CN103936832A - Antibacterial peptide derived from anchovy cooking waste liquid and antibacterial peptide separation method - Google Patents

Abstract

The invention relates to an antibacterial peptide derived from anchovy cooking waste liquid and a separation method thereof. Food-grade enzyme preparations are used to enzymatically hydrolyze the concentrate of anchovy cooking waste liquid, and simulated cell membrane equilibrium dialysis coupled with high-performance liquid chromatography is used to treat the antibacterial peptide. The antimicrobial peptides are separated, the amino acid sequence of the purified peptides is analyzed and their activity is verified. The results showed that the antimicrobial peptide was a decapeptide, and its amino acid sequence was GLSRLFTALK. The peptide has antibacterial activity against seven indicator bacteria including Staphylococcus aureus and Escherichia coli. GLSRLFTALK is obtained by enzymatic hydrolysis, has the characteristics of high safety and strong antibacterial properties, and has potential application value in the pharmaceutical, food and feed industries. This method utilizes the imitating cell membrane binding properties of antimicrobial peptides to achieve targeted separation and overcomes the shortcomings of conventional peptide separations that involve many steps and take a long time. The invention uses fish processing wastewater as raw material, reduces environmental pollution and increases added value of products.

Description

An antimicrobial peptide derived from anchovy cooking waste liquid and its separation method

technical field

The invention relates to an antibacterial peptide derived from cooking waste liquid of kun fish and a separation method thereof, belonging to the field of food biotechnology. the

Background technique

Kunyu is a small and low-value marine fish in the middle and upper layers of the cluster. It is widely distributed in the Yellow Sea and East China Sea. It is rich in protein and oil and has high nutritional value. It contains a lot of proteolytic enzymes in its body, and it is prone to autolysis and rot after fishing, so it is commonly known as "rotten from water" and "rotten boat". After the anchovy is caught, it needs to be steamed immediately, and then processed into the traditional dried anchovy product - sea anchovy. The cooking waste liquid contains nutrients and flavor substances such as protein, amino acid and nucleotide, and has great development and utilization prospects. However, at present, the processing and utilization of the cooking waste liquid of kun fish is less, and the main disposal method is direct discharge and discarding, resulting in waste of resources and environmental pollution. Recycling and high-value utilization of anchovy cooking waste liquid is of great significance for fully developing marine resources, realizing high-value processing of modern seafood and solving environmental pollution problems. the

The emergence of multidrug-resistant bacteria caused by the irrational use of traditional antibiotics has become a global public health problem. As a new member of the antibiotic family, antimicrobial peptides can specifically act on pathogenic bacteria and are non-toxic or less toxic to the host. It has broad-spectrum antibacterial activity, and also has inhibitory activity against bacteria, fungi, and even some cancer cells and viruses. And because of its unique antibacterial mechanism, antimicrobial peptides are not easy to produce drug resistance. A large number of related studies have shown that antimicrobial peptides are expected to become efficient and safe new antibacterial agents, and have great application potential in medicine, food, cosmetics, feed and other industries. the

The preparation methods of antimicrobial peptides mainly include direct extraction method, genetic engineering method, chemical synthesis method and proteolysis method. Because the proteolysis method has the advantages of mild reaction conditions, easy control of the process, convenient and easy-to-get raw materials, and high safety, researchers believe that it may be one of the most promising methods for mass production of antimicrobial peptides. However, the components of proteolysis products are complex, and the traditional separation method of active single peptide often adopts the method of multi-step chromatography combined with activity identification, that is, the activity of each component must be verified in each separation process, which is cumbersome, time-consuming and cost high. Previous studies have shown that it is feasible to rapidly screen antimicrobial peptides based on the affinity between antimicrobial peptides and cell membranes using cell membrane affinity chromatography (a method for quickly and accurately discovering, identifying and preparing protein hydrolyzate-derived antimicrobial peptides, patent application number 201110406867.3 ). However, the cell membrane in cell membrane affinity chromatography needs to be immobilized on a specific carrier to prepare a corresponding stationary phase. The combination with the carrier will interfere with the combination of the cell membrane and the target peptide to a certain extent in space, so it is limited in practical applications. limits. Traditional equilibrium dialysis is a classic method to study the binding balance between small molecules and biomacromolecules. Biomacromolecules and their potential binding targets are placed in the dialysis bag together. The small molecules bound by the macromolecules are bound in the dialysis bag, while the unbound small molecules are free to pass through the dialysis bag. So far, there have been no reports on the separation of antimicrobial agents by equilibrium dialysis. the

Contents of the invention

The technical problem to be solved by the present invention is to provide an antimicrobial peptide derived from anchovy cooking waste liquid, which can be used in medicine, food, cosmetics and feed industries. the

The second technical problem to be solved by the present invention is to provide a method for separating antimicrobial peptides derived from the cooking waste liquid of squid fish in view of the above background technology. the

The technical scheme adopted by the present invention to solve the above-mentioned first technical problem is: the antimicrobial peptide derived from the cooking waste liquid of kun fish is characterized in that it is a decapeptide compound, and its amino acid sequence is GLSRLFTALK (Gly-Leu-Ser- Arg-Leu-Phe-Thr-Ala-Leu-Lys), its molecular ion peak m/z1105.67Da[M+H] ⁺ was detected by mass spectrometry.

The technical solution adopted by the present invention to solve the above-mentioned second technical problem is: a method for separating antimicrobial peptides derived from cooking waste liquid of kun fish, which is characterized in that it comprises the following steps:

1) The cooking waste liquid of kun fish is concentrated by rotary steaming to 1/10-1/20 of the original volume, and then hydrolyzed with protease (0.02-0.04g enzyme/g crude protein) at a temperature of 55-65°C for 4-6 hours. After the solution is completed, boil at 90-100°C for 8-10 minutes to inactivate the enzyme, and then quickly cool it down to room temperature; centrifuge at 8000-10000g for 20-30 minutes to take the supernatant, put it in a -86°C ultra-low temperature refrigerator, and pre-freeze it for 4-30 minutes. After 6 hours, carry out vacuum freeze-drying (-55～-45℃, 0.03～0.2mBar), and store at -18～-20℃ after freeze-drying for future use;

2) The hydrolyzate obtained in step 1) is concentrated to form a 10-25 mg/ml aqueous solution with ultrapure water, and its relative molecular weight distribution is determined by high performance liquid gel permeation chromatography;

3) Cultivate the bacteria to the logarithmic phase, take the bacteria by centrifugation, resuspend them in Tris-HCl buffer (10-25mM, pH7.5), and extract them with chloroform-methanol (1:2, V/V) solution. Cell membrane lipids were suspended in chloroform and steamed to form a film, then hydrated with Tris-HCl buffer (10-25mM, pH7.2), and ultrasonically treated until transparent to prepare a bacterial simulated cell membrane suspension;

4) Put the bacterial simulated cell membrane suspension (50-60 mg/ml) obtained in step 3) and the concentrated hydrolyzate (10-25 mg/ml) obtained in step 1) into a dialysis bag Dialyze in Tris-HCl buffer (10-25mM, pH7.2); use bacteria to simulate cell membrane suspension (50-60mg/ml) and Tris-HCl buffer (10-25mM, pH7.2), etc. The volume was placed in the same dialysis bag for dialysis as a control;

5) Analyze the sample equilibrium dialysate obtained in step 4) and the contrast equilibrium dialysate through reversed-phase high performance liquid chromatography, collect and prepare the difference peaks of the two, and verify its antibacterial ability with Staphylococcus aureus as the indicator bacteria , the amino acid sequence of the component with the strongest antibacterial activity was analyzed by mass spectrometry. the

The selection of the molecular weight cut-off of the dialysis bag in step 4) needs to be determined according to the molecular weight distribution of the hydrolyzate measured in step 2), and the molecular weight cut-off of the dialysis bag needs to be greater than the maximum molecular weight of the hydrolyzate. the

Preferably, the protease described in step 1) is a composite protease Protamex, the enzyme activity is ≥90 U/mg, the hydrolysis condition is pH6.5, the temperature is 55±2°C, and the hydrolysis time is 6h. the

As preferably, the chromatographic conditions of the high performance liquid gel permeation chromatography described in step 2) are: the gel column is a TSKgel2000SWXL post, 300 × 7.5mm; mobile phase: acetonitrile/water/trifluoroacetic acid, 45/55/ 0.1(V/V); Elution rate: 0.5ml/min; Column temperature: 30°C; UV detection wavelength: 220nm. The chromatographic conditions of the reversed-phase high performance liquid chromatography described in step 5) are: chromatographic column is Waters symmetry C18, 250 * 4.6mm; Mobile phase: A water (containing 0.1% trifluoroacetic acid) and B acetonitrile; Elution Procedure: the concentration of acetonitrile is increased from 10% to 80% within 0-26 minutes, the elution rate is 0.5ml/min; the wavelength of ultraviolet detection: 215nm. the

Compared with prior art, the advantage of the inventive method is reflected in:

1. This patented technology uses food processing by-products as raw materials, which can realize the value-added effect of turning waste into treasure. the

2. This patented technology takes the simulated cell membrane equilibrium dialysis technology as the core technology. The simulated cell membrane is not bound by any constraints in the separation system, which overcomes the shortcomings of the traditional cell membrane chromatography that the combination of the membrane and the carrier interferes with its binding to the target peptide. Responses to the interaction between antimicrobial peptides and bacterial phospholipid membranes. The method simplifies the targeted separation steps of the antimicrobial peptides and reduces the production cost. the

3. The antibacterial test proves that the patented product has antibacterial activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Streptococcus pneumoniae, Shigella dysenteriae, Pseudomonas aeruginosa and Salmonella typhimurium. the

Description of drawings

Fig. 1 is a graph showing the molecular weight distribution of the hydrolyzate after the cooking waste liquid of anchovy is concentrated. the

Fig. 2 is the RP-HPLC comparison chart of the equilibrium dialysate of the hydrolyzate and the simulated cell membrane interaction sample and the control equilibrium dialysate after the concentrated cooking waste liquid of kun fish is concentrated. the

Figure 3 is the amino acid analysis mass spectrum of GLSRLFTALK. the

Figure 4 is a flow chart of the separation process of antimicrobial peptides from anchovy cooking waste liquid. the

Detailed ways

The present invention will be further described below in conjunction with the embodiments of the accompanying drawings. the

1) The cooking waste liquid of kun fish was concentrated to 1/10 of the original volume by rotary steaming, and hydrolyzed for 6 hours with the complex protease Protamex (0.02 enzyme/g crude protein) at pH 6.5 and temperature 55±2°C. , boiled at 100°C for 10min to inactivate the enzyme, then rapidly cooled to room temperature; centrifuged at 8000g for 20min to take the supernatant, pre-frozen in -86°C ultra-low temperature refrigerator for 6h, and then vacuum freeze-dried (-55°C, 0.2mBar), After freeze-drying, the anchovy cooking waste liquid was concentrated and then hydrolyzed, which was stored at -20°C for future use;

2) The hydrolyzate was concentrated with ultrapure water to prepare a 10mg/ml aqueous solution, and its relative molecular weight distribution was determined by high performance liquid gel permeation chromatography (Figure 1), and its maximum molecular weight was measured to be 21687Da. The chromatographic conditions are: the gel column is TSKgel2000SWXL column, 300×7.5mm; mobile phase: acetonitrile/water/trifluoroacetic acid, 45/55/0.1 (V/V); elution rate: 0.5ml/min; column temperature : 30°C; UV detection wavelength: 220nm;

3) Cultivate Staphylococcus aureus to the logarithmic phase, centrifuge the bacteria, resuspend in Tris-HCl buffer (25mM, pH7.5), and extract with chloroform-methanol (1:2, V/V) solution Its cell membrane lipid, suspended in chloroform and steamed to form a film, was hydrated with Tris-HCl buffer solution (10mM, pH7.2), and ultrasonically treated until transparent to prepare a simulated cell membrane suspension of Staphylococcus aureus;

4) Put equal volumes of Staphylococcus aureus simulated cell membrane suspension (60 mg/ml) and concentrated anchovy cooking waste liquid (25 mg/ml) in a dialysis bag with a molecular weight cut-off of 25,000, in Tris-HCl buffer solution (10mM, pH7.2) for dialysis; put the same volume of Staphylococcus aureus simulated cell membrane suspension (60mg/ml) and Tris-HCl buffer (10mM, pH7.2) in a dialysis bag with a molecular weight cut-off of 25000 Carry out dialysis as a control sample;

5) The sample equilibrium dialysate and the reference equilibrium dialysate are analyzed by reversed-phase high-performance liquid chromatography, and the chromatographic conditions are as follows: the chromatographic column is Waters symmetry C18, 250 × 4.6mm; mobile phase: A water (containing 0.1% three Fluoroacetic acid) and B acetonitrile; elution procedure: the concentration of acetonitrile increases from 10% to 80% within 0-26min, and the elution rate is 0.5ml/min; UV detection wavelength: 215nm. Collect and prepare both differential peaks (Fig. 2), and use Staphylococcus aureus as indicator bacteria to verify its antibacterial ability;

6) The amino acid sequence of the component with the strongest antibacterial activity was analyzed by mass spectrometry, and its amino acid sequence was GLSRLFTALK (Gly-Leu-Ser-Arg-Leu-Phe-Thr-Ala-Leu-Lys), and its molecular ion Peak m/z 1105.67 Da[M+H] ⁺ (Figure 3).

7) Using Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Streptococcus pneumoniae, Shigella dysenteriae, Pseudomonas aeruginosa, and Salmonella typhimurium as indicator bacteria, the antibacterial activity of GLSRLFTALK was tested, and the results showed that the peptide had a positive effect on the above All bacteria have antibacterial activity, and the minimum inhibitory concentration is between 16 and 256 μg/ml, which shows that it has strong antibacterial activity and wide antibacterial spectrum. the

Claims (4)

1. An antimicrobial peptide derived from the cooking waste liquid of kun fish is characterized in that the antimicrobial peptide is a decapeptide compound, and its amino acid sequence is GLSRLFTALK (Gly-Leu-Ser-Arg-Leu-Phe-Thr-Ala-Leu- Lys), its molecular ion peak m/z1105.67Da[M+H] ⁺ was detected by mass spectrometry, and this antimicrobial peptide sequence is a new antimicrobial peptide sequence. 2. A method for separating antimicrobial peptides derived from cooking waste liquid of kun fish, characterized in that the hydrolyzate after concentration of waste cooking liquid of kun fish is subjected to targeted separation by using simulated cell membrane equilibrium dialysis coupled with high performance liquid chromatography , to collect the components with the strongest antibacterial activity to obtain the target single peptide. The specific steps are: 1) The cooking waste liquid of kun fish is concentrated by rotary steaming to 1/10-1/20 of the original volume, and then hydrolyzed with protease (0.02-0.04g enzyme/g crude protein) at a temperature of 55-65°C for 4-6 hours. After the solution is completed, boil at 90-100°C for 8-10 minutes to inactivate the enzyme, and then quickly cool it down to room temperature; centrifuge at 8000-10000g for 20-30 minutes to take the supernatant, put it in a -86°C ultra-low temperature refrigerator, and pre-freeze it for 4-30 minutes. After 6 hours, carry out vacuum freeze-drying (-55~-45°C, 0.03~0.2mBar), and store it at -18~-20°C after freeze-drying; 2) Concentrating the anchovy cooking waste liquid obtained in step 1) and then concentrating the hydrolyzate into a 10-25 mg/ml aqueous solution with ultrapure water, and measuring its relative molecular weight distribution by high performance liquid gel permeation chromatography; 3) Cultivate the bacteria to the logarithmic phase, take the bacteria by centrifugation, resuspend them in Tris-HCl buffer (10-25mM, pH7.5), and extract them with chloroform-methanol (1:2, V/V) solution. Cell membrane lipids were suspended in chloroform and steamed to form a film, then hydrated with Tris-HCl buffer (10-25mM, pH7.2), and ultrasonically treated until transparent to prepare a bacterial simulated cell membrane suspension; 4) Put the bacterial simulated cell membrane suspension (50-60 mg/ml) obtained in step 3) and the concentrated hydrolyzate (10-25 mg/ml) obtained in step 1) into a dialysis bag Dialyze in Tris-HCl buffer (10-25mM, pH7.2); use bacteria to simulate cell membrane suspension (50-60mg/ml) and Tris-HCl buffer (10-25mM, pH7.2), etc. The volume was placed in the same dialysis bag for dialysis as a control; 5) Analyze the sample equilibrium dialysate obtained in step 4) and the contrast equilibrium dialysate through reversed-phase high performance liquid chromatography, collect and prepare the difference peaks of the two, and verify its antibacterial ability with Staphylococcus aureus as the indicator bacteria , the amino acid sequence of the component with the strongest antibacterial activity was analyzed by mass spectrometry. 3. The antimicrobial peptide according to claim 1, characterized in that it has antibacterial activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Streptococcus pneumoniae, Shigella dysenteriae, Pseudomonas aeruginosa and Salmonella typhimurium. 4. The separation method of antimicrobial peptide according to claim 2, characterized in that the cooking waste liquid of the kun fish is the waste cooking liquid of the yellow sea kun fish (Engraulis japonicus).