Antibacterial bioactive peptide as well as preparation method and application thereof

Abstract

The invention discloses an antibacterial bioactive peptide as well as a preparation method and application thereof, and belongs to the technical field of biology. The sea cucumber antibacterial bioactive peptide is prepared from sea cucumbers, and the amino acid sequence of the sea cucumber antibacterial bioactive peptide is shown as SEQ ID NO.1. Through detection, the bioactive peptide has a remarkable inhibiting effect on growth of gram-positive bacteria staphylococcus aureus and gram-negative bacteria escherichia coli, the minimum inhibitory concentration (MIC) value is low, and a strong antibacterial effect is shown; the method has a wide application prospect in preparation of products with an antibacterial effect, such as food, medicines, health care products, cosmetics or animal feed.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an antibacterial bioactive peptide and a preparation method and application thereof. Background Art

[0002] Antibiotics have long been the primary means of preventing and treating bacterial diseases. However, the surge in drug-resistant strains caused by overuse of antibiotics has created a severe global public health crisis, necessitating the development of new antimicrobial drugs. Antimicrobial peptides are a class of natural antimicrobial substances found widely in organisms. They possess broad-spectrum antimicrobial activity, are less susceptible to developing drug resistance, and possess structural diversity, offering broad application prospects.

[0003] Marine bioactive peptides are peptide compounds extracted, isolated, or obtained through bioengineering techniques from marine organisms. These peptides, typically composed of a few to dozens of amino acids and possessing specific physiological activities, have attracted considerable attention due to their diverse sources, strong specificity, and low toxicity. Bioactive peptides derived from sea cucumbers have demonstrated remarkable properties, including lowering blood pressure, combating fatigue, enhancing immunity, combating tumors, and delaying aging. However, limited research has linked sea cucumber peptides to their antimicrobial effects, leading to a lack of clarity regarding the correlation between product function and composition. This has made it difficult to establish scientifically-based product quality standards, further limiting further research and product development. Summary of the Invention

[0004] The present invention aims to provide an antimicrobial bioactive peptide, its preparation method, and its application to address the problems of the prior art described above. The present invention prepares an antimicrobial bioactive peptide from sea cucumbers, the amino acid sequence of which is shown in SEQ ID NO. 1. Testing has shown that the bioactive peptide exhibits significant inhibitory effects on the growth of both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, with low minimum inhibitory concentration (MIC) values, demonstrating a strong antimicrobial effect. The peptide has broad application prospects in the preparation of antimicrobial products such as food, medicine, health products, cosmetics, or animal feed.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an antibacterial bioactive peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a method for preparing the above-mentioned antibacterial bioactive peptide, comprising the following steps:

[0008] S1, mixing sea cucumber powder with water and ultrasonically treating;

[0009] S2, enzymatically treating the mixture after ultrasonic treatment with pepsin, papain and alkaline protease in sequence, and collecting the enzymatic supernatant;

[0010] S3. Treating the enzymatic hydrolysis supernatant with ethanol, ultrafiltration, and further separation by silica gel column chromatography and high performance liquid chromatography to obtain the antibacterial bioactive peptide.

[0011] Optionally, the solid-liquid ratio of the sea cucumber powder to water is 1 g:6-10 mL; the power of the ultrasonic treatment is 200-400 W, and the time is 15-30 min.

[0012] Optionally, the pH of the pepsin hydrolysis is 2.0, the temperature is 40° C., and the time is 4-6 hours; the final concentration of the pepsin is 3-5 kU / mL;

[0013] The pH of the papain hydrolysis is 7.0, the temperature is 40° C., and the time is 4-6 hours; the final concentration of the papain is 3-5 kU / mL;

[0014] The pH value of the alkaline protease during enzymatic hydrolysis is 8.0, the temperature is 40° C., and the time is 4-6 hours; the final concentration of the alkaline protease is 3-5 kU / mL.

[0015] Optionally, the ethanol extraction method is to mix anhydrous ethanol and enzymatic supernatant in a volume ratio of 2-4:1, and extract at 4°C for 10-16 hours; the molecular retention capacity of the ultrafiltration is 3kDa.

[0016] Optionally, the chromatographic column for the high performance liquid chromatography separation is C18; the mobile phase A is 0.1% trifluoroacetic acid-acetonitrile, and the mobile phase B is 0.1% trifluoroacetic acid-water; the elution gradient is 0→5 min, 5% A+95% B (v / v), 5→20 min, 50% A+50% B (v / v), 20→30 min, 90% A+10% B (v / v); the flow rate is 1 mL / min; and the detection wavelength is 214 nm.

[0017] The present invention also provides the use of the above antibacterial bioactive peptide in the preparation of antibacterial products.

[0018] Furthermore, the antibacterial product has the effect of inhibiting the growth of Staphylococcus aureus and Escherichia coli.

[0019] The present invention also provides an antibacterial product, wherein the effective ingredient of the antibacterial product includes the above-mentioned antibacterial bioactive peptide.

[0020] Furthermore, the antibacterial product has the effect of inhibiting the growth of Staphylococcus aureus and Escherichia coli.

[0021] The present invention discloses the following technical effects:

[0022] The present invention prepares an antibacterial bioactive peptide from sea cucumbers through pretreatment, combined enzymatic hydrolysis, separation and purification, and other methods. The amino acid sequence of the peptide is shown in SEQ ID NO. 1. Testing has shown that the bioactive peptide prepared in the present invention has a significant inhibitory effect on the growth of both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, with low minimum inhibitory concentration (MIC) values, demonstrating a strong antibacterial effect. The bioactive peptide of the present invention has broad application prospects in the preparation of products with antibacterial effects, such as food, medicine, health products, cosmetics, or animal feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 is a high performance liquid chromatogram of the bioactive peptide component of the present invention;

[0025] Figure 2 is the infrared spectrum of the bioactive peptide of the present invention;

[0026] Figure 3 This is a mass spectrometry detection diagram of the bioactive peptide of the present invention. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] This embodiment provides a method for preparing a bioactive peptide with antibacterial efficacy, comprising the following steps:

[0034] S1, clean the fresh sea cucumber from mud and sand, remove the internal organs, wash, cut into small pieces, treat with liquid nitrogen, and quickly grind into powder to obtain a pre-treated material;

[0035] S2. The pre-treated material was suspended in distilled water at a solid-liquid ratio of 1 g:8 mL, and ultrasonicated at 300 W for 20 min in an ultrasonic instrument. The pH was adjusted to 2.0 with 1 mol / L HCl, pepsin (enzyme concentration of 4 kU / mL) was added, and enzymolysis was carried out at 40 ° C for 5 h; the pH was adjusted to 7.0 with 1 mol / L NaOH, papain (enzyme concentration of 4 kU / mL) was added, and enzymolysis was carried out at 40 ° C for 5 h; the pH was adjusted to 8.0 with 1 mol / L NaOH, alkaline protease (enzyme concentration of 4 kU / mL) was added, and enzymolysis was carried out at 40 ° C for 5 h; the temperature was raised to 95 ° C for 15 min to inactivate the enzyme activity, cooled, and centrifuged at 12000g at 4 ° C for 20 min to obtain the enzymatic supernatant;

[0036] S3. Add anhydrous ethanol to the enzymatic supernatant (the volume ratio of anhydrous ethanol to enzymatic supernatant is 3:1) and store at 4°C for 12 h; centrifuge at 12000g for 20 min at 4°C, take the supernatant and ultrafilter (ultrafiltration membrane molecular cutoff capacity 3 kDa), concentrate by rotary evaporation, and perform initial separation by silica gel column chromatography and then further separation by high performance liquid chromatography. The HPLC conditions were as follows: chromatographic column: Kromasil C18 (4.6×150 mm, 5 μm); mobile phase: A: acetonitrile (containing 0.1% (v / v) trifluoroacetic acid), B: water (containing 0.1% (v / v) trifluoroacetic acid); elution gradient: 0→5 min, 5% A+95% B (v / v); 5→20 min, 50% A+50% B (v / v); 20→30 min, 90% A+10% B (v / v); flow rate: 1 mL / min; detection wavelength: 214 nm; sample load: 20 μL. The chromatographic peaks obtained after elution were as follows: Figure 1 As shown, the main chromatographic peak (retention time 19.82 min) was collected and freeze-dried to obtain a bioactive peptide with antibacterial effect. Amino acid sequence analysis was performed using an amino acid automatic analyzer to obtain the amino acid sequence of the bioactive peptide as shown in SEQ ID NO.1.

[0037] SEQ ID NO. 1: Gln Phe Leu Arg Val Ser Gly Pro Leu Leu Lys Tyr Pro ValVal Gly Pro Gln (QFLRVSGPLLKYPVVGPQ).

[0038] Example 2

[0039] This example performs the following tests on the bioactive peptides of Example 1:

[0040] 1. Fourier transform infrared (FT-IR) spectroscopy

[0041] Infrared spectroscopy is sensitive to the chemical structure of molecules and is suitable for the determination of proteins and peptides in different states, concentrations, and environments. It is a useful tool for determining the structure of proteins and peptides. In this example, the infrared spectrum of the sample was collected using a KBr pellet method using a Fourier transform infrared (FT-IR) spectrometer (Bruker ALPHA, Germany). Test conditions: wave number range 4000-400 cm -1 , scan times 32, resolution 0.5cm -1 .

[0042] The results are as follows Figure 2 As shown, 3303cm -1 and 3061cm -1 The obvious absorption peaks at 2964 and 2931 cm are typical characteristic peaks of amide A and B.-1 The absorption band at is attributed to the CH stretching vibration of the saturated carbon chain (methyl / methylene, -CH3 / -CH2-). The characteristic absorption peaks of peptide amide I, II and III bands are located at 1651, 1534 and 1246 cm -1 1450 and 1393cm -1 The absorption bands at are attributed to the CH bending vibrations of saturated carbon chains (methyl / methylene, -CH3 / -CH2-).

[0043] 2. Mass spectrometry identification

[0044] The mass spectrometry of bioactive peptide samples was tested using the MS (ESI) method. The results are shown in Figure 3 The base peak in the mass spectrum of the active peptide is m / z 1998.44 (M+), which is similar to the active peptide (C 95 H 152 N 24 O 23 ) was consistent with the calculated molecular weight value of 1998.37. Therefore, the amino acid sequence structure of the active peptide was further confirmed.

[0045] 3. Minimum inhibitory concentration determination

[0046] The minimum inhibitory concentration (MIC) of the bioactive peptides was tested by the micro-two-fold gradient dilution method. The Gram-positive bacteria Staphylococcus aureus (E. coli) and Gram-negative bacteria Escherichia coli (S. aureus) were used as experimental strains. The active peptide solution was serially diluted in a 96-well plate and the bacterial solution (the final concentration of the bacterial solution was 5×10 4 The minimum inhibitory concentration (MIC) was determined by observing bacterial growth for 20 hours. The results showed that the MIC values ​​of the bioactive peptide against Staphylococcus aureus (E. coli) and Escherichia coli (S. aureus) were 64 μg / mL and 128 μg / mL, respectively, demonstrating strong antibacterial effects.

[0047] 4. Cytotoxicity test

[0048] The cytotoxicity of the samples was tested using the CCK-8 method. Human immortalized keratinocytes (HaCaT) in the logarithmic growth phase were taken and diluted to 1×10 5 Cells were plated at 100 μL / well in a 96-well culture plate (100 μL per well) and cultured overnight in an incubator. 100 μL of DMEM culture medium containing different sample concentrations was added, and a control group containing complete culture medium without sample was used. Cultures were continued for 24 or 48 hours. The culture medium was removed, washed with PBS, and culture medium containing 10% CCK-8 was added at 100 μL / well. Cultures were continued for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated as follows.

[0049] Cell survival rate = (absorbance of experimental well - absorbance of blank well) / (absorbance of control well - absorbance of blank well) × 100%

[0050] The results of the cytotoxicity test are shown in Table 1. The experimental results show that the bioactive peptide did not show cytotoxicity when treated with HaCaT cells for 24 hours and 48 hours within the test concentration range (25-800 μg / mL), indicating that the bioactive peptide can be used to prepare antibacterial products in the fields of food, medicine, health products, cosmetics or animal feed, has good safety and good application prospects.

[0051] Table 1 Effects of active peptide samples on HaCaT cell viability (n=5)

[0052]

[0053] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An antimicrobial bioactive peptide, characterized in that: The amino acid sequence of the antibacterial bioactive peptide is shown in SEQ ID NO.

1.

2. The method for preparing the antimicrobial bioactive peptide according to claim 1, characterized in that: The steps include: S1, mixing sea cucumber powder with water and ultrasonically treating; S2, enzymatically treating the mixture after ultrasonic treatment with pepsin, papain and alkaline protease in sequence, and collecting the enzymatic supernatant; S3. Treating the enzymatic hydrolysis supernatant with ethanol, ultrafiltration, and further separation by silica gel column chromatography and high performance liquid chromatography to obtain the antibacterial bioactive peptide.

3. The preparation method according to claim 2, characterized in that The solid-liquid ratio of the sea cucumber powder to water is 1g:6-10mL; the power of the ultrasonic treatment is 200-400W, and the time is 15-30min.

4. The preparation method according to claim 2, characterized in that The pH of the pepsin hydrolysis is 2.0, the temperature is 40° C., and the time is 4-6 hours; the final concentration of the pepsin is 3-5 kU / mL; The pH of the papain hydrolysis is 7.0, the temperature is 40°C, and the time is 4-6 hours; the final concentration of the papain is 3-5 kU / mL; The pH value of the alkaline protease during enzymatic hydrolysis is 8.0, the temperature is 40° C., and the time is 4-6 hours; the final concentration of the alkaline protease is 3-5 kU / mL.

5. The preparation method according to claim 2, characterized in that The ethanol extraction method comprises mixing anhydrous ethanol and enzymatic supernatant in a volume ratio of 2-4:1, and extracting at 4° C. for 10-16 hours; and the molecular retention capacity of the ultrafiltration is 3 kDa.

6. The preparation method according to claim 2, characterized in that The chromatographic column for the high performance liquid chromatography separation is C18; the mobile phase A is 0.1% trifluoroacetic acid-acetonitrile, and the mobile phase B is 0.1% trifluoroacetic acid-water; the elution gradient is 0→5 min, 5% A+95% B (v / v), 5→20 min, 50% A+50% B (v / v), 20→30 min, 90% A+10% B (v / v); the flow rate is 1 mL / min; and the detection wavelength is 214 nm.

7. Use of the antibacterial bioactive peptide according to claim 1 in the preparation of antibacterial products.

8. The use according to claim 7, characterized in that The antibacterial product has the effect of inhibiting the growth of Staphylococcus aureus and Escherichia coli.

9. An antibacterial product, characterized in that: The active ingredient of the antibacterial product includes the antibacterial bioactive peptide according to claim 1.

10. The antibacterial product according to claim 9, characterized in that: The antibacterial product has the effect of inhibiting the growth of Staphylococcus aureus and Escherichia coli.

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