An antibacterial bioactive peptide and a preparation method and application thereof

Abstract

The application discloses an antibacterial bioactive peptide and a preparation method and application thereof, and belongs to the technical field of biology. The application prepares a sea cucumber antibacterial bioactive peptide from sea cucumber, and the amino acid sequence of the bioactive peptide is shown as SEQ ID NO. 1. The bioactive peptide has a significant inhibiting effect on the growth of gram-positive bacteria Staphylococcus aureus and gram-negative bacteria Escherichia coli, has a low minimum inhibitory concentration (MIC) value, shows a strong antibacterial effect, and has a wide application prospect in the preparation of products with antibacterial effect, such as food, medicine, health care product, cosmetic or animal feed.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an antibacterial bioactive peptide, its preparation method, and its application. Background Technology

[0002] For a long time, antibiotics have been the primary means of preventing and treating bacterial diseases. However, with the surge in drug-resistant strains due to antibiotic overuse, the world faces a severe public health crisis, urgently requiring the development of new antibacterial drugs. Antimicrobial peptides are a class of naturally occurring antimicrobial substances widely found in organisms. They possess broad-spectrum antibacterial properties, are less prone to inducing drug resistance, and exhibit structural diversity, making them promising for a wide range of applications.

[0003] Marine bioactive peptides refer to peptide compounds extracted and isolated from marine organisms or obtained through bioengineering techniques. These peptides, typically composed of several to dozens of amino acids, possess specific physiological activities and have attracted considerable attention due to their diverse sources, high specificity, and low toxicity. Bioactive peptides from sea cucumbers exhibit numerous remarkable properties, including lowering blood pressure, combating fatigue, enhancing immunity, anti-tumor activity, and delaying aging. However, research on the correlation between sea cucumber peptides and their antibacterial properties is limited, leading to unclear correlations between product functions and components. This makes it difficult to establish scientifically based product quality standards and further restricts in-depth research and product development. Summary of the Invention

[0004] The purpose of this invention is to provide an antimicrobial bioactive peptide, its preparation method, and its application, to solve the problems existing in the prior art. This invention prepares an antimicrobial bioactive peptide from sea cucumber, the amino acid sequence of which is shown in SEQ ID NO. 1. Testing revealed that this bioactive peptide has a significant inhibitory effect on the growth of both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, exhibiting a low minimum inhibitory concentration (MIC) value and demonstrating a strong antimicrobial effect. It has broad application prospects in the preparation of products with antimicrobial activity, such as food, pharmaceuticals, health products, cosmetics, or animal feed.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] The present invention provides an antimicrobial 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 antimicrobial bioactive peptides, comprising the following steps:

[0008] S1. Mix sea cucumber powder with water and then ultrasonically process it.

[0009] S2. The ultrasonically treated mixture is sequentially hydrolyzed by pepsin, papain and alkaline protease, and the hydrolysate is collected.

[0010] S3. The enzymatic hydrolysis supernatant is treated with ethanol, ultrafiltered, and then separated 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 1g:6-10mL; the ultrasonic treatment power is 200-400W, and the time is 15-30min.

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

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

[0014] The alkaline protease was hydrolyzed at a pH of 8.0, a temperature of 40°C, and a time of 4-6 hours; the final concentration of the alkaline protease was 3-5 kU / mL.

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

[0016] Optionally, the chromatographic column used for high-performance liquid chromatography is C18; mobile phase A is 0.1% trifluoroacetic acid-acetonitrile, and 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 application of the above-mentioned antimicrobial bioactive peptides in the preparation of antimicrobial 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] This invention utilizes pretreatment, complex enzymatic hydrolysis, separation, and purification methods to prepare an antibacterial bioactive peptide from sea cucumber, the amino acid sequence of which is shown in SEQ ID NO.1. Testing revealed that the bioactive peptide prepared by this invention exhibits significant inhibitory effects on the growth of both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, with low minimum inhibitory concentrations (MICs), demonstrating strong antibacterial efficacy. This bioactive peptide has broad application prospects in the preparation of antibacterial products such as food, pharmaceuticals, health products, cosmetics, or animal feed. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a high-performance liquid chromatogram of the bioactive peptide components of this invention;

[0025] Figure 2 The infrared spectrum of the bioactive peptide of this invention;

[0026] Figure 3 This is a mass spectrometry diagram of the bioactive peptides of this invention. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0032] Example 1

[0033] This embodiment provides a method for preparing bioactive peptides with antibacterial effects, including the following steps:

[0034] S1. Clean the fresh sea cucumbers of mud and sand, remove the internal organs, wash them clean, cut them into small pieces, treat them with liquid nitrogen, and grind them into powder to obtain the pre-treated material.

[0035] S2. The pretreatment material was suspended in distilled water at a solid-liquid ratio of 1g:8mL. The mixture was ultrasonicated at 300W for 20min. The pH was adjusted to 2.0 with 1mol / L HCl, and pepsin (4kU / mL) was added. The mixture was then hydrolyzed at 40℃ for 5h. The pH was adjusted to 7.0 with 1mol / L NaOH, and papain (4kU / mL) was added. The mixture was then hydrolyzed at 40℃ for 5h. The pH was adjusted to 8.0 with 1mol / L NaOH, and alkaline protease (4kU / mL) was added. The mixture was then hydrolyzed at 40℃ for 5h. The enzyme activity was inactivated by heating to 95℃ for 15min, cooling, and centrifuging at 12000g for 20min at 4℃ to obtain the enzymatic supernatant.

[0036] S3. Add anhydrous ethanol to the enzymatic hydrolysis supernatant (the volume ratio of anhydrous ethanol to enzymatic hydrolysis supernatant is 3:1) and store at 4℃ for 12h; centrifuge at 12000g for 20min at 4℃, take the supernatant for ultrafiltration (ultrafiltration membrane molecular cutoff is 3kDa), concentrate by rotary evaporation, first separate by silica gel column chromatography, and then further separate by high performance liquid chromatography. The high-performance liquid chromatography (HPLC) conditions were as follows: Column: Kromasil C18 (4.6 × 150 mm, 5 μm); Mobile phase: A was acetonitrile (containing 0.1% (v / v) trifluoroacetic acid), B was 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 loading volume: 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, freeze-dried, and the resulting bioactive peptide with antibacterial activity was obtained. Amino acid sequence analysis was performed using an automated amino acid analyzer, and the amino acid sequence of the bioactive peptide is 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] In this embodiment, the bioactive peptides of Example 1 were tested as follows:

[0040] 1. Fourier Transform Infrared (FT-IR) Spectroscopy Analysis

[0041] Infrared spectroscopy is sensitive to the chemical structure of molecules and is suitable for determining proteins and peptides in different states, concentrations, and environments, making it a useful tool for determining protein and peptide structures. This example uses the KBr pellet method, and the infrared spectra of the samples were acquired using a Fourier transform infrared (FT-IR) spectrometer (Bruker ALPHA, Germany). Test conditions: wavenumber range 4000–400 cm⁻¹. -1 32 scans, 0.5cm resolution -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⁻¹ are typical characteristic peaks of amide A and B bands, respectively.-1 The absorption band at this point is attributed to the CH stretching vibration of the saturated carbon chain (methyl / methylene, -CH3 / -CH2-). The characteristic absorption peaks of peptide amide bands I, II, and III are located at 1651, 1534, and 1246 cm⁻¹, respectively. -1 1450 and 1393cm -1 The absorption bands at the locations are attributed to the CH bending vibrations of the saturated carbon chains (methyl / methylene, -CH3 / -CH2-).

[0043] 2. Mass spectrometry identification

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

[0045] 3. Determination of minimum inhibitory concentration

[0046] The minimum inhibitory concentration (MIC) of bioactive peptides was determined using a micro-secondary dilution method. Gram-positive Staphylococcus aureus (E. coli) and Gram-negative Escherichia coli (S. aureus) were used as experimental strains. The bioactive peptide solution was serially diluted twofold in 96-well plates, and bacterial suspension was added (final bacterial concentration 5 × 10⁻⁶). 4 The bioactive peptide was cultured for 20 h at a concentration of CFU / mL, and the minimum inhibitory concentration (MIC) was determined by observing the bacterial growth. The results showed that the MIC values ​​against Staphylococcus aureus (E. coli) and Escherichia coli (S. aureus) were 64 μg / mL and 128 μg / mL, respectively, both exhibiting strong antibacterial effects.

[0047] 4. Cytotoxicity test

[0048] Cytotoxicity of the samples was tested using the CCK-8 assay. Human immortalized keratinocytes (HaCaT) in logarithmic growth phase were taken and diluted to 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated overnight. 100 μL of DMEM medium containing different concentrations of the sample was added to each well, with a control group containing only the complete medium without the sample. Incubation continued for 24 or 48 hours. After removing the medium and washing with PBS, 100 μL of medium containing 10% CCK-8 was added to each well, and incubation continued for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula.

[0049] Cell viability = (Absorbance of experimental wells - Absorbance of blank wells) / (Absorbance of control wells - Absorbance of blank wells) × 100%

[0050] The results of the cytotoxicity experiment are shown in Table 1. The results show that the bioactive peptide did not exhibit cytotoxicity when HaCaT cells were treated with the experimental concentration range (25-800 μg / mL) for 24 h and 48 h. This indicates that the bioactive peptide can be used to prepare antimicrobial products for use in food, pharmaceuticals, health products, cosmetics or animal feed, and has good safety and promising application prospects.

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

[0052]

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

Claims

1. An antimicrobial bioactive peptide, characterized in that, The amino acid sequence of the antimicrobial 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, Includes the following steps: S1. Mix sea cucumber powder with water and then ultrasonically process it. S2. The ultrasonically treated mixture is sequentially hydrolyzed by pepsin, papain and alkaline protease, and the hydrolysate is collected. S3. The enzymatic hydrolysis supernatant is treated with ethanol, ultrafiltered, and then separated 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 ultrasonic treatment power is 200-400W, and the time is 15-30 min.

4. The preparation method according to claim 2, characterized in that, The pepsin hydrolysis was performed at a pH of 2.0, a temperature of 40°C, and a time of 4-6 h; the final concentration of the pepsin was 3-5 kU / mL. The papain was hydrolyzed at a pH of 7.0, a temperature of 40°C, and a time of 4-6 h; the final concentration of the papain was 3-5 kU / mL. The alkaline protease was hydrolyzed at a pH of 8.0, a temperature of 40°C, and a time of 4-6 h; the final concentration of the alkaline protease was 3-5 kU / mL.

5. The preparation method according to claim 2, characterized in that, The ethanol extraction method is as follows: anhydrous ethanol and enzymatic hydrolysis supernatant are mixed at a volume ratio of 2-4:1 and extracted at 4°C for 10-16 h; the molecular cutoff of the ultrafiltration is 3 kDa.

6. The preparation method according to claim 2, characterized in that, The high-performance liquid chromatography (HPLC) separation used a C18 column; mobile phase A was 0.1% trifluoroacetic acid-acetonitrile, and mobile phase B was 0.1% trifluoroacetic acid-water; the elution gradient was 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 was 1 mL / min; and the detection wavelength was 214 nm.

7. The application of the antimicrobial bioactive peptide according to claim 1 in the preparation of antimicrobial products, characterized in that, The antibacterial product has the effect of inhibiting the growth of Staphylococcus aureus and Escherichia coli.

8. An antibacterial product, characterized in that, The active ingredient of the antibacterial product includes the antibacterial bioactive peptide as described in claim 1.

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

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