, Xu Xin, Liu Ju. Study on the disinfection effect and stability of 75% ethanol [J]. International Journal of Biologicals, 2013, 36 (04): 182-184.).

[0005] It can be seen that the main concentration of ethanol as a disinfectant component of disinfection products is high-concentration ethanol greater than 60%, while low-concentration ethanol is generally not used as a separate active ingredient in disinfection products due to its poor bactericidal effect. It is generally used for organic phase in liquid-liquid extraction technology (Lei Zhigang, Wen Cuiping, Li Qunsheng, Zhu Jiqin, Wu Xiaohua, Zhao Yujing. Application of liquid-liquid extraction technology in pretreatment of low-concentration ethanol aqueous solution samples [J]. Modern Industry, 2011, 31(02): 53-5.), catalytic hydrogen production, catalytic dehydration to produce ethylene and other chemical development (Chen Yu, Wu Yulong, Dai Bin, et al. Research progress in the catalytic preparation of downstream chemicals with low-concentration ethanol [J]. Chemical Industry Progress, 2009, 028(010): 1742-1749.), wiping the skin to lower body temperature, etc.

[0006] According to the provisions of GB12268-2012 Dangerous Goods List, ethanol concentration exceeding 24% is considered dangerous goods, which poses certain difficulties in logistics and transportation. At the same time, ethanol has the best antibacterial effect at a concentration of 75%, and low concentrations of ethanol have almost no antibacterial effect. This poses a huge challenge to alcohol disinfectants, so it is urgent to appropriately reduce the ethanol concentration and ensure its antibacterial effect.

[0007] On the other hand, with the growing demand for disinfection products, people have been looking for and trying safer, more environmentally friendly and effective ingredients in addition to traditional antibacterial ingredients. As a new type of bioactive ingredient, polypeptides have broad-spectrum antibacterial properties, inhibiting or killing bacteria, fungi, viruses, parasites, etc., and are mild and non-irritating, and have incomparable advantages in drug resistance. Polypeptides can interact with microbial cell membranes to form holes, affect the osmotic pressure inside and outside the cells, and then cause cell death. Since the cell membrane structure of microorganisms has evolved over hundreds of millions of years, its structure will not change significantly in a short period of time, so this antibacterial mechanism of polypeptides is not easy to make bacteria resistant. Therefore, polypeptide peptides are also increasingly favored as the main active ingredient of disinfection products.

[0008] However, since the polypeptides induced in vivo have problems such as low yield and high technical requirements; and artificially synthesized polypeptides have low purity and unsatisfactory effects, the production cost of polypeptides is relatively high. The current market price ranges from 1,000 yuan / g to 2,000 yuan / g, which significantly increases the production cost of polypeptide substances, and further makes the price of polypeptide disinfection products more expensive than ordinary compound disinfection products, which is not conducive to market promotion and increase in market share.

[0009] Based on the above problems, some literature reports that low-concentration ethanol has a synergistic effect when used in combination with other antimicrobial ingredients. For example, Drake David R et al. disclosed that the combined use of ethanol and saponin acid has a synergistic effect. The two alone have no antibacterial effect on MRSA, but the combination of the two can achieve a complete killing effect (Drake David R, Brogden Kim A, Dawson Deborah V, Wertz Philip W. Thematic review series: skinlipids. Antimicrobial lipids at the skin surface. [J]. Journal of lipidresearch, 2008, 49 (1).).

[0010] However, this document only discloses the synergistic effect of two antibacterial ingredients on a certain bacterium, and does not mean that the combined use of the two antibacterial substances has a synergistic effect on all bacteria. For example, Theansungnoen T et al. disclosed that the combination of lysozyme and crocodile leukocyte extract had no synergistic effect on Bifidobacterium breve, but had a good synergistic effect on Vibrio cholerae (Theansungnoen T, Tastub P, Jangpromma N, et al. Antimicrobial efficacy of a combination of crocodile (Crocodylus siamensis) leukocyte extract and hen egg lysozyme [J]. Chiang Mai Journal of Science, 2018, 45 (2): 797-810.); Cao, Y et al. disclosed that the combination of antimicrobial peptide ZXR-2.3 and antimicrobial peptide KT2 had no synergistic effect on Streptococcus mutans, while the combination of antimicrobial peptide ZXR-2.3 and broad-spectrum anti-biofilm peptide IDR-1018 had a synergistic effect on Streptococcus mutans (Cao, Y., Yin, H., Wang, W. et al. Killing Streptococcus mutans in mature biofilm with a combination ofantimicrobial and antibiofilm peptides[J].Amino Acids,2020,52(1):1-14.).

[0011] In summary, not all two antibacterial substances have a synergistic effect when used in combination, and not all two antibacterial substances have a synergistic effect on all bacteria when used in combination.

[0012] The inventors unexpectedly discovered during their research that low-concentration ethanol mixed with the polypeptide of the present invention can effectively reduce the concentration of the polypeptide and ethanol while not reducing the antibacterial effect, and has a synergistic effect on microorganisms. It is also easy to transport, saves costs, and has good market application prospects. Summary of the invention

[0013] In view of the deficiencies in the prior art, one object of the present invention is to provide a polypeptide composition, and a second object is to provide a use of the polypeptide composition, characterized in that the composition comprises a polypeptide with an amino acid sequence of SEQ ID NO.1 and low-concentration ethanol.

[0014] The amino acid sequence of the polypeptide of the present invention is SEQ ID: NO.1.

[0015] The ethanol described in the present invention is low-concentration ethanol.

[0016] Preferably, the ethanol concentration is ≤24%.

[0017] Preferably, the composition further comprises other auxiliary materials, carriers or auxiliary ingredients.

[0018] Preferably, the polypeptide composition is used in disinfectants, antibacterial products and cosmetics.

[0019] The present invention provides a polypeptide composition and use thereof. The polypeptide of the present invention can be compounded with low-concentration ethanol to have a synergistic effect. After the two are compounded, they also have a certain killing effect on bacteria, especially spores, and the respective use concentrations are reduced without reducing the antibacterial effect. The composition is highly safe and contains no hormones or antibiotics. Meanwhile, in actual production, the production cost of polypeptides is relatively high, resulting in a high selling price for polypeptide products. The composition of the polypeptide of the present invention and low-concentration ethanol can effectively reduce the use concentration of the polypeptide and save costs. The low-concentration ethanol is also convenient for transportation, and has broad commercial application prospects. DETAILED DESCRIPTION

[0020] One object of the present invention is to provide a polypeptide composition, and the second object is to provide a use of the polypeptide composition, characterized in that the composition comprises a polypeptide with an amino acid sequence of SEQ ID NO.1 and low-concentration ethanol.

[0021] The amino acid sequence of the polypeptide of the present invention is SEQ ID: NO.1.

[0022] The ethanol described in the present invention is low-concentration ethanol.

[0023] Preferably, the ethanol concentration is ≤24%.

[0024] Preferably, the composition further comprises other auxiliary materials, carriers or auxiliary ingredients.

[0025] The other excipients, carriers or auxiliary ingredients described in the present invention may contain formulation materials for changing, maintaining or preserving, for example, the pH, osmotic volume, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissociation or release rate, adsorption or penetration of the composition.

[0026] Other auxiliary materials, carriers or auxiliary components are not listed one by one, and ordinary technicians in this field can make specific selections based on common knowledge.

[0027] Preferably, the primary vehicle or carrier of the composition may be aqueous or non-aqueous in nature, such as a suitable vehicle or carrier may be purified water.

[0028] A person skilled in the art can select suitable excipients according to the liquid dosage form to be prepared, according to the general technical knowledge and requirements of the dosage form in the art, add appropriate excipients and additives on the basis of the polypeptide, and prepare it according to conventional formulation technology.

[0029] The preparation of the present invention can be prepared by any suitable method known in the art, and can be deleted or adjusted according to the requirements of sterilization.

[0030] The preferred concentration for a particular situation can be determined routinely by one skilled in the art.

[0031] Preferably, the polypeptide composition is used in disinfectants, antibacterial products and cosmetics.

[0032] The polypeptide of the present invention can be obtained by chemical synthesis, expression, separation and purification through genetic engineering technology (for specific methods, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0033] As used in the present invention, the terms "preparation", "polypeptide composition" and "composition" are interchangeable unless otherwise specified, and refer to a preparation containing two active ingredients, polypeptide and low-concentration ethanol, and may also contain other excipients, carriers or auxiliary ingredients.

[0034] As used in the present invention, the term "other excipients, carriers or auxiliary ingredients" refers to non-toxic liquid fillers, diluents or other formulation excipients and carriers, and the composition can be made into various dosage forms according to actual needs.

[0035] The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the disclosure which is defined only by the claims and the specification.

[0036] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. In the examples, the reagents added are all commercially available unless otherwise specified.

[0037] The embodiments of this part further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention.

[0038] In order to illustrate that the polypeptide of the present invention has a synergistic effect on microorganisms when used in combination with low-concentration ethanol, Staphylococcus aureus, a representative of Gram-positive bacteria, Escherichia coli, a representative of Gram-negative bacteria, and Bacillus subtilis with spores were selected for antibacterial performance tests.

[0039] Example 1 Anti-Escherichia coli performance test of polypeptide and ethanol complex

[0040] The fractional inhibitory concentration (FIC) index is one of the pharmacodynamic (PD) parameters of antibiotics. It is the combined drug sensitivity of two antibiotics, that is, when two antibiotics are used simultaneously, four situations may occur: synergy, antagonism, irrelevance, and addition.

[0041] Calculation of FIC index: FIC index = MIC (group A combined) / MIC (group A alone) + MIC (group B combined) / MIC (group B alone).

[0042] FIC index interpretation criteria: when the FIC index is ≤0.5, the two drugs have a synergistic effect; when the FIC index is 0.5-1, the two drugs have an additive effect; when the FIC index is 1-2, the two drugs have an unrelated effect; when the FIC index is >2, the two drugs have an antagonistic effect.

[0043] Since both peptides and ethanol are antibacterial substances, the FIC index was selected to evaluate the effect of their combination.

[0044] Perform operations according to the provisions of 2.1.8.4 of the Disinfection Technical Specifications (2002).

[0045] Preparation of bacterial suspension: Take the activated bacterial suspension, adjust the OD value, and prepare the initial bacterial suspension. The OD value of the bacterial suspension of Escherichia coli and Staphylococcus aureus is 600 is 0.8, which corresponds to a bacterial solution concentration of about 10 8 cfu / mL, and then diluted with (2X) LB medium to a concentration of 5.0×10 5 -5.0×10 6 cfu / mL as the test bacterial suspension.

[0046] The polypeptide and ethanol were diluted with sterile ultrapure water to different concentrations of test solution, 50 μL of each was added to a 96-well plate, and then 100 μL of the diluted bacterial suspension was added.

[0047] The polypeptide was diluted with sterile ultrapure water to a concentration of 0 mg / L, 1.25 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, and 40 mg / L, and ethanol was diluted with sterile ultrapure water to a concentration of 0%, 1%, 3%, 6%, 9%, 12%, 15%, 18%, 21%, and 24%. 50 μL of the above polypeptides and ethanol concentrates of different concentrations were taken and placed in a 96-well plate, and 100 μL of Escherichia coli suspension was added to each well, and positive and negative controls were set up, wherein the positive control was 100 μL sterile water + 100 μL bacterial suspension; the negative control was 100 μL sterile water + 100 μL sterile (2X) LB medium. Two parallel experiments were set up, named group 1 and group 2.

[0048] Place the 96-well plate in a 37±2℃ incubator, incubate for 48h, and observe the results.

[0049] When the positive control has bacterial growth (turbidity) and the negative control has no bacterial growth (transparent), the concentration of the test bacterial suspension is 5×10 5 -5×10 6 cfu / mL, the concentration of antibacterial (inhibitory) agent corresponding to the highest dilution of sterile growth in the test group is the MIC of the sample against the test bacteria.

[0050] According to the results of 96-well plate, the specific values ​​are shown in Table 1.

[0051] Table 1 Determination of MIC value and FIC index of peptide-ethanol complex against Escherichia coli

[0052]

[0053] According to the FIC index calculation formula, the FIC value of group 1 is 0.46, and the FIC value of group 2 is 0.46. Therefore, the FIC index of ethanol and polypeptide combined with Escherichia coli is less than 0.5, and the two have a synergistic effect.

[0054] Example 2 Anti-Staphylococcus aureus performance test of polypeptide and ethanol compound

[0055] The polypeptide was diluted with sterile ultrapure water to a concentration of 0 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, and 300 mg / L, and ethanol was diluted with sterile ultrapure water to a concentration of 0%, 1%, 3%, 6%, 9%, 12%, 15%, 18%, 21%, and 24%. 50 μL of the above polypeptides and ethanol concentrates of different concentrations were placed in a 96-well plate, and 100 μL of Escherichia coli suspension was added to each well, and positive and negative controls were set, wherein the positive control was 100 μL sterile water + 100 μL bacterial suspension; the negative control was 100 μL sterile water + 100 μL sterile (2X) LB medium. Two parallel experiments were set up, named group 3 and group 4.

[0056] Place the 96-well plate in a 37±2℃ incubator, incubate for 48h, and observe the results.

[0057] When the positive control has bacterial growth (turbidity) and the negative control has no bacterial growth (transparent), the concentration of the test bacterial suspension is 5×10 5 -5×10 6 cfu / mL, the concentration of antibacterial (inhibitory) agent corresponding to the highest dilution of sterile growth in the test group is the MIC of the sample against the test bacteria.

[0058] According to the results of 96-well plate, the specific values ​​are shown in Table 2.

[0059] Table 2 Determination of MIC value and FIC index of peptide-ethanol complex against Staphylococcus aureus

[0060]

[0061] According to the FIC index calculation formula, the FIC value of group 3 is 0.39, and the FIC value of group 4 is 0.27. Therefore, the FIC index of ethanol and polypeptide combined with Staphylococcus aureus is less than 0.5, and the two have a synergistic effect.

[0062] Example 3 Anti-Bacillus subtilis performance test of polypeptide and ethanol compound

[0063] Perform operations according to the provisions of 2.1.8.4 of the Disinfection Technical Specifications (2002).

[0064] Preparation of bacterial suspension: Take the activated bacterial suspension and adjust the OD value to make the initial bacterial suspension so that the OD600 of the Bacillus subtilis bacterial suspension is 0.8, which corresponds to a bacterial suspension concentration of about 10 8 cfu / mL, and then diluted with (2X) LB medium to a concentration of 5.0× 10 5 -5.0×10 6 cfu / mL as the test bacterial suspension.

[0065] The polypeptide and ethanol were diluted with sterile ultrapure water to different concentrations of test solution, 50 μL of each was added to a 96-well plate, and then 100 μL of the diluted bacterial suspension was added.

[0066] The polypeptide was diluted with sterile ultrapure water to a concentration of 0 mg / L, 0.2 mg / L, 0.39 mg / L, 0.78 mg / L, 1.56 mg / L, 3.13 mg / L, 6.25 mg / L, 12.5 mg / L, 25 mg / L, and 50 mg / L, and ethanol was diluted with sterile ultrapure water to a concentration of 0%, 5%, 10%, 15%, 20%, and 24%. 50 μL of the above polypeptides and ethanol concentrates of different concentrations were taken and placed in a 96-well plate, and 100 μL of Escherichia coli suspension was added to each well, and positive and negative controls were set up, wherein the positive control was 100 μL sterile water + 100 μL bacterial suspension; the negative control was 100 μL sterile water + 100 μL sterile (2X) LB medium. Two parallel experiments were set up, named group 5 and group 6.

[0067] Place the 96-well plate in a 37±2℃ incubator, incubate for 48h, and observe the results.

[0068] When the positive control has bacterial growth (turbidity) and the negative control has no bacterial growth (transparent), the concentration of the test bacterial suspension is 5×10 5 -5×10 6 cfu / mL, the concentration of antibacterial (inhibitory) agent corresponding to the highest dilution of sterile growth in the test group is the MIC of the sample against the test bacteria.

[0069] Since ethanol alone has no killing effect on Bacillus subtilis, it is impossible to make a judgment using the FIC index.

[0070] According to the results of 96-well plate, the specific values ​​are shown in Table 3.

[0071] Table 3 Determination of MIC values ​​of peptides and ethanol complexes against Bacillus subtilis

[0072]

[0073] According to Table 3, the MIC value of the polypeptide of group 5 was reduced from the original 3.13 mg / L to 0.2 mg / L after compounding with ethanol, and the ethanol concentration was changed from low concentration (≤24%) without antibacterial effect to 10% concentration; the MIC value of the polypeptide of group 6 was reduced from the original 3.13 mg / L to 0.2 mg / L after compounding with ethanol, and the ethanol concentration was changed from low concentration (≤24%) without antibacterial effect to 5%. The compounding of the two can effectively reduce the use concentration of polypeptide and low concentration ethanol in inhibiting Bacillus subtilis, and at the same time reduce the use concentration of ethanol on Bacillus subtilis.

[0074] The polypeptide of the present invention is composed of 19 amino acids, is non-toxic and non-irritating, and its degradation products are natural amino acids, without producing drug residues. At the same time, since the bactericidal mechanism of the polypeptide is that it carries a strong positive charge and is adsorbed on the surface of the bacteria, the local potential of the cell wall changes suddenly to form a high potential difference, and finally the cell wall is broken down to form a perforation. When the polypeptide molecules accumulate to a certain number, the holes that have been formed are further enlarged, and the integrity of the cell membrane is destroyed, so that the intracellular substances flow out and the bacteria die. Its unique bactericidal mechanism determines that the polypeptide is not easy to produce drug resistance, and it has no biological toxicity and no irritation.

[0075] Taking all factors into consideration, after the polypeptide of the present invention is compounded with low-concentration ethanol, the antibacterial concentration of the polypeptide is effectively reduced, which saves production costs without reducing its antibacterial effect. At the same time, low-concentration ethanol is also convenient for transportation. No harmful substances are produced after the polypeptide composition is combined. Therefore, the polypeptide composition has a good advantage in disinfection products and has broad application prospects.

[0076] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention. Sequence Listing <110> Jiangsu Jirui Biotechnology Co., Ltd. <120> A polypeptide composition and its use <141> 2020-12-31 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Arg Phe Lys Arg Phe Arg Lys Lys Phe Lys Lys Leu Phe Lys 1 5 10 15 Lys Leu Ser

Claims

1. An antibacterial composition, characterized in that The active ingredients of the composition are: a polypeptide with an amino acid sequence of SEQ ID NO.1 and ethanol, wherein the polypeptide is 0.2 mg / L and the volume percentage of ethanol is 5%; or the polypeptide is 0.2 mg / L and the volume percentage of ethanol is 10%; or the polypeptide is 2.5 mg / L and the volume percentage of ethanol is 6%; or the polypeptide is 25 mg / L and the volume percentage of ethanol is 3%.

2. The composition according to claim 1, characterized in that The composition includes an auxiliary material.

3. A use of the composition as claimed in claim 1 or 2, characterized in that The composition is used in preparing disinfectants and cosmetics.

4. A use of the composition as claimed in claim 1 or 2, characterized in that The composition is used in preparing antibacterial products.

Images