Antibacterial protein secreted by lactic acid tablets and separation and identification method thereof

Abstract

The invention discloses antibacterial protein secreted by a lactic acid tablet and a separation and identification method of the antibacterial protein. The preparation method of the lactic acid tablet specifically comprises the following steps: preparing fermentation supernate of pediococcus acidilactici YH-15; comprising the following steps: preparing a whey culture medium, inoculating and culturing YH-15, and preparing and storing fermentation supernatant. Carrying out separation and purification and characterization detection on the bacteriocin-like substance; comprising the steps of salting-out of saturated ammonium sulfate protein, purification of protein through hydrophobic column chromatography, dialysis and freeze-drying, and relates to the technical field of protein spectrum identification, according to the antibacterial protein secreted by the lactic acid tablet and the separation and identification method thereof, a protein sample is subjected to enzymolysis through an FASP enzymolysis method, and after reduction and alkylation centrifugal treatment is conducted by adding a reagent, the protein sample is subjected to freeze-drying; ammonium bicarbonate and pancreatin are used for enzymolysis, peptide fragments after enzymolysis are collected in a centrifugal mode, high-quality peptide fragment raw materials are provided for identification of the protein spectrum, impurity dry burning is reduced, and meanwhile the identification precision of the protein spectrum of a protein sample is effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of protein spectrum identification, in particular to an antibacterial protein secreted by a lactic acid tablet and a separation and identification method thereof. Background Art

[0002] The basic principle of protein spectrum identification is to digest the protein sample into a mixture of peptides after a series of treatments using proteases, then ionize the peptides using a mass spectrometer, and separate the peptide ions with a specific mass-to-charge ratio using a mass analyzer. Finally, the protein is identified by comparing the actual spectrum with the primary mass spectrum peaks and secondary mass spectrum peaks theoretically generated after protein digestion with proteases. In order to achieve high-quality identification of protein samples, traditional protein spectrum identification methods place higher requirements on the identification accuracy of protein samples. Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention provides an antibacterial protein secreted by a lactic acid tablet and a method for separating and identifying the protein, thereby solving the problems raised in the above-mentioned background technology.

[0004] To achieve the above objectives, the present invention is implemented by the following technical scheme: an antibacterial protein secreted by a lactic acid tablet and a method for separating and identifying the same. The method for preparing the lactic acid tablet specifically comprises the following steps: Preparation of fermentation supernatant of Pediococcus acidilactici YH-15; including whey culture medium configuration, inoculation and cultivation of YH-15, and preparation and storage of fermentation supernatant; Isolation, purification and characterization of bacteriocin-like substances; including salting out of saturated ammonium sulfate protein, protein purification by hydrophobic column chromatography, dialysis and freeze-drying.

[0005] Preferably, the separation method specifically comprises the following steps: The protein content was determined by Bradford method; Color development by sulfuric acid-phenol method and biuret reaction; Ultraviolet and Fourier transform infrared spectroscopy analysis.

[0006] Preferably, the whey culture medium is specifically configured as follows: 20 grams of whey powder, 4 grams of glucose and 0.75 grams of potassium dihydrogen phosphate are weighed, and these ingredients are dissolved in distilled water to prepare a culture medium with a total volume of 1000 milliliters; the pH value is within the range of 6.0-6.5, the pH value of the culture medium is accurately measured using a pH meter, 1M sodium hydroxide or 1M hydrochloric acid is added to adjust until the ideal pH value is reached, and the culture medium is placed in a high-pressure steam sterilizer and sterilized at 121 degrees Celsius for 15 to 20 minutes to ensure that the culture medium is sterile. After sterilization, the culture medium is cooled to room temperature or a suitable temperature for inoculation. If it is not used temporarily, the culture medium can be stored in a refrigerator at 4°C to maintain its freshness and prevent contamination; The inoculation method of YH-15 is specifically as follows: after the culture medium is cooled, 600 ml of whey culture medium is taken into a conical flask, 4% of the bacterial culture with OD600=1.5 is inoculated into 600 mL of whey culture medium, and the bacterial suspension with OD600=1.5 corresponds to approximately 1.5×109 bacteria, 1.5×4×108, cells / mL=6×106, cells / mL; a total of approximately 3.6×109 cells of bacteria, sealed with a semipermeable membrane, placed in a constant temperature shaker, and fermented and cultured at 37°C and an appropriate shaking speed for 36 hours; The preparation and storage of culture and fermentation supernatants are as follows: after the fermentation is completed, the fermented bacterial liquid is centrifuged, and the bacteria and the culture medium are separated by centrifugation at 10,000 rpm for 15 minutes. After centrifugation, the supernatant is collected and filtered through a 0.22 / μm microporous filter membrane to remove possible bacterial debris and other particulate matter to obtain the CFS mother liquor; the obtained CFS is stored at 4°C for future use to maintain its biological activity and stability.

[0007] Preferably, the salting out of the saturated ammonium sulfate protein specifically includes taking 600 mL of free extract, adjusting the pH value to 6.5, then placing it on a shaker, adding ammonium sulfate powder until the ammonium sulfate saturation in the CFS reaches 50%, and the protein will begin to precipitate due to reduced solubility, shaking on a shaker for 30 minutes after adding ammonium sulfate to ensure sufficient mixing, and letting the CFS stand overnight at 4°C so that the protein precipitate has enough time to settle, and after standing overnight, separating the precipitate from the supernatant by centrifugation, and after the centrifugation is completed, taking the supernatant and placing it on a shaker again, and continuing to add ammonium sulfate powder, this time the saturation is increased to 80% to precipitate more protein, and the remaining operations are the same as the initial precipitation, and standing and centrifuging at 4°C again, the precipitate obtained after centrifugation contains the target protein, and the precipitate is redissolved using 6 mL of 0.2 mol / L^ phosphate buffer with a pH value of 6.5, and the precipitated protein is redissolved into a solution state, and the redissolved protein solution is stored at 4°C to maintain its stability and activity for further experimental research; The hydrophobic column chromatography protein purification specifically includes: loading 10mL of octyl-agarose gel CL-4B into the chromatography column, washing with ultrapure water with 5 column volumes to remove impurities, balancing the column with 3 column volumes of 2mol / LNaCl solution to ensure a consistent chemical environment, loading a protein precipitation complex solution containing the target molecule and pre-added with solid NaCl to a concentration of 2mol / L onto the column, performing preliminary elution with 1mol / LNaCl solution to remove non-specifically bound molecules, and then further eluting with 0.5mol / LNaCl solution and ultrapure water, each 30mL, to reduce the salt concentration to promote desorption of the target molecule. During the whole process, a centrifuge tube is used to collect the eluate in the 0.5mol / L-1NaCl elution stage, which may contain the desired hydrophobic molecules. The collected eluate should then be monitored by an appropriate analytical method to evaluate the purity and yield of the target molecule; The specific steps of dialysis and freeze-drying are: loading the eluate with antibacterial activity into a dialysis bag with a molecular weight cutoff of 500 to 1000, and then placing the dialysis bag into a 3-liter beaker containing 2 liters of ultrapure water, using a magnetic stirrer to stir at a low speed to ensure that the water is replaced every 4 to 6 hours, and continuing the dialysis for 48 hours. During this period, a conductivity meter is used to regularly detect the conductivity of the dialysate and ultrapure water until the two are the same, indicating that the dialysis is completed. The dialyzed liquid is vacuum freeze-dried for 4 to 5 days to finally obtain a solid powder of about.

[0008] Preferably, the Bradford method for determining protein content is as follows: in the process of preparing the BCA protein quantitative detection kit, firstly, it is necessary to prepare the BCA protein quantitative detection working solution according to the specified ratio according to the number of samples, which includes a mixture of BCA solution and Cu2+ solution. Then, the 10 mg / ml protein standard is diluted to 1000 μg / ml. First, 6 clean test tubes and the required chemical reagents are prepared, and a series of BSA standard solutions with equal gradient concentrations are prepared, and then multiple dilutions are made into 500, 250, 125, 62.5, and 1000 μg / ml of the protein standard. , 31.2μg / ml and other concentration gradients are used for standard curve determination to form a standard product series for constructing a standard curve. In a 96-well plate, blank control, standard product and sample are added respectively and mixed with BCA protein quantitative detection working solution. Then, incubate at 37°C for 1 hour, or increase to 60°C to shorten the incubation time. After the incubation, use an enzyme reader to measure the absorbance of each well, adjust the dilution multiple of the sample according to the absorbance value, and ensure that the measurement result is within the effective range of the standard curve. Finally, calculate the protein concentration in the sample according to the standard curve and the absorbance value of the sample; The ultraviolet spectrum and Fourier transform infrared spectrum analysis are specifically as follows: the ultraviolet spectrum analysis is to dilute the freeze-dried sample solution and use an ultraviolet spectrophotometer to measure the ultraviolet absorption spectrum in the wavelength range of 190 to 400 nm, while the Fourier transform infrared spectrum analysis uses the potassium bromide tablet method to prepare the sample and measure the infrared spectrum of the sample in the range of 500 to 4000 cm^-1.

[0009] Preferably, the separation and identification method specifically comprises the following steps: Step 1: Digest the protein sample into peptides using trypsin; Step 2: Detecting the peptide samples by liquid chromatography-tandem mass spectrometry; Step 3: Perform qualitative analysis on the DDA data to obtain the peptide ion library.

[0010] Preferably, in step 1, the method of digesting the protein sample into peptides by trypsin comprises: The protein sample was enzymatically hydrolyzed using the FASP enzymatic hydrolysis method. 100 μL of 8M Urea, 50 mM DTT was added to the protein sample by volume at 56°C for 40 min to obtain a diluted protein sample. Place the ultrafiltration tube into the collection tube and add the diluted protein sample into the ultrafiltration tube, centrifuge at 12000g for 15 min; Then add 200 μL of Urea buffer to the ultrafiltration tube and centrifuge at 12000 g for 15 min; Add 100 μL of Urea buffer containing 50 mM IAA to the ultrafiltration tube, incubate in the dark for 20 min, and centrifuge at 12000 g for 10 min; Add 100 μL Urea buffer and centrifuge at 12000 g for 10 min. Repeat twice. Add 100 μL of 50 mM ammonium bicarbonate to the ultrafiltration tube and centrifuge at 12,000 g for 10 min. Repeat twice. Add 80 μL of 50 mM pancreatin in ammonium bicarbonate, shake for 1 min, and place the ultrafiltration tube in a 37 °C water bath for enzymatic hydrolysis for 16-18 h; The ultrafiltration tube was transferred to a new collection tube and centrifuged at 12000 g for 10 min to collect; Add another 50 μL of ABC to the ultrafiltration tube and collect by centrifugation at 12000 g for 10 min; The collected peptides were freeze-dried and stored.

[0011] Preferably, the method of performing liquid chromatography-tandem mass spectrometry detection on the peptide sample in step 2 includes: The lyophilized peptides were fully dissolved in solution A and centrifuged at 16000g for 15 min. The supernatant was added to the built-in tube and placed in the automatic sampling device; The sample was injected into the C18 trapping column from the autosampler at a flow rate of 3 μL / min using an EASY-nLC 1200 liquid chromatograph, adjusted to a flow rate of 300 nL / min, and then eluted onto the C18 analytical column; Elution with solution B for 45 min; Thermo Orbitrap Tribid Fusion mass spectrometer was used to collect mass spectrometry data and output raw files.

[0012] Preferably, in step 3, the method of performing qualitative analysis on the DDA data to obtain the peptide ion library includes: The raw files generated by mass spectrometry were searched using MaxQuant version 1.6.5, using the Pediococcus protein sequence downloaded from uniprot as the database. Trypsin was used for digestion, the maximum number of missed cuts allowed was 2, methionine oxidation and protein N-terminal acetylation were set as variable modifications, carbamidomethyl was set as a fixed modification, the false positive rate (FDR) of proteins and peptides was < 0.01, and all other settings were set as default.

[0013] The present invention provides an antibacterial protein secreted by a lactic acid tablet and a method for separating and identifying the protein. The invention has the following beneficial effects: The present invention uses the FASP enzymatic hydrolysis method to enzymatically hydrolyze the protein sample, adds reagents for reduction and alkylation, and then uses ammonium bicarbonate and pancreatin for enzymatic hydrolysis after centrifugation to collect the peptides after enzymatic hydrolysis, thereby providing high-quality peptide raw materials for protein spectrum identification, reducing impurity dry burning, and effectively improving the identification accuracy of the protein spectrum of the protein sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram of the identification process of the present invention; Figure 2 A schematic diagram of the bacteriocin of the present invention; Figure 3 It is a schematic diagram of the bacteriocin molecule of the present invention; Figure 4 Schematic diagram of the antibacterial effect of the protein (bacteriocin) of the present invention. DETAILED DESCRIPTION

[0015] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] See also Figure 1 The embodiment of the present invention provides the following technical solution: an antibacterial protein secreted by a lactic acid tablet and a method for separating and identifying the same, which specifically comprises the following steps: Step 1: Digest the protein sample into peptides using trypsin: The protein sample was enzymatically hydrolyzed using the FASP enzymatic hydrolysis method. 100 μL of 8M Urea, 50 mM DTT was added to the protein sample by volume at 56°C for 40 min to obtain a diluted protein sample. Place the ultrafiltration tube into the collection tube and add the diluted protein sample into the ultrafiltration tube, centrifuge at 12000g for 15 min; Then add 200 μL of Urea buffer to the ultrafiltration tube and centrifuge at 12000 g for 15 min; Add 100 μL of Urea buffer containing 50 mM IAA to the ultrafiltration tube, incubate in the dark for 20 min, and centrifuge at 12000 g for 10 min; Add 100 μL Urea buffer and centrifuge at 12000 g for 10 min. Repeat twice. Add 100 μL of 50 mM ammonium bicarbonate to the ultrafiltration tube and centrifuge at 12,000 g for 10 min. Repeat twice. Add 80 μL of 50 mM trypsin in ammonium bicarbonate containing 500 ng trypsin, shake for 1 min, and place the ultrafiltration tube in a 37 °C water bath for enzymatic hydrolysis for 16-18 h; The ultrafiltration tube was transferred to a new collection tube and centrifuged at 12000 g for 10 min to collect; Add another 50 μL of ABC to the ultrafiltration tube and collect by centrifugation at 12000 g for 10 min; The collected peptides were freeze-dried and stored.

[0017] Step 2: Detect the peptide samples by liquid chromatography-tandem mass spectrometry: The lyophilized peptides were fully dissolved in solution A, i.e. 0.1% FA, and centrifuged at 16000g for 15 min. The supernatant was added to the built-in tube and placed in the automatic sampling device; The sample was injected into the C18 trapping column from the autosampler at a flow rate of 3 μL / min using an EASY-nLC 1200 liquid chromatograph, adjusted to a flow rate of 300 nL / min, and then eluted to a C18 analytical column with an inner diameter of 75 μm. Elution with solution B, which is 95% ACN and 0.1% FA, for 45 minutes; Thermo Orbitrap Tribid Fusion mass spectrometer was used to collect mass spectrometry data and output raw files.

[0018] Figure 2 A is the mass spectrometry peak diagram of the bacteriocin, B is the amino acid sequence of the bacteriocin, C is the secondary structure ratio of the bacteriocin, D is the secondary structure heat map of the bacteriocin, and E is the molecular formula of the bacteriocin; Figure 3 A is the molecular planar primary structure of the bacteriocin, B is the tertiary spatial structure predicted by the SWISS website of the bacteriocin and its similarity with other substances, C is the three-dimensional tertiary structure of the bacteriocin, and D is the relative molecular mass of the bacteriocin.

[0019] To further illustrate, the mass spectrometer parameter settings include: The spray voltage of the ion source was set to 2.1 kV, the primary scanning range was 350-1500 m / z, the resolution was 120 K (@ m / z 200), the AGC target was 3e6, and the Maximum IT:80 ms; the secondary resolution was 15 K (@ m / z 200), the isolation window was 1.6 Th, the AGC target was 5e4, the maximum IT was 40 ms, and the MS2 Activation was HCD (collision energy:28).

[0020] Step 3: Perform qualitative analysis on DDA data to obtain peptide ion library: The raw files generated by mass spectrometry were searched using MaxQuant version 1.6.5, using the Pediococcus protein sequence downloaded from uniprot as the database. Trypsin was used for digestion, the maximum number of missed cuts allowed was 2, methionine oxidation (M) and protein N-terminal acetylation (Acetyl) were used as variable modifications, carbamidomethyl (C) was used as a fixed modification, the false positive rate (FDR) for proteins and peptides was <0.01, and all other settings were as default.

[0021] In summary, the above reagents and consumables mainly include formic acid and ammonium bicarbonate (NH4HCO3) purchased from Sigma, trypsin purchased from Promega; mass spectrometry grade acetonitrile and water purchased from Thermo; other reagents are domestic analytical grade.

[0022] The main instruments and equipment include: EASY-nLC 1200 liquid chromatography, Thermo Orbitrap Tribid Fusion mass spectrometer, Eppendorf refrigerated centrifuge and vacuum freeze dryer.

[0023] A bacteriostatic protein secreted by a lactic acid tablet and a method for separating and identifying the same. The method for preparing the lactic acid tablet specifically comprises the following steps: Preparation of fermentation supernatant of Pediococcus acidilactici YH-15; including whey culture medium configuration, inoculation and cultivation of YH-15, and preparation and storage of fermentation supernatant; Isolation, purification and characterization of bacteriocin-like substances; including salting out of saturated ammonium sulfate protein, protein purification by hydrophobic column chromatography, dialysis and freeze-drying.

[0024] In the present invention, the separation method specifically comprises the following steps: The protein content was determined by Bradford method; Color development by sulfuric acid-phenol method and biuret reaction; Ultraviolet and Fourier transform infrared spectroscopy analysis.

[0025] In the present invention, the whey culture medium is specifically configured as follows: 20 grams of whey powder, 4 grams of glucose and 0.75 grams of potassium dihydrogen phosphate are weighed, and these components are dissolved in distilled water to prepare a culture medium with a total volume of 1000 milliliters; the pH value is within the range of 6.0-6.5, the pH value of the culture medium is accurately measured using a pH meter, 1M sodium hydroxide or 1M hydrochloric acid is added to adjust until the ideal pH value is reached, and the culture medium is placed in a high-pressure steam sterilizer and sterilized at 121 degrees Celsius for 15 to 20 minutes to ensure that the culture medium is sterile. After sterilization, the culture medium is cooled to room temperature or a suitable temperature for inoculation. If it is not used temporarily, the culture medium can be stored in a refrigerator at 4°C to maintain its freshness and prevent contamination; The inoculation method of YH-15 is specifically as follows: after the culture medium is cooled, 600 ml of whey culture medium is taken into a conical flask, 4% of the bacterial culture with OD600=1.5 is inoculated into 600 mL of whey culture medium, and the bacterial suspension with OD600=1.5 corresponds to approximately 1.5×109 bacteria, 1.5×4×108, cells / mL=6×106, cells / mL; a total of approximately 3.6×109 cells of bacteria, sealed with a semipermeable membrane, placed in a constant temperature shaker, and fermented and cultured at 37°C and an appropriate shaking speed for 36 hours; The preparation and storage of culture and fermentation supernatants are as follows: after the fermentation is completed, the fermented bacterial liquid is centrifuged, and the bacteria and the culture medium are separated by centrifugation at 10,000 rpm for 15 minutes. After centrifugation, the supernatant is collected and filtered through a 0.22 / μm microporous filter membrane to remove possible bacterial debris and other particulate matter to obtain the CFS mother liquor; the obtained CFS is stored at 4°C for future use to maintain its biological activity and stability.

[0026] In the present invention, the salting out of saturated ammonium sulfate protein specifically includes taking 600 mL of free extract, adjusting the pH value to 6.5, and then placing it on a shaker, adding ammonium sulfate powder until the ammonium sulfate saturation in the CFS reaches 50%, and the protein will begin to precipitate due to reduced solubility. After adding ammonium sulfate, shake on a shaker for 30 minutes to ensure sufficient mixing, and let the CFS stand overnight at 4°C so that the protein precipitate has enough time to settle. After standing overnight, the precipitate is separated from the supernatant by centrifugation. After the centrifugation is completed, the supernatant is taken and placed on a shaker again, and ammonium sulfate powder is continued to be added. This time, the saturation is increased to 80% to precipitate more protein. The remaining operations are the same as the initial precipitation, and the precipitate is again stood and centrifuged at 4°C. The precipitate obtained after centrifugation contains the target protein, and the precipitate is redissolved using 6 mL of 0.2 mol / L^ phosphate buffer with a pH value of 6.5, and the precipitated protein is redissolved into a solution state. The redissolved protein solution is stored at 4°C to maintain its stability and activity for further experimental research; The hydrophobic column chromatography protein purification specifically includes: loading 10mL of octyl-agarose gel CL-4B into the chromatography column, washing with ultrapure water with 5 column volumes to remove impurities, balancing the column with 3 column volumes of 2mol / LNaCl solution to ensure a consistent chemical environment, loading a protein precipitation complex solution containing the target molecule and pre-added with solid NaCl to a concentration of 2mol / L onto the column, performing preliminary elution with 1mol / LNaCl solution to remove non-specifically bound molecules, and then further eluting with 0.5mol / LNaCl solution and ultrapure water, each 30mL, to reduce the salt concentration to promote desorption of the target molecule. During the whole process, a centrifuge tube is used to collect the eluate in the 0.5mol / L-1NaCl elution stage, which may contain the desired hydrophobic molecules. The collected eluate should then be monitored by an appropriate analytical method to evaluate the purity and yield of the target molecule; The specific steps of dialysis and freeze-drying are: loading the eluate with antibacterial activity into a dialysis bag with a molecular weight cutoff of 500 to 1000, and then placing the dialysis bag into a 3-liter beaker containing 2 liters of ultrapure water, using a magnetic stirrer to stir at a low speed to ensure that the water is replaced every 4 to 6 hours, and continuing the dialysis for 48 hours. During this period, a conductivity meter is used to regularly detect the conductivity of the dialysate and ultrapure water until the two are the same, indicating that the dialysis is completed. The dialyzed liquid is vacuum freeze-dried for 4 to 5 days to finally obtain a solid powder of about.

[0027] In the present invention, the Bradford method for determining protein content is specifically as follows: in the process of preparing the BCA protein quantitative detection kit, firstly, it is necessary to prepare the BCA protein quantitative detection working solution according to the specified ratio according to the number of samples, which includes the mixture of BCA solution and Cu2+ solution. Then, the 10 mg / ml protein standard is diluted to 1000 μg / ml. First, 6 clean test tubes and the required chemical reagents are prepared, and a series of BSA standard solutions with equal gradient concentrations are prepared, and then multiple dilutions are made into 500, 250, 125, 62.5, and 1000 μg / ml of the protein standard. , 31.2μg / ml and other concentration gradients are used for standard curve determination to form a standard product series for constructing a standard curve. In a 96-well plate, blank control, standard product and sample are added respectively and mixed with BCA protein quantitative detection working solution. Then, incubate at 37°C for 1 hour, or increase to 60°C to shorten the incubation time. After the incubation, use an enzyme reader to measure the absorbance of each well, adjust the dilution multiple of the sample according to the absorbance value, and ensure that the measurement result is within the effective range of the standard curve. Finally, calculate the protein concentration in the sample according to the standard curve and the absorbance value of the sample; The ultraviolet spectrum and Fourier infrared spectrum analysis are specifically as follows: the ultraviolet spectrum analysis is to dilute the freeze-dried sample solution and use an ultraviolet spectrophotometer to measure the ultraviolet absorption spectrum in the wavelength range of 190 to 400 nm, while the Fourier infrared spectrum analysis uses the potassium bromide tablet method to prepare the sample and measure the infrared spectrum of the sample in the range of 500 to 4000 cm^-1 In the embodiments provided by the present invention, it should be understood that the disclosed devices, apparatuses and methods may be implemented in other ways.

[0028] It is also obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention.

[0029] Therefore, no matter from which point of view, the embodiments should be regarded as illustrative and non-restrictive, and the scope of the present invention is limited by the appended claims rather than the above description, so it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any attached figure mark in the claims should not be regarded as limiting the claims involved.

[0030] In addition, it is clear that the word "comprising" does not exclude other units or steps, and the singular does not exclude the plural. Multiple units or devices stated in the system claim can also be implemented by one unit or device through software or hardware. The second and other words are used to indicate names, but not to indicate any particular order.

[0031] Finally, it should be noted that the above embodiments are only used to illustrate the technical method of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical method of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical method of the present invention.

Claims

1. An antibacterial protein secreted by a lactic acid tablet and a method for separating and identifying the protein, characterized in that: The preparation method of the lactic acid tablet specifically comprises the following steps: Preparation of fermentation supernatant of Pediococcus acidilactici YH-15; including whey culture medium configuration, inoculation and cultivation of YH-15, and preparation and storage of fermentation supernatant; Isolation, purification and characterization of bacteriocin-like substances; including salting out of saturated ammonium sulfate protein, protein purification by hydrophobic column chromatography, dialysis and freeze-drying.

2. The antibacterial protein secreted by a lactic acid tablet and the method for separating and identifying the protein according to claim 1, characterized in that: The separation method specifically comprises the following steps: The protein content was determined by Bradford method; Color development by sulfuric acid-phenol method and biuret reaction; Ultraviolet and Fourier transform infrared spectroscopy analysis.

3. The antibacterial protein secreted by a lactic acid tablet and the method for separating and identifying the same according to claim 1, characterized in that: The whey culture medium is specifically configured as follows: 20 grams of whey powder, 4 grams of glucose and 0.75 grams of potassium dihydrogen phosphate are weighed, and these ingredients are dissolved in distilled water to prepare a culture medium with a total volume of 1000 milliliters; the pH value is within the range of 6.0-6.5, the pH value of the culture medium is accurately measured using a pH meter, 1M sodium hydroxide or 1M hydrochloric acid is added to adjust until the ideal pH value is reached, and the culture medium is placed in a high-pressure steam sterilizer and sterilized at 121 degrees Celsius for 15 to 20 minutes to ensure that the culture medium is sterile. After sterilization, the culture medium is cooled to room temperature or a suitable temperature for inoculation. If it is not used temporarily, the culture medium can be stored in a refrigerator at 4°C to maintain its freshness and prevent contamination; The inoculation method of YH-15 is specifically as follows: after the culture medium is cooled, 600 ml of whey culture medium is taken into a conical flask, 4% of the bacterial culture with OD600=1.5 is inoculated into 600 mL of whey culture medium, and the bacterial suspension with OD600=1.5 corresponds to approximately 1.5×109 bacteria, 1.5×4×108, cells / mL=6×106, cells / mL; a total of approximately 3.6×109 cells of bacteria, sealed with a semipermeable membrane, placed in a constant temperature shaker, and fermented and cultured at 37°C and an appropriate shaking speed for 36 hours; The preparation and storage of culture and fermentation supernatants are as follows: after the fermentation is completed, the fermented bacterial liquid is centrifuged, and the bacteria and the culture medium are separated by centrifugation at 10,000 rpm for 15 minutes. After centrifugation, the supernatant is collected and filtered through a 0.22 / μm microporous filter membrane to remove possible bacterial debris and other particulate matter to obtain the CFS mother liquor; the obtained CFS is stored at 4°C for future use to maintain its biological activity and stability.

4. The antibacterial protein secreted by a lactic acid tablet and the method for separating and identifying the same according to claim 1, characterized in that: The salting out of the saturated ammonium sulfate protein specifically includes taking 600 mL of the free extract, adjusting the pH value to 6.5, and then placing it on a shaker, adding ammonium sulfate powder until the ammonium sulfate saturation in the CFS reaches 50%, and the protein will begin to precipitate due to reduced solubility. After adding ammonium sulfate, shake on a shaker for 30 minutes to ensure sufficient mixing, and let the CFS stand overnight at 4°C so that the protein precipitate has enough time to settle. After standing overnight, the precipitate is separated from the supernatant by centrifugation. After the centrifugation is completed, the supernatant is placed on a shaker again, and ammonium sulfate powder is continued to be added. This time, the saturation is increased to 80% to precipitate more protein. The remaining operations are the same as the initial precipitation, and the precipitate is again stood and centrifuged at 4°C. The precipitate obtained after centrifugation contains the target protein, and the precipitate is redissolved using 6 mL of 0.2 mol / L^ phosphate buffer with a pH value of 6.5, and the precipitated protein is redissolved into a solution state. The redissolved protein solution is stored at 4°C to maintain its stability and activity for further experimental research; The hydrophobic column chromatography protein purification specifically includes: loading 10mL of octyl-agarose gel CL-4B into the chromatography column, washing with ultrapure water with 5 column volumes to remove impurities, balancing the column with 3 column volumes of 2mol / LNaCl solution to ensure a consistent chemical environment, loading a protein precipitation complex solution containing the target molecule and pre-added with solid NaCl to a concentration of 2mol / L onto the column, performing preliminary elution with 1mol / LNaCl solution to remove non-specifically bound molecules, and then further eluting with 0.5mol / LNaCl solution and ultrapure water, each 30mL, to reduce the salt concentration to promote desorption of the target molecule. During the whole process, a centrifuge tube is used to collect the eluate in the 0.5mol / L-1NaCl elution stage, which may contain the desired hydrophobic molecules. The collected eluate should then be monitored by an appropriate analytical method to evaluate the purity and yield of the target molecule; The specific steps of dialysis and freeze-drying are: loading the eluate with antibacterial activity into a dialysis bag with a molecular weight cutoff of 500 to 1000, and then placing the dialysis bag into a 3-liter beaker containing 2 liters of ultrapure water, using a magnetic stirrer to stir at a low speed to ensure that the water is replaced every 4 to 6 hours, and continuing the dialysis for 48 hours. During this period, a conductivity meter is used to regularly detect the conductivity of the dialysate and ultrapure water until the two are the same, indicating that the dialysis is completed. The dialyzed liquid is vacuum freeze-dried for 4 to 5 days to finally obtain a solid powder of about.

5. The antibacterial protein secreted by a lactic acid tablet and the method for separating and identifying the same according to claim 1, characterized in that: The Bradford method for determining protein content is as follows: in the process of preparing the BCA protein quantitative detection kit, firstly, the BCA protein quantitative detection working solution needs to be prepared according to the specified ratio according to the number of samples, which includes the mixture of BCA solution and Cu2+ solution. Then, the 10mg / ml protein standard is diluted to 1000μg / ml. First, 6 clean test tubes and the required chemical reagents are prepared to prepare a series of BSA standard solutions with equal gradient concentrations, and then multiple dilutions are made into 500, 250, 125, 62.5, 3 1.2μg / ml isoconcentration gradient is used for standard curve determination to form a standard series for constructing a standard curve. In a 96-well plate, blank control, standard and sample are added respectively and mixed with BCA protein quantitative detection working solution. Then, incubate at 37℃ for 1 hour, or you can choose to increase to 60℃ to shorten the incubation time. After the incubation, use an enzyme reader to measure the absorbance of each well, adjust the dilution multiple of the sample according to the absorbance value, and ensure that the measurement result is within the effective range of the standard curve. Finally, calculate the protein concentration in the sample according to the absorbance value of the standard curve and the sample; The ultraviolet spectrum and Fourier transform infrared spectrum analysis are specifically as follows: the ultraviolet spectrum analysis is to dilute the freeze-dried sample solution and use an ultraviolet spectrophotometer to measure the ultraviolet absorption spectrum in the wavelength range of 190 to 400 nm, while the Fourier transform infrared spectrum analysis uses the potassium bromide tablet method to prepare the sample and measure the infrared spectrum of the sample in the range of 500 to 4000 cm^-1.

6. The antibacterial protein secreted by a lactic acid tablet and the method for separating and identifying the protein according to claim 1, characterized in that: The separation and identification method specifically comprises the following steps: Step 1: Digest the protein sample into peptides using trypsin; Step 2: Perform liquid chromatography-tandem mass spectrometry on the peptide samples; Step 3: Perform qualitative analysis on the DDA data to obtain the peptide ion library.

7. The antibacterial protein secreted by a lactic acid tablet and the method for separating and identifying the protein according to claim 6, characterized in that: The method of digesting the protein sample into peptides by trypsin in step 1 includes: The protein sample was enzymatically hydrolyzed using the FASP enzymatic hydrolysis method. 100 μL of 8M Urea, 50mM DTT was added to the protein sample by volume at 56°C for 40 min to obtain a diluted protein sample. Place the ultrafiltration tube into the collection tube and add the diluted protein sample into the ultrafiltration tube, centrifuge at 12000g for 15 min; Then add 200 μL of Urea buffer to the ultrafiltration tube and centrifuge at 12000 g for 15 min; Add 100 μL of Urea buffer containing 50 mM IAA to the ultrafiltration tube, incubate in the dark for 20 min, and centrifuge at 12000 g for 10 min; Add 100 μL Urea buffer and centrifuge at 12000 g for 10 min. Repeat twice. Add 100 μL of 50 mM ammonium bicarbonate to the ultrafiltration tube and centrifuge at 12,000 g for 10 min. Repeat twice. Add 80 μL of 50 mM pancreatin in ammonium bicarbonate, shake for 1 min, and place the ultrafiltration tube in a 37 °C water bath for enzymatic hydrolysis for 16-18 h; The ultrafiltration tube was transferred to a new collection tube and centrifuged at 12000 g for 10 min to collect; Add another 50 μL of ABC to the ultrafiltration tube and collect by centrifugation at 12000 g for 10 min; The collected peptides were freeze-dried and stored.

8. The antibacterial protein secreted by a lactic acid tablet and the method for separating and identifying the same according to claim 6, characterized in that: The method of performing liquid chromatography-tandem mass spectrometry detection on the peptide sample in step 2 includes: The lyophilized peptides were fully dissolved in solution A and centrifuged at 16000g for 15 min. The supernatant was added to the built-in tube and placed in the automatic sampling device; The sample was injected into the C18 trapping column from the autosampler at a flow rate of 3 μL / min using an EASY-nLC 1200 liquid chromatograph, adjusted to a flow rate of 300 nL / min, and then eluted onto the C18 analytical column; Elution with solution B for 45 min; Thermo Orbitrap Tribid Fusion mass spectrometer was used to collect mass spectrometry data and output raw files.

9. The antibacterial protein secreted by a lactic acid tablet and the method for separating and identifying the protein according to claim 6, characterized in that: In step 3, the method of performing qualitative analysis on the DDA data to obtain the peptide ion library includes: The raw files generated by mass spectrometry were searched using MaxQuant version 1.6.5, using the Pediococcus protein sequence downloaded from uniprot as the database. Trypsin was used for digestion, the maximum number of missed cuts allowed was 2, methionine oxidation and protein N-terminal acetylation were set as variable modifications, carbamidomethyl was set as a fixed modification, the false positive rate of proteins and peptides was <0.01, and all other settings were set as default.

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