Lactobacillus paracasei bacteriocin for inhibiting multi-drug-resistant pseudomonas aeruginosa and preparation method of lactobacillus paracasei bacteriocin

Abstract

The invention discloses a lactobacillus paracasei bacteriocin for inhibiting multi-drug-resistant pseudomonas aeruginosa and a preparation method of the lactobacillus paracasei bacteriocin, and belongs to the field of fermentation engineering. According to the invention, a multi-drug-resistant pseudomonas aeruginosa inhibition zone and lactic acid bacteria growth and acid production are taken as indexes, the casei paracasei KD10 capable of remarkably inhibiting multi-drug-resistant pseudomonas aeruginosa is screened and separated, fermentation conditions are optimized, bacteriocin is obtained by separation and purification from supernate of fermentation liquor, and the amino acid sequence of the bacteriocin is identified. Bacteriostatic experiments show that the lactobacillus paracasei KD10 bacteriocin has broad-spectrum bacteriostasis, and has a certain bacteriostatic effect on various clinically common gram-positive bacteria and gram-negative bacteria. In addition, bacteriocin has good stability to temperature, pH, non-digestive enzymes, storage temperature and ultraviolet rays, and is sensitive to digestive enzymes. The invention provides theoretical and technical references for the development of bacteriocin required for treating and preventing multi-drug-resistant pseudomonas aeruginosa infection.

Description

Technical Field

[0001] The invention relates to the field of fermentation engineering, in particular to a Lactobacillus paracasei bacteriocin for inhibiting multi-drug resistant Pseudomonas aeruginosa and a preparation method thereof. Background Art

[0002] Pseudomonas aeruginosa (P.aeruginosa), also known as Pseudomonas aeruginosa, is a common conditional pathogen and a non-fermenting Gram-negative bacillus. Pseudomonas aeruginosa is a pathogen shared by humans and livestock and can cause various chronic infections. In hospital infection monitoring, it was found that Gram-negative bacteria are the most important pathogens, among which Pseudomonas aeruginosa is one of the most common pathogens. Pseudomonas aeruginosa is highly infectious and is most common in lung infections, urinary tract infections, burns on tissue or skin surfaces, and bloodstream infections. Due to the high isolation rate, Pseudomonas aeruginosa has become the main target of prevention and treatment.

[0003] At present, antibiotics are mainly used to control Pseudomonas aeruginosa, which has led to the increasing problem of drug resistance of Pseudomonas aeruginosa. Sun Jun et al. analyzed the distribution and drug resistance of Pseudomonas aeruginosa in clinical infection and found that 342 strains of Pseudomonas aeruginosa had different degrees of resistance to antibiotics, among which the resistance rates of cotrimoxazole, chloramphenicol and ceftriaxone exceeded 60%. He Guohua et al. analyzed the clinical distribution and drug resistance of 805 strains of Pseudomonas aeruginosa and found that Pseudomonas aeruginosa was most sensitive to polymyxin B and amikacin, with sensitivity rates of 98.01% (789 / 805) and 96.64% (778 / 805), respectively. It was more sensitive to aztreonam and ticarcillin / clavulanic acid, with sensitivity rates of 76.89% (619 / 805) and 54.29% (437 / 805), respectively. The sensitivity rate to other common antibiotics (except for natural resistance) was above 80.00%.

[0004] Bacteriocins produced by lactic acid bacteria are a class of peptide substances with antibacterial activity. They have a certain antibacterial effect on Pseudomonas aeruginosa, but there are few reports on whether they are effective against multidrug-resistant Pseudomonas aeruginosa. The reason is that the research on bacteriocins of Pseudomonas aeruginosa mainly focuses on single resistant strains. Therefore, it is necessary to develop bacteriocins against multidrug-resistant Pseudomonas aeruginosa. Summary of the invention

[0005] The invention aims to provide a Lactobacillus paracasei bacteriocin for inhibiting multidrug-resistant Pseudomonas aeruginosa and a preparation method thereof, so as to solve the problems existing in the above-mentioned prior art. The Lactobacillus paracasei KD10 obtained by screening significantly inhibits multidrug-resistant Pseudomonas aeruginosa, and the bacteriocin is separated and purified from the supernatant of the bacterial fermentation broth, and the amino acid sequence of the bacteriocin is identified, so as to provide a theoretical and technical reference for the development of bacteriocins required for treating and preventing multidrug-resistant Pseudomonas aeruginosa infection.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a Lacticaseibacillus paracasei KD10 producing a bacteriocin capable of inhibiting multidrug-resistant Pseudomonas aeruginosa. The Lacticaseibacillus paracasei has a preservation number of CCTCC NO: M20232074, a preservation time of October 30, 2023, a preservation unit of the China Center for Type Culture Collection, and a preservation address of Wuhan University, Wuhan, China.

[0008] The invention also provides a Lactobacillus paracasei bacteriocin for inhibiting multi-drug resistant Pseudomonas aeruginosa. The Lactobacillus paracasei bacteriocin is obtained from the supernatant of fermentation liquid after Lactobacillus paracasei is fermented in MRS broth. The preservation number of the Lactobacillus paracasei is CCTCC NO: M20232074.

[0009] Preferably, the amino acid sequence of the Lactobacillus paracasei bacteriocin includes the sequences shown in SEQ ID NOs. 1-15.

[0010] The present invention also provides a method for preparing the Lactobacillus paracasei bacteriocin, comprising the following steps:

[0011] S1: The culture solution of Lactobacillus paracasei activated in MRS broth was inoculated into the fermentation medium at an inoculation rate of 2-4%, and cultured at 35-39° C. for 30-42 h, and refrigerated centrifuged at 4° C.-8° C. at 6000-8000 rpm for 10 min-15 min to obtain the supernatant;

[0012] S2: The supernatant is concentrated to 1 / 4 of the volume on a rotary evaporator (60°C-75°C, speed 100-120rpm), and then 3 times the volume of ethyl acetate is added and mixed thoroughly, and the mixture is placed in a shaker and shaken at 120rpm for 12h, and then allowed to stand for 2h and the ethyl acetate layer is poured out, and the raffinate is extracted twice with ethyl acetate, and the three ethyl acetate extracts are combined and then evaporated to dryness on a rotary evaporator (60°C-75°C, speed 100-120rpm), and then purified water is added for re-dissolution to obtain a bacteriocin aqueous solution;

[0013] S3: Ultrafiltration of the bacteriocin aqueous solution through 10K, 5K, and 3K filter membranes respectively, collecting the components less than 3K for freeze-drying to obtain the Lactobacillus paracasei bacteriocin.

[0014] The minimum inhibitory concentration and minimum bactericidal concentration of the bacteriocin obtained by the above preparation method against multidrug-resistant Pseudomonas aeruginosa are 0.78 mg / mL and 3.12 mg / mL respectively.

[0015] The bacteriocin has good stability. When kept at 20-100°C for 30 minutes, its activity loss is less than 10%, and when stored at 25°C for 90 days, its activity loss is less than 8%. Ultraviolet irradiation has no effect on its activity.

[0016] The bacteriocin remained stable at pH 2.0-11.0 with an activity loss of less than 8%.

[0017] The present invention also provides application of the Lactobacillus paracasei or its fermentation liquid in preparing a medicine for inhibiting multi-drug resistant Pseudomonas aeruginosa.

[0018] The present invention also provides the use of the Lactobacillus paracasei bacteriocin in any of the following items:

[0019] (1) Application in the preparation of drugs for inhibiting multidrug-resistant Pseudomonas aeruginosa;

[0020] (2) Use in the preparation of drugs for inhibiting Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Proteus mirabilis and / or Acinetobacter baumannii.

[0021] The present invention also provides an antibacterial agent, which comprises the Lactobacillus paracasei bacteriocin, and the antibacterial agent comprises a bacterial agent for inhibiting multi-drug resistant Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Proteus mirabilis and / or Acinetobacter baumannii.

[0022] The present invention discloses the following technical effects:

[0023] The present invention screens and separates a strain of Lactobacillus paracasei KD10 that inhibits multidrug-resistant Pseudomonas aeruginosa. The fermentation broth supernatant of the bacteria contains bacteriocin with good antibacterial effect. It is found through experiments that the bacteriocin contains 15 peptides, and the bacteriocin of Lactobacillus paracasei KD10 has a broad-spectrum antibacterial property, and has a certain antibacterial effect on common clinical Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis); the MIC of Lactobacillus paracasei KD10 bacteriocin to multidrug-resistant Pseudomonas aeruginosa is 0.78 mg / ml, and the MBC is 3.12 mg / ml. The Lactobacillus paracasei KD10 bacteriocin provided by the present invention provides a theoretical and technical reference for the development of bacteriocins required for treating and preventing multidrug-resistant Pseudomonas aeruginosa infection.

[0024] The Lactobacillus paracasei KD10 bacteriocin separated by the invention has good stability to temperature, pH, non-digestive enzymes, storage temperature and ultraviolet rays, is sensitive to digestive enzymes, and provides basic data and theoretical basis for developing clinical drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0026] Figure 1 is the MALDI-TOF-MS mass spectrum of strain KD10;

[0027] Figure 2 The antibacterial effect of lactic acid bacteria fermentation supernatant and bacteria on multi-drug resistant Pseudomonas aeruginosa;

[0028] Figure 3 The effect of culture time on the growth, antibacterial effect, pH and acidity of lactic acid bacteria; A: diameter of the inhibition zone and bacterial sludge weight; B: pH value and acidity;

[0029] Figure 4 The effect of culture time on bacteriocin production by Lactobacillus paracasei KD10;

[0030] Figure 5 The effect of inoculum size on bacteriocin production by Lactobacillus paracasei KD10;

[0031] Figure 6 The effect of culture temperature on bacteriocin production by Lactobacillus paracasei KD10;

[0032] Figure 7 The antibacterial activity of each component of ultrafiltration KD10;

[0033] Figure 8 is the liquid chromatogram of KD10 bacteriocin;

[0034] Fig. 9 This is the secondary mass spectrum of the peptide VISAVAQTNA;

[0035] Fig.10 is the secondary mass spectrum of the peptide PQGPQGPRG;

[0036] Fig.11 This is the secondary mass spectrum of the peptide GPSGFYRIN;

[0037] Fig.12 is the secondary mass spectrum of the peptide GVQDFDRLR;

[0038] Fig.13 is the concentration of bacteriocin produced by KD10 and its inhibitory effect on multidrug-resistant Pseudomonas aeruginosa;

[0039] Fig.14 is the inhibition curve of KD10 bacteriocin concentration against multidrug-resistant Pseudomonas aeruginosa;

[0040] Fig.15 is the effect of temperature on the activity of KD10 bacteriocin;

[0041] Fig.16 The effect of acid and alkali on the activity of KD10 bacteriocin;

[0042] Fig.17 is the effect of enzyme on the activity of KD10 bacteriocin;

[0043] Fig.18 The effect of storage conditions on the activity of KD10 bacteriocin;

[0044] Fig.19 This is the effect of ultraviolet light on the activity of KD10 bacteriocin. DETAILED DESCRIPTION

[0045] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0047] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content of this specification shall prevail.

[0048] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0049] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0050] The experimental strains involved in the following examples: multidrug-resistant Pseudomonas aeruginosa (P.aeruginosa, which is resistant to tazobactam (TZP), ceftazidime (TAZ), cefepime (FEP), aztreonam (ATM), imipenem (IPM), meropenem (MEM), tobramycin (TOB), amikacin (AK), ciprofloxacin (CIP) and levofloxacin (LEV), and is sensitive to polymyxin B (PMB), Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter baumannii, Albicans Saccharomyces, Candida acruseii. The above strains can be purchased through conventional commercial channels, and the above strains are all stored in the laboratory of Xianyang Central Hospital.

[0051] Lactic acid bacteria: The four lactic acid bacteria numbered KD10, KD5, L57, and 7649 were all preserved by Laboratory 2065, Department of Bioengineering, College of Food Science and Engineering, Shaanxi University of Science and Technology.

[0052] Culture medium: (1) Lactic acid bacteria activation and fermentation medium (MRS broth medium): 2% glucose, 0.8% beef extract, 1% peptone, 0.5% yeast powder, 0.2% diammonium citrate, 0.2% potassium dihydrogen phosphate, 0.5% anhydrous sodium acetate, 0.005% manganese sulfate, 0.01% magnesium sulfate, 0.1% Tween-80; pH 6.2±0.2; sterilize with high pressure steam at 121°C for 20 min.

[0053] (2) Antibacterial punch test medium (MRS broth agar medium): 1.5% agar powder was added to MRS broth, pH was 6.2-6.4, and sterilized at 121°C for 20 min.

[0054] Example 1 Isolation and Identification of Lactobacillus paracasei KD10

[0055] 1. Isolation of Lactobacillus paracasei KD10

[0056] Weigh a certain amount of whole goat milk powder, add distilled water in a ratio of 1:8 (w / v) to make reconstituted goat milk, sterilize at 115°C for 10 minutes, add milk kefir grains (purchased from the Taobao store Tibet Linzhi Natural Snow Lotus Mushroom, the website is: https: / / item.taobao.com / item.htm?_u=u3mt71o1c4a&id=16772532386&spm=a1z09.2.0.0.7f4d2e8dwTV9kw), ferment at room temperature for 22 hours to obtain goat milk kefir, then filter out the kefir grains, rinse with sterile saline to activate once, repeat the operation and continue to activate 3 times, stir and crush the goat milk kefir obtained after the third activation, take 1mL and inject it into 9mL of sterile saline to mix well to make a diluent. Repeat the above steps until the dilution multiple reaches 10 -6 ~10 -8 . Pipette 0.1mL of diluent and spread it on the sterilized and cooled MRS agar plate aseptically, and culture it at 37℃ for 48h. After obvious single colonies are formed, select a plate with 30 to 80 single colonies, pick typical colonies, and purify them by streaking on MRS agar medium several times until the colony morphology on the entire plate is consistent. Select single colonies and inoculate them into MRS broth and culture them at 37℃ for 20h. Then, aseptically pipette 0.1mL of KD10 culture medium into a lyophilization tube, add 0.1mL of sterile skim milk, mix well, freeze-dry, and store frozen.

[0057] 2. Identification of Lactobacillus paracasei KD10

[0058] 2.1 Identification by fully automatic microbial mass spectrometer (VITEKMS, BioMérieux, France)

[0059] Strain KD10 was inoculated on Columbia blood agar plates and cultured at 37°C for 24 h. Pinpoint-sized colonies were evenly spread on the target plate, and 1 μL of matrix solution was added and mixed. After drying, the sample was loaded onto an automatic microbial mass spectrometer for sample analysis. The strain name was determined based on the characteristic peaks after comparison with the VITEK-MS mass spectrometer research library. The MALDI-TOF-MS identification results are shown in Figure 1 .

[0060] 2.2 Identification by fully automatic microbial identification instrument (VITEK 2, BioMérieux, France)

[0061] Take the colony of strain KD10, add 3 ml of 0.45% saline, adjust to a bacterial suspension with a McFarland turbidity of 0.5, insert the bacterial suspension into a Gram-positive bacillus identification card and place it on a microbial identification instrument. After the bacterial suspension is put on the machine, the biochemical reaction results are obtained. According to the biochemical results, the ASE8.01 database is compared to identify and determine the name of the bacterial species. The biochemical reaction identification results are shown in Table 1.

[0062] Table 1 Biochemical reaction identification results of strain KD10

[0063]

[0064] Note: +. Biochemical reaction positive; -. Biochemical reaction negative.

[0065] The mass spectrometry characteristic peaks of KD10 were analyzed using the Launch pad software of the VITEK-MS mass spectrometer research library (RUO). Figure 1 is the mass spectrum result of KD10. The mass spectrum peaks of KD10 m / z 2036, 4448, 6964, and 9396 are characteristic peaks of Lactobacillus paracasei. The identification result meets the credibility of more than 99%. Table 1 shows the biochemical reaction results of KD10. After comparison with the AES8.01 database, KD10 is Lactobacillus paracasei, and the identification result meets the credibility of more than 99%. In summary, the two methods identified strain KD10 as Lactobacillus paracasei, and it was deposited in the China Center for Type Culture Collection on September 7, 2023, with the accession number of CCTCC NO: M20232074.

[0066] Example 2 Optimization of fermentation conditions of Lactobacillus paracasei KD10 and analysis of fermentation product components

[0067] 1. Experimental methods

[0068] 1.1 Activation of lactic acid bacteria

[0069] The freeze-dried lactic acid bacteria powders numbered KD10, KD5, L57, and 7649 stored at low temperature (-18°C) were dissolved in 1 mL of sterile saline, inoculated into MRS broth culture medium, and kept at 37°C for 24 hours to obtain the first generation of activated lactic acid bacteria; then, they were inoculated into MRS broth culture medium at a 5% inoculation rate, and cultured at 37°C for 24 hours. The activation was repeated 3 times and the culture was refrigerated for later use.

[0070] 1.2 Preparation of fermentation supernatant and collection of bacterial cells

[0071] The activated lactic acid bacteria strains KD10, KD5, L57, and 7649 were inoculated into MRS broth culture medium at a 5% inoculation rate, cultured at 37°C for 24 hours, centrifuged at a speed of 10,000 r / m and a temperature of 4°C for 20 minutes, and the supernatant was filtered using a filter membrane with a pore size of 0.45 μm to obtain the fermentation broth supernatant. At the same time, the precipitated bacteria were collected and placed in a 4°C refrigerator for standby use.

[0072] 1.3 Determination of inhibition zone of bacteria and fermentation supernatant

[0073] Take an appropriate amount of bacteria and put it into a sterile test tube, add 1 mL of sterile saline, and mix thoroughly to make the viable count of 1×10 8 cfu / mL of bacterial suspension was used as standby. MRS agar culture dish was used as the test medium, and the bacterial suspension and lactic acid bacteria fermentation supernatant were used to perform punch method inhibition zone test respectively.

[0074] 1.4 Screening of lactic acid bacteria producing multidrug-resistant Pseudomonas aeruginosa inhibitors

[0075] Prepare MRS broth agar medium, use multidrug-resistant Pseudomonas aeruginosa as indicator bacteria, take 100 μL of indicator bacteria liquid, evenly spread it on the surface of the agar medium, and place the sterilized Oxford cup ( The wells were punched in MRS broth agar medium (7 mm), and the fermentation supernatants of lactic acid bacteria strains KD10, KD5, L57, and 7649 were used as antibacterial agents. Then 100 μL of the antibacterial agent was injected into the wells and cultured at 37 °C for 24 h. The diameter of the inhibition zone was observed and the length of the diameter was measured with a vernier caliper. The antibacterial effect was determined based on the pore diameter. Finally, the pH, acidity, and OD were used to determine the antibacterial effect. 600 , and the diameter of the inhibition zone were used as the determination indicators to further compare the antibacterial effects of the four lactic acid bacteria, and the strains with better antibacterial effects were selected through comprehensive judgment.

[0076] 1.5 Lactic acid bacteria growth assay

[0077] The growth of lactic acid bacteria was determined by turbidimetry. After the lactic acid bacteria were fermented, 3 mL of the suspension was placed in a cuvette. At the same time, another cuvette was prepared and poured with 3 mL of uninoculated culture medium as a control. The absorbance of the fermentation liquid was measured at a wavelength of 600 nm using an ultraviolet spectrophotometer to obtain the OD value. 600 The numerical value of .

[0078] 1.6 pH determination

[0079] The pH value of the fermentation broth was measured using a pH meter at 25°C.

[0080] 1.7 Optimization of fermentation conditions for the production of antibacterial agents by Lactobacillus paracasei KD10

[0081] Using MRS broth as fermentation medium, single factor experiments were conducted to study the effects of culture time, culture temperature and inoculation size on the production of antibacterial agents and determine the appropriate range of each factor.

[0082] 2. Experimental results

[0083] 2.1 Comparison of the antibacterial properties of lactic acid bacteria and supernatant against multidrug-resistant Pseudomonas aeruginosa

[0084] The results are as follows Figure 2As shown in the figure, the diameter of the inhibition zone of the lactic acid bacteria suspension against multidrug-resistant Pseudomonas aeruginosa is 7.9-8.7 mm. Since the pore size of the Oxford cup is 7 mm, the net diameter of the inhibition zone of the lactic acid bacteria suspension is 0.9-1.7 mm. It can be seen that the inhibition of the bacteria against Pseudomonas aeruginosa is extremely insignificant and has almost no antibacterial effect. However, the lactic acid bacteria fermentation supernatant has a good antibacterial effect. Among them, there was no significant difference in the diameter of the inhibition zone of lactic acid bacteria strains KD10 and KD5 against Pseudomonas aeruginosa (P>0.05), and there was a significant difference in the diameter of the inhibition zone of lactic acid bacteria strains L57 and 7649 against Pseudomonas aeruginosa (P<0.05), with the diameters being 18.34±0.11mm, 16.55±0.59mm, 14.57±0.25mm, and 14.29±0.34mm, respectively. The order of the diameters of the inhibition zones was KD10>KD5>L57>7469. The above results showed that the effective antibacterial components of lactic acid bacteria were mainly distributed in the fermentation supernatant, and there were almost no antibacterial components in the bacterial sludge. In addition, the supernatant of the fermentation broth of strains KD10 and KD5 had a better effect.

[0085] 2.2 Effect of culture time on lactic acid bacteria growth, antibacterial effect and acid production

[0086] On the basis of the preliminary screening, multidrug-resistant Pseudomonas aeruginosa was used as the indicator bacteria to rescreen the antibacterial effects of KD10, KD5, 7469, and L574 strains. The wet weight of the bacteria, pH, acidity, and diameter of the inhibition zone were used as detection indicators. The four strains were inoculated with 5% of the inoculation amount into MRS broth medium and cultured at a constant temperature of 37°C. The samples were tested at 12h, 18h, and 24h, respectively. The results are as follows: Figure 3 As shown. Figure 3It can be seen that with the increase of culture time, pH showed a downward trend, acidity showed an upward trend, bacterial weight continued to increase, and the diameter of the inhibition zone continued to increase. After 24 hours of fermentation, all indicators reached the upper limit. The pH of KD10, KD5, 7469, and L57 had significant differences (P<0.05), which were 3.48, 3.57, 3.75, and 3.78, respectively; the wet weight of bacterial cells had significant differences (P<0.05), which were 1. 052g, 0.977g, 0.723g, 0.566g; acidity had significant differences (P<0.05), which were 194°T, 182°T, 177°T, 162°T; inhibition zone diameter had significant differences (P<0.05), which were 18.22mm, 17.44mm, 16.91mm, 14.43mm, and the antibacterial effect was KD10>KD5>7469>L57. After adding protective agents and freeze-dried, lactic acid bacteria can be used as food functional factors, and lactic acid can also be extracted and utilized as a by-product of fermentation. Therefore, the inhibition zone diameter, cell weight and acidity were used as the main screening reference indicators to screen out KD10 with better antibacterial effect, fast growth rate and high acid production for subsequent research.

[0087] 2.3 Effects of fermentation conditions on bacteriocin production by Lactobacillus paracasei KD10

[0088] (1) Cultivation time

[0089] Under the condition of ensuring that the inoculation amount is 5% and the culture temperature is constant at 37℃, the culture time is used as a variable to study the effect on the growth of Lactobacillus paracasei KD10. The pH value of the MRS broth medium was adjusted to 7.0, and KD10 was inoculated into it according to the inoculation amount of 5%, and cultured at 37℃ for 12h, 18h, 24h, 30h, 36h, 42h, and 48h, respectively. The OD value of each culture time was measured. 600 and pH, and then centrifuged at 8000r / min for 15min to obtain the supernatant, and the antibacterial test was performed. The results are shown in Figure 4 .

[0090] Depend on Figure 4 It can be seen that the OD of KD10 600 As the culture time increases, it increases synchronously, and finally tends to be flat, reaching a peak of 1.896 at 42h; pH decreases with the increase of culture time, and tends to be stable at 30h; the diameter of the inhibition zone of the fermentation supernatant of Lactobacillus paracasei KD10 against multidrug-resistant Pseudomonas aeruginosa increases first and then decreases. When cultured for 42h, the diameter of the inhibition zone reaches the maximum, which is 19.11mm.

[0091] (2) Inoculation volume

[0092] On the basis of the single factor study on culture time, the culture time was kept at 42 h and the culture temperature was kept at 37 °C. KD10 was inoculated into MRS broth medium at inoculation amounts of 1.5%, 3%, 4.5%, and 6% for 42 h. The OD values ​​of each inoculation amount were measured. 600 and pH, and then centrifuged at 8000r / min for 15min to obtain the supernatant, and the inhibition zone test was performed. The results are shown in Figure 5 .

[0093] Depend on Figure 5 It can be seen that KD10 OD 600 There was little change in pH, OD 600 The values ​​were all between 1.765-1.823, and the pH was between 3.89-3.92; the diameter of the inhibition zone increased first and then decreased. When the inoculation amount was 3%, the diameter of the inhibition zone was the largest, which was 18.52 mm.

[0094] (3) Culture temperature

[0095] Keeping the culture time at 42h and the inoculum at 3%, KD10 was inoculated into MRS broth medium for culture at 33℃, 35℃, 37℃, 39℃, and 41℃, and the OD at each culture temperature was measured. 600 and pH, and then centrifuged at 8000r / min for 15min to obtain the supernatant, and the inhibition zone test was performed. The results are shown in Figure 6 .

[0096] Depend on Figure 6 It can be seen that as the culture temperature increases, KD10 OD 600 The diameter of the inhibition zone showed a trend of increasing first and then decreasing, reaching the maximum value at 37℃. 600 and inhibition zone diameters were 1.813 and 19.61 mm, respectively.

[0097] (4) Determination of the fermentation conditions for the production of antibacterial agents by Lactobacillus paracasei KD10

[0098] According to the single factor test results of culture temperature, culture time and inoculum size, the culture temperature was selected as 35℃, 37℃ and 39℃, the culture temperature was 30h, 36h and 42h, and the inoculum size was 2%, 3% and 4% for further experiments to determine the appropriate range. 600 The value (R1) and the diameter of the inhibition zone of multidrug-resistant Pseudomonas aeruginosa were taken as indicators (R2). The test results are shown in Table 2.

[0099] Table 2 Experimental results of fermentation conditions on bacteriocin production by Lactobacillus paracasei KD10

[0100]

[0101] As shown in Table 2, when the culture temperature is 35°C-39°C, the culture temperature is 30h-42h and the inoculation amount is 2%-4%, the OD value of the fermentation liquid is 1.8213-2.1372, and the diameter of the inhibition zone is 17.22-20.2mm.

[0102] Example 3 Extraction of bacteriocin from fermentation product of Lactobacillus paracasei KD10

[0103] 1. Bacteriocin Extraction

[0104] The fermentation liquid obtained under the optimal conditions selected by Lactobacillus paracasei KD10 according to Example 2 was centrifuged at a rotation speed of 10000 r / m and a temperature of 4° C. for 20 min to obtain a cell-free fermentation supernatant (CFS, Cell free supernatants).

[0105] Alcohol precipitation extraction: Use a vacuum rotary evaporator to concentrate CFS 10 times, add 95% anhydrous ethanol, mix thoroughly and adjust the anhydrous ethanol concentration to 75%, precipitate at 4℃ for 12h, centrifuge (10000r / m, 20min) to obtain the precipitate and aqueous phase respectively, and determine their antibacterial effects. Use a Roche biochemical analyzer to determine the protein concentration of the supernatant and precipitate, and calculate the specific activity, total activity and recovery rate through the relationship (1), (2), and (3).

[0106] Organic solvent extraction: Based on the principle of like dissolves like, CFS was concentrated 4 times using a vacuum rotary evaporator, fully mixed with ethyl acetate at a ratio of 1:3, placed in a shaker at a speed of 120 r / min for 12 hours, then left to rest for 2 hours, and the extraction was repeated 3 times to obtain an organic phase and an aqueous phase, respectively. The organic phase was then concentrated using a rotary evaporator at a temperature of 60°C and a speed of 90 r / min until the ethyl acetate was completely evaporated, and then redissolved in pure water to determine its antibacterial effect. The protein concentrations of the organic and aqueous phases were determined using a Roche biochemical analyzer, and the specific activity, total activity, and recovery rate were calculated using equations (1), (2), and (3).

[0107] Ammonium sulfate precipitation method: add 60%, 70%, 80%, 90%, 100% saturation ammonium sulfate solution to CFS, stir for 2 hours using a magnetic stirrer, leave for 12 hours, centrifuge (10000r / m, 20min) to obtain precipitate and supernatant, and measure their antibacterial effect. Use Roche biochemical analyzer to measure the protein concentration of precipitate and water, and calculate the specific activity, total activity and recovery rate by equations (1), (2), and (3).

[0108] Specific activity (AU / mg) = titer (AU / mL) / protein concentration (mg / mL) (1);

[0109] Total activity (AU) = specific activity (AU / mg) × volume of protein-containing solution (mL) (2);

[0110] Recovery rate (%) = total activity (AU) / total activity of supernatant (AU) (3).

[0111] 2. Ultrafiltration purification of bacteriocin KD10 and activity determination of each component

[0112] Ultrafiltration technology is used to pressurize the sample through filter membranes of different pore sizes through pumping, so as to achieve the purpose of purifying, separating and concentrating the original solution. Filter membranes with cutoff molecular weights of less than 3k, 3-5k, 5-10k and greater than 10k are selected for filtration respectively, and antibacterial tests are carried out on different components obtained by ultrafiltration to determine their activity.

[0113] 3. Preparation of bacteriocin

[0114] Firstly, the fermentation broth prepared by Lactobacillus paracasei KD10 under the optimal fermentation conditions was centrifuged to obtain the fermentation supernatant, and then ethyl acetate was added to extract the obtained organic phase, which was then concentrated at a temperature of 60°C and a rotation speed of 90r / min until the ethyl acetate was completely evaporated, and then it was redissolved in pure water, and finally ultrafiltration was used to obtain the component with the best antibacterial effect. After freezing at -80°C for 24 hours, it was freeze-dried in a vacuum freeze dryer for 48 hours to obtain bacteriocin freeze-dried powder, which was stored at -20°C for future use.

[0115] 4. Determination of bacteriocin inhibition spectrum

[0116] The bacteriostatic test was carried out using the indicator bacteria shown in Example 2.

[0117] 5. LC-MS / MS identification and structure prediction of bacteriocins

[0118] The bacteriocin lyophilized powder was prepared into a solution with a concentration of 0.1 g / mL, and then separated, purified and identified by LC-MS / MS. The identified peptides were compared in UniProtKB, and the three-dimensional structure of the bacteriocin was predicted using Swiss-Mode.

[0119] 6. Determination of minimum inhibitory concentration and minimum bactericidal concentration of KD10 bacteriocin

[0120] Determine the minimum inhibitory concentration (MIC) of the bacteriocin freeze-dried powder, take the indicator bacteria in the logarithmic phase, and prepare a bacterial suspension (the bacterial suspension concentration is 10 6CFU / mL), weigh 100 mg of KD10 bacteriocin freeze-dried powder, dilute with 4 mL of nutrient broth and add to 96 microwell plates, add 300 μL of the diluted stock solution to the first well, add 150 μL of nutrient broth to the 2nd to 11th wells, aspirate 150 μL from the first well, add to the second well and mix evenly, aspirate 150 μL into the third well, and so on, aspirate 150 μL from the 10th well and discard, the 11th well is the positive control. The bacterial suspension and nutrient broth medium without adding bacteriocin are the negative control group. Culture at 35°C for 24 hours for the determination of MIC value. Determine the OD of the test group and the control group 600 The difference is taken as the mass concentration of bacteriocin freeze-dried powder, OD 600 The difference is used as the vertical axis to draw a curve, the inflection point is MIC, and the concentration corresponding to the dilution with the minimum inhibitory effect is the minimum inhibitory concentration MIC.

[0121] Based on the MIC determination, the concentrations of bacteriocin were set to MIC, 2MIC, 4MIC, and 8MIC, respectively. 100 μL was mixed with the indicator bacteria, spread on broth agar medium, and cultured at 37°C for 24 hours. The number of colonies on the plate was observed. If the number of colonies was less than 5, the inhibitory concentration was the minimum bactericidal concentration (MBC).

[0122] 7. Study on the stability of antibacterial agents

[0123] The effects of temperature, pH, enzyme, storage temperature and ultraviolet radiation on the antibacterial activity of bacteriocins produced by Lactobacillus paracasei KD10 were studied respectively.

[0124] 8. Results and Analysis

[0125] 8.1 Effect of extraction method on antibacterial effect and recovery rate of bacteriocin

[0126] Using multidrug-resistant Pseudomonas aeruginosa as the indicator bacteria, the bacteriocins in the fermentation broth of Lactobacillus paracasei KD10 were extracted according to the above three extraction methods. The results are shown in Table 3.

[0127] As shown in Table 3, when the alcohol precipitation method is used for crude extraction, relatively more bacteriocin remains in the aqueous phase, and the activity of bacteriocin is lost to a certain extent during the ethanol extraction process; when the ammonium sulfate precipitation method is used, the effect is relatively good when the concentration is 70% and 80%, but there is also a certain antibacterial effect in the supernatant, indicating that the extraction of bacteriocin is incomplete; when ethyl acetate is used for extraction, only a small amount of bacteriocin is contained in the aqueous phase, and the antibacterial effect is the best. As shown in Table 4, the total activity and recovery rate of ethyl acetate extraction are the highest, and the effects of ammonium sulfate precipitation and alcohol precipitation are equivalent. In summary, ethyl acetate is used to extract bacteriocin in the subsequent process.

[0128] Table 3 Effect of extraction method on the antibacterial activity of bacteriocin of KD10

[0129]

[0130] Table 4 Effect of extraction method on bacteriocin recovery of KD10

[0131]

[0132] 8.2 Effect of ultrafiltration on the activity and recovery of bacteriocin components

[0133] KD10 was extracted with ethyl acetate to obtain fractions less than 3k, 3-5k, 5-10k, and greater than 10k. The antibacterial activity against multidrug-resistant Pseudomonas aeruginosa was determined using the fermentation supernatant as a control. Figure 7 As shown in the figure, the diameters of the inhibition zones of the supernatant solution, less than 3k, 3-5k, 5-10k, and greater than 10k of KD10 are 28.69±0.14mm, 28.58±0.13mm, 15.18±0.22mm, 17.62±0.21mm, and 7.28±0.27mm, respectively. It can be seen that the antibacterial effect of the supernatant and the supernatant solution after being treated with an ultrafiltration membrane less than 3k has no significant change, while the antibacterial effect of 3-5k, 5-10k, and greater than 10k has decreased. KD10 was ultrafiltered on the basis of ethyl acetate extraction and rehydration. According to Table 5, the total activity and recovery rate of less than 3k are the best, which are 8.48×10 4 , 53.5%, 8.08×10 4 , 49.50%, indicating that the substances with relatively good antibacterial effects are mainly concentrated in small molecules. After ultrafiltration, the KD10 solution with a concentration of less than 3k was observed. The supernatant became clear visibly. The main reason was that a large amount of impurities in the supernatant solution were filtered out after ultrafiltration. By calculating the volume ratio of the supernatant solution to the ultrafiltration liquid, the ultrafiltration purification multiple of KD10 was 1.98 times. The supernatant after ultrafiltration was retained for later use, concentrated by rotary evaporation, and further separated and purified. The protein concentration of the precipitate and water was determined using a Roche biochemical analyzer, and the specific activity, total activity, and recovery rate were calculated using the relationship (1), (2), and (3).

[0134] Table 5 Effect of ultrafiltration on the recovery rate of KD10 bacteriocin

[0135]

[0136] 8.3 LC-MS / MS purification and identification of bacteriocins

[0137] The bacteriocin of Lactobacillus paracasei (components with less than 3k) with the strongest antibacterial effect on multidrug-resistant Pseudomonas aeruginosa obtained in 8.2 was purified and its amino acid sequence was identified by LC-MS / MS. The liquid chromatogram is shown in Figure 8As shown, the amino acid series of bacteriocin is shown in Table 6. The peptide sequences corresponding to bacteriocin No. 1-5 in Table 6 are numbered as SEQ ID NO: 1-SEQ ID NO: 15, respectively.

[0138] Table 6 Amino acid sequence of bacteriocin

[0139]

[0140]

[0141] In the antibacterial test of each component of ultrafiltration, it was found that bacteriocins with a molecular weight less than 3k had the best antibacterial effect. The main reason is that low molecular weight peptides have good structural stability. Therefore, the molecular weight of the peptide will be used as an important reference for the screening of bacteriocin segments; studies have shown that bacteriocins have hydrophilic and hydrophobic groups that can form an amphiphilic structure. When entering the hydrophobic area of ​​the bacterial cell membrane, they can destroy the bacterial cell membrane and cause the bacteria to die. Therefore, hydrophobicity is also an important reference for screening peptides; the positive charge of bacteriocins plays a core role in the electrostatic interaction between bacterial cell membranes. It can change the charge number of the bacterial cell membrane, causing cell membrane damage and death. Most bacteriocins have positive charges ranging from +2 to +9. Therefore, the number of charges is also an important reference for screening. In summary, considering the molecular weight, hydrophobicity, and charge number at the same time, peptides with 8-10 amino acids were selected, namely VISAVAQTNA, PQGPQGPRG, GPSGFYRIN, and GVQDFDRLR. The secondary mass spectrum of the selected peptide KD10 is shown in Figure 9-12 .

[0142] Swiss-Mode (lhttps: / / swissmodel.expasy.org / interactive / cJcUUM / models / ) was used to predict the three-dimensional structure of bacteriocins. The bacterial cell membrane is destroyed by bacteriocins to a large extent due to the secondary structure. The most common secondary structures in bacteriocins are α-helix, β-fold and irregular curl. The above peptides less than 3k all have α-helix structure. Studies have shown that the hydrophilic and hydrophobic structures of α-helix can cause deformation of bacterial cell membranes, or attach to negatively charged cell membranes through electrostatic interactions, thereby interacting with the membrane.

[0143] 8.4 Antibacterial spectrum of bacteriocin produced by Lactobacillus paracasei KD10

[0144] The antibacterial results of bacteriocin (concentration of 0.1 g / mL) produced by Lactobacillus paracasei KD10 against 7 common clinical pathogens (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter baumannii, Candida albicans, and Candida krusei) are shown in Table 7.

[0145] The results showed that KD10 had a positive effect on Staphylococcus aureus (G + ), Klebsiella pneumoniae (G - ), Escherichia coli (G - ), Proteus mirabilis (G - ), Acinetobacter baumannii (G - ) all had significant antibacterial effects, with the diameter of the inhibition zone ranging from 20 to 29 mm, among which Acinetobacter baumannii had the best antibacterial effect; it had no antibacterial effect on yeasts such as Candida albicans and Candida krusei, with the diameter of the inhibition zone ranging from 7.2 to 7.3 mm, which was close to the diameter of the Oxford cup ( 7mm). In summary, this shows that the bacteriocin produced by KD10 has a certain broad-spectrum antibacterial effect on both Gram-positive and Gram-negative bacteria, which can provide a reference for subsequent development and utilization.

[0146] Table 7 Antibacterial spectrum of KD10 bacteriocin

[0147]

[0148]

[0149] 8.5 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of bacteriocins against multidrug-resistant Pseudomonas aeruginosa

[0150] The MIC and MBC of the antibacterial agent produced by Lactobacillus paracasei KD10 against multidrug-resistant Pseudomonas aeruginosa were determined by the two-fold dilution method. Figure 13 to Figure 14 shown.

[0151] from Figure 13 to Figure 14 It can be seen that the bacteriocin produced by KD10 still has a significant antibacterial effect after 6 consecutive dilutions, but after the 7th dilution, the antibacterial effect is significantly weakened, and its MIC is 0.78 mg / mL. Therefore, the concentration is greater than the MIC in OD within 24 h. 600 The value remained at 0; in the 5 groups of experiments where the bacteriocin concentration was less than the MIC, the OD 600 Mainly kept at 0, the main reason is that the bacteria are in the delayed period, 6-14h OD 600 Logarithmic growth trend, 14-24h OD 600 Maintain a high trend. Based on the MIC determination, the two-fold dilution method was used to dilute the bactericidal concentrations with different concentrations. The results showed that the MIC of KD10 bacteriocin was 0.78 mg / mL and the MBC was 3.12 mg / mL.

[0152] 8.6 Effect of temperature on the stability of KD10 bacteriocin

[0153] KD10 bacteriocin was treated at -20℃, 0℃, 20℃, 40℃, 60℃, 80℃, 100℃, and 120℃ for 30min, and multidrug-resistant Pseudomonas aeruginosa was used as the indicator bacteria. The test results are shown in Fig.15 .

[0154] Depend on Fig.15 It can be seen that there is no significant difference between the experimental group and the control group (P>0.05). Compared with the control group without any treatment, the diameter of the inhibition zone of KD10 was shortened by 19.7% after being treated at -20℃; when treated at 100℃, the diameter of the inhibition zone of KD10 was shortened by 9.9%; when treated at 121℃, the diameter of the inhibition zone of KD10 was shortened by 12.2%. It can be seen that KD10 bacteriocin has good thermal stability. Therefore, KD10 bacteriocin has good heat resistance and is not significantly affected by temperature, especially high temperature, in the application of food industry or medicine.

[0155] 8.7 Effects of acid and base on the stability of KD10 bacteriocin

[0156] The pH of the KD10 bacteriocin solution was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, respectively. The multidrug-resistant Pseudomonas aeruginosa was used as the indicator bacteria. The test results are shown in Fig.16 .

[0157] Depend on Fig.16 It can be seen that there is no significant difference between the experimental group and the control group (P>0.05). Compared with the control group without any treatment, the diameter of the inhibition zone of KD10 is shortened by 7.84% at pH 2; and by 7.3% at pH 11. It can be seen that the KD10 bacteriocin is not greatly affected by pH. Therefore, it has a good reference value for providing preservatives for the food industry, medicine and health, animal husbandry and other industries.

[0158] 8.8 Effect of enzymes on the stability of KD10 bacteriocin

[0159] The KD10 bacteriocin solution was treated by adding pepsin, trypsin, papain, catalase, and α-amylase, respectively. The optimum pH of each protease was adjusted using 1 mol / L HCl and 2 mol / L NaOH. The pH of pepsin, trypsin, papain, catalase, and α-amylase was adjusted to 2.0, 7.0, 5.7, 7.0, and 7.0, respectively. 1 mL / L of each of the five proteases was added to the bacteriocin and mixed. The mixture was placed in a water bath at 37°C for 2 hours, and then treated at 80°C for 10 minutes to eliminate the activity of the enzyme. The pH was then adjusted to be consistent with the original supernatant. Multidrug-resistant Pseudomonas aeruginosa was used as the indicator bacteria. The test results are shown in Fig.17 .

[0160] Depend on Fig.17 It can be seen that after adding trypsin, there was a significant difference between the experimental group and the control group (P<0.05), and KD10 decreased by 42.2%; after adding pepsin, there was a significant difference between the experimental group and the control group (P<0.05), and KD10 decreased by 38.4%, indicating that the bacteriocin produced by KD10 can be decomposed by pepsin and trypsin, indicating that bacteriocin is a type of protein. There was no significant difference between the experimental group with added papain, catalase, and α-amylase and the control group (P>0.05). Among them, KD10 decreased by 9.2% after adding papain, decreased by 7.8% after adding catalase, and decreased by 1.6% after adding α-amylase, indicating that the bacteriocin produced by KD10 has a strong tolerance to papain, catalase, and α-amylase. Among them, papain contains non-protein amino acids and is not greatly affected. Bacteriocins are substances with small molecular weight and stable structure, and are not greatly affected by catalase and α-amylase.

[0161] 8.9 Effect of storage conditions on the stability of KD10 bacteriocin

[0162] KD10 bacteriocin was stored at -20℃, 4℃, and 25℃ for 0d, 15d, 30d, 60d, and 90d. The multidrug-resistant Pseudomonas aeruginosa was used as the indicator bacteria. The test results are shown in Table 1. Fig.18 .

[0163] Depend on Fig.18 It can be seen that the diameter of the inhibition zone of KD10 bacteriocin stored under different conditions showed a gradual decrease trend. Among them, the change rate at -20℃ and 4℃ was relatively slow. After 90 days of storage at -20℃, the diameter of the inhibition zone did not change significantly (P>0.05), and KD10 decreased by 3.2%. After 90 days of storage at 4℃, KD10 decreased by 4.3%. It can be seen that it has good stability in low-temperature freezing and refrigeration. After 90 days of storage at 25℃, KD10 decreased by 7.9%.

[0164] 8.10 Effect of UV light on the stability of KD10 bacteriocin

[0165] KD10 bacteriocin was pretreated with ultraviolet light and irradiated under 20W ultraviolet light for 0h, 6h, 12h, 18h, 24h, and 48h, respectively. Multidrug-resistant Pseudomonas aeruginosa was used as the indicator bacteria. The test results are shown in Fig.19 .

[0166] Depend on Fig.19 It can be seen that there is no significant difference between the experimental group and the control group (P>0.05), and the KD10 bacteriocin has almost no effect on the antibacterial effect of Pseudomonas aeruginosa after ultraviolet irradiation. It can be seen that the antibacterial agent has good stability under ultraviolet irradiation.

[0167] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Lactobacillus paracasei that produces bacteriocins that inhibit multidrug-resistant Pseudomonas aeruginosa, characterized in that: The preservation number of the Lactobacillus paracasei is CCTCC NO: M20232074.

2. A Lactobacillus paracasei bacteriocin for inhibiting multidrug-resistant Pseudomonas aeruginosa, characterized in that: The Lactobacillus paracasei bacteriocin is obtained from the supernatant of the fermentation liquid of Lactobacillus paracasei after fermentation in MRS broth, wherein the preservation number of the Lactobacillus paracasei is CCTCC NO: M20232074.

3. The Lactobacillus paracasei bacteriocin as claimed in claim 2, characterized in that The amino acid sequence of the Lactobacillus paracasei bacteriocin includes the sequences shown in SEQ ID NOs. 1-15.

4. A method for preparing a Lactobacillus paracasei bacteriocin according to claim 2 or 3, characterized in that: The following steps are involved: S1: The culture solution of Lactobacillus paracasei activated in MRS broth was inoculated into the fermentation medium at an inoculation rate of 2-4%, and cultured at 35-39° C. for 30-42 hours, and then centrifuged at room temperature to obtain the supernatant; S2: After concentrating the supernatant, add ethyl acetate, mix thoroughly, place on a shaker for extraction, then let stand and pour out the ethyl acetate layer, add ethyl acetate to the raffinate for extraction twice, combine the three ethyl acetate extracts, evaporate to dryness, add purified water for re-dissolution, and obtain a bacteriocin aqueous solution; S3: ultrafiltering the bacteriocin aqueous solution through 10K, 5K and 3K filter membranes respectively, collecting the components less than 3K for freeze-drying, and obtaining the Lactobacillus paracasei bacteriocin.

5. The bacteriocin according to claim 2, characterized in that In S1, the culture conditions are: inoculation amount 2-4%, culture temperature 35-39°C, and culture time 30-42h.

6. The bacteriocin according to claim 2, characterized in that In S2, the amount of ethyl acetate added is 3 times the volume of the concentrated supernatant; the conditions for the oscillation extraction are: 120 rpm oscillation extraction for 12 hours; and the standing time is 2 hours.

7. Use of Lactobacillus paracasei or its fermented liquid as claimed in claim 1 in preparing a medicine for inhibiting multidrug-resistant Pseudomonas aeruginosa.

8. Use of the Lactobacillus paracasei bacteriocin as claimed in claim 2 or 3 in any one of the following: (1) Application in the preparation of drugs for inhibiting multidrug-resistant Pseudomonas aeruginosa; (2) Use in the preparation of drugs for inhibiting Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Proteus mirabilis and / or Acinetobacter baumannii.

9. An antibacterial agent, characterized in that It comprises the Lactobacillus paracasei bacteriocin according to claim 2 or 3, and the antibacterial agent comprises a bacterial agent that inhibits multi-drug resistant Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Proteus mirabilis and / or Acinetobacter baumannii.

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