Lactococcus lactis capable of producing nisin as well as construction and application of engineering bacteria of lactococcus lactis

Abstract

The invention relates to lactococcus lactis capable of producing nisin as well as construction and application of engineering bacteria of the lactococcus lactis. The invention provides a Lactococcus lactis ZLL111 strain, which is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No.31943. The Lactococcus lactis ZLL111 strain has the advantages that the Lactococcus lactis ZLL111 strain is prepared from Lactococcus lactis; the lactococcus lactis provided by the invention has a relatively strong antibacterial effect, produces nisin, has the characteristics of acid resistance, high temperature resistance, protease resistance and the like, and has a relatively strong anti-salmonella effect in vitro. Genetically engineered bacteria are constructed by using the bacteriocin, and the expression quantity is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to Lactococcus lactis producing nisin, construction of its secretory engineering bacteria and applications thereof. Background Art

[0002] Salmonella, as a zoonotic Gram-negative pathogen, is extremely widespread in the global farm environment, posing a continuous health threat. In livestock and poultry farming, this bacterium is one of the main pathogens causing septicemia, enteritis and severe diarrhea in piglets, not only resulting in high mortality, growth retardation and decreased production performance of young animals, but also bringing significant economic losses. More worryingly, Salmonella has shown a high degree of drug resistance in the farm environment, and multi-drug resistance is particularly common and severe, making it a very challenging common pathogen in veterinary clinical practice.

[0003] The harm of Salmonella is not limited to the breeding industry. As an important zoonotic pathogen, it can enter the food chain through contaminated livestock and poultry products (such as meat, eggs, milk), and then infect humans. According to statistics, Salmonella infection accounts for an alarming 70%-80% of foodborne diseases caused by bacteria globally, and is one of the main culprits leading to human food poisoning and gastroenteritis, posing a major threat to public health safety. Therefore, effectively controlling Salmonella infection in the breeding link is of double significance for ensuring animal health, food safety and human health.

[0004] For a long time, antibiotics have been the main means for preventing and treating Salmonella diseases. However, the long-term, extensive and often unscientific use of antibiotics in the breeding industry (such as sub-therapeutic doses used as growth promoters) has directly led to the continuous emergence, screening and spread of drug-resistant Salmonella strains. A number of domestic and foreign monitoring data clearly show that the overall drug resistance rate of Salmonella to various commonly used antibiotics has climbed above 80%, and there have even been reports of drug resistance to some antibiotics regarded as the "last line of defense" (such as carbapenems). The spread of drug resistance has made the clinical treatment of Salmonella infection increasingly difficult, with decreased efficacy, extended treatment cycles, increased costs, and even the risk of running out of drugs.

[0005] Bacteriocins, as a class of potential alternatives to antibiotics, have received increasing attention in recent years. They are proteins or polypeptides with antibacterial activity produced by certain bacteria through ribosomal synthesis mechanisms. Different from traditional antibiotics (usually secondary metabolites of microorganisms that exert antibacterial effects by interfering with biosynthetic pathways such as cell wall synthesis, protein synthesis, or nucleic acid replication), the mechanism of action of bacteriocins is often more direct and physical. Many bacteriocins (especially those targeting Gram-negative bacteria) can specifically bind to the cell membranes of target bacteria, causing cell content leakage and transmembrane potential collapse by perforating or disrupting membrane integrity, ultimately rapidly killing the bacteria. This unique "punching" mechanism enables bacteriocins to also have a powerful inhibitory or killing effect on many pathogenic bacteria that have developed resistance to traditional antibiotics, demonstrating the potential to overcome drug resistance. Bacteriocins have the characteristics of high safety, good stability, and no residue, and are currently attracting increasing attention in the field of food preservation. Currently reported bacteriocins are mainly applied to food preservation and mainly used to inhibit Gram-positive bacteria, while most of these pathogenic bacteria such as Salmonella in aquaculture production are Gram-negative pathogenic bacteria. There are very few bacteriocin products that are truly applied in large-scale aquaculture production. The structural and functional diversity of bacteriocins and large-scale preparation are still bottlenecks restricting their application in aquaculture production. Summary of the Invention

[0006] The first object of the present invention is to provide a Lactococcus lactis that produces nisin.

[0007] The second object of the present invention is to provide the use of the Lactococcus lactis.

[0008] The third object of the present invention is to provide a genetically engineered bacterium based on the bacteriocin of the Lactococcus lactis and its application.

[0009] The present invention screened a Lactococcus lactis ZLL111 with excellent growth performance and acid and bile salt tolerance from fecal samples and deposited it in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 31943.

[0010] The Lactococcus lactis ZLL111 of the present invention can produce class I bacteriocin (nisin), and this bacteriocin still has antibacterial activity after being treated at pH 4.1 and with catalase, still has antibacterial activity after being treated at high temperature (40 - 100 °C), and still has antibacterial activity after being treated with proteases (trypsin, papain, proteinase K, pepsin).

[0011] Furthermore, the present invention provides a bacterial agent containing the above-mentioned strain. The bacterial agent can be a single agent composed of the above-mentioned strain and a carrier, or a compound bacterial agent composed of other probiotics. Here, the probiotics preferably have the property of being resistant to the bacteriocin.

[0012] Furthermore, the present invention also provides the bacteriocin produced by the Lactococcus lactis. Its sequence is shown in SEQ ID No.2. The specific sequence is as follows: MLSLKLAANNIKKGFKSFAPFLMASVTMFVMLFVTASIALSPSIHKLRGGSSLSQIMSFALIVLSIFAVLILIYSYRFLQTQRSKEFGLYDILGFGKTRIVGVAFLELLLSYIITFVVGTICGIAFSKFLFLVKHFMIGGNYFNLVISPTAILLLAILFFVFFLVLMMIGVWIIWRSSSLDLLREESKGEKEPKSNLFFAIAAVIRPNNFAGAALTVEDPMAAIMKFFIAVLLVIFGTYLFYISFTVWYLKLKKKRPSYYKPNNFITTSSMLYRMKANAVGLGNITILLSMTIVTVVVSLGVFLGTENSVKTYYTREAKTYSVANNTDVKQDIERIKSAAASKNIEIKNISDMYYAQDLQADRVKSSKDQFITSNKQGVMFDKNSYFATLTTAQTLKSLGNKDIPSLKDNQVLLVDISKNKTSFDSKIKSIQWYGETYQVADTLNSVKNFPSSSVVTVSSKVMMIVFANNQAFDKGLANLNKTISGDQGFSNLASTSIIFDIKPVDEKRFTKAFKEEFKDNKDLSISYRSEALQDQRAQIGGLVFVGFVLGISFILGAALIIYYKQLSEGAQDKRSFKILQEVGLSKEEVQKTIKSQVRLIFFLPLVITIAHFAGAYLMIEKIIMLFEINDRSVIFTISLATIAILAIIYYLIYKATSRVYYKIRIYW

[0013] The bacteriocin of the present invention also includes bacteriocins with equivalent activity formed by substituting, deleting or adding one or several amino acids in the above sequence.

[0014]

[0015] Furthermore, the present invention also includes complementary sequences of the above-mentioned coding genes, transcribed RNAs, and the like.

[0016] Furthermore, the present invention also provides a recombinant vector containing the above-mentioned coding gene. The vector can be a cloning vector or an expression vector.

[0017] Furthermore, the present invention also provides a genetically engineered bacterium transformed with the above-mentioned vector. The starting bacterium of the genetically engineered bacterium can be Saccharomyces cerevisiae. In one embodiment of the present invention, the starting bacterium of the genetically engineered bacterium is a yeast.

[0018] Furthermore, the present invention also provides a product containing the above-mentioned Lactococcus lactis, the above-mentioned bacterial agent, the above-mentioned bacteriocin, or the genetically engineered bacterium. The products include, but are not limited to: feed additives, feeds, food additives, foods, preservatives, drugs, or health products.

[0019] Furthermore, the present invention also provides uses of the above-mentioned Lactococcus lactis, the above-mentioned bacterial agent, the above-mentioned bacteriocin, or the genetically engineered bacterium, including but not limited to applications in the preparation of antibacterial products.

[0020] Furthermore, the present invention also provides a method for preparing a bacteriocin, which is obtained by culturing the above-mentioned Lactococcus lactis or genetically engineered bacterium.

[0021] The Lactococcus lactis (Lactococcus lactis) ZLL111 of the present invention has good culture characteristics and good antibacterial ability. The bacteriocin has the characteristics of acid resistance and high temperature resistance, and still has antibacterial activity after being treated with protease. The genetically engineered bacterium constructed by the present invention can further improve the expression of the bacteriocin and enhance the antibacterial ability. Description of the Drawings

[0022] Figure 1 . is the culture growth curve of the strain of the present invention;

[0023] Figure 2. is the stability test result of the bacteriocin of the strain of the present invention, wherein Figure 2A shows the influence of different protease treatments, Figure 2B shows the influence of different temperature treatments, Figure 2C shows the influence of different pH treatments;

[0024] Figure 3 is the electrophoresis diagram of the bacteriocin induced and expressed by the genetically engineered bacterium of the present invention. Detailed Embodiments

[0025] The following specific embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0026] Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art.

[0027] Example 1. Strain screening and identification

[0028] Using MRS medium as the basal medium, pig fecal samples were collected. Weigh 25 g and place it in a triangular flask with glass beads containing 225 mL of sterile normal saline, and shake well to obtain a bacterial solution. Pipette 1.0 mL of the bacterial solution into a test tube containing 9.0 mL of sterile normal saline and mix well. This dilution is 10 -1 , repeat the above operation, and successively prepare bacterial solutions of 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 . Select 10 -4 , 10 -5 , 10 -6 Three dilutions, and pipette 0.1 mL of the bacterial solution for each dilution and drop it on the MRS medium plate (Beijing Aoboxing) respectively. Incubate at 37 °C for 24 - 48 h under anaerobic conditions, pick typical colonies, and observe the growth performance and acid production performance. A total of 11 strains with better performance were isolated. An antibacterial test was carried out on the 11 strains (after activating the Lactococcus lactis ZLL111 strain, inoculate it into MRS liquid medium (Beijing Aoboxing) according to an inoculation amount of 1% (v / v), and after static culture at 37 °C for 24 h, take an appropriate amount of the fermentation broth, centrifuge at 6000 rpm for 10 min and take the supernatant. Adjust the viable cell counts of the indicator bacteria Escherichia coli, Salmonella, Staphylococcus aureus, and Shigella to 10 7 CFU / mL, dilute the fermentation broth, supernatant and dilute 2 times respectively. Pipette 180 μL of each dilution and add it into the Oxford cups respectively. Incubate Escherichia coli statically at 37 °C and measure the antibacterial diameter), and the results are shown in Table 1. Among them, the highest OD600nm value of ZLL111 after growing for 24 h is 2.021, and the viable cell count is 1.51×10 9CFU / g, the lowest pH value is 4.05, and the highest antibacterial diameter against Salmonella is 28.5 mm. The 16S rDNA of the strain was amplified by PCR and sequenced. After the above tests, a strain with excellent growth performance and acid and bile salt tolerance was screened, which is ZLL111. Through colony morphology observation, physiological and biochemical characteristic analysis, and 16S DNA molecular identification. The 16S rDNA of the strain was amplified by PCR (primers are 27F SEQ ID NO.4: 5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (SEQ ID NO.5: 5’-TACGACTTAACCCCAATCGC-3’) and sequenced, and the sequence after the sequencing result of the amplification product. Lactococcus lactis grows in MRS medium, anaerobically cultured at 37 °C for 24 h, the colonies are white, round, with a moist surface and opaque. The cells are spherical, 0.8 - 1.0 μm, arranged singly or in pairs, and Gram-positive. It was determined that the strain ZLL111 is Lactococcus lactis.

[0029] The strain ZLL111 was sent to the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) for patent deposit on September 11, 2024. The deposit number is CGMCC No. 31943, the deposit name is ZLL111, and the taxonomic name is Lactococcus lactis.

[0030] Table 1 Results of growth characteristics and antibacterial screening tests

[0031] Bacterial name <![CDATA[24hOD 600nm value]]> Inhibitory diameter against Escherichia coli (mm) Inhibitory diameter against Salmonella (mm) Inhibitory diameter against Staphylococcus aureus (mm) Inhibitory diameter against Shigella (mm) ZLP101 1.568 15.50 13.00 15.45 16.50 ZLR102 1.785 16.50 15.55 16.50 14.50 ZLA103 1.852 18.00 16.00 14.50 18.20 ZLP104 2.001 19.50 14.55 16.50 15.00 ZLC105 1.985 17.55 17.25 14.55 16.50 ZLF106 1.547 18.60 18.50 16.40 17.50 ZLA107 1.459 15.20 14.65 18.50 16.55 ZLC108 1.857 11.50 12.55 17.85 18.20 ZLL109 1.245 14.30 19.45 19.00 19.00 ZLP110 1.554 15.55 18.50 15.85 18.25 ZLL111 2.021 22.75 23. 50 21.50 20.50

[0032] 16S rDNA: GTTGAGCGCTGAAGGTTGGTACTTGTACCGACTGGATGAGCAGCGAACGG

[0033] GTGAGTAACGCGTGGGGAATCTGCCTTTGAGCGGGGGACAACATTTGGAA

[0034] ACGAATGCTAATACCGCATAACAACTTTAAACACAAGTTTTAAGTTTGAA

[0035] AGATGCAATTGCATCACTCAAAGATGATCCCGCGTTGTATTAGCTAGTTG

[0036] GTGAGGTAAAGGCTCACCAAGGCGATGATACATAGCCGACCTGAGAGGGT

[0037] GATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAG

[0038] CAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCG

[0039] TGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGGTAGAGAAGAACG

[0040] TTGGTGAGAGTGGAAAGCTCATCAAGTGACGGTAACTACCCAGAAAGGGA

[0041] CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTG

[0042] TCCGGATTTATTGGGCGTAAAGCGAGCGCAGGTGGTTTATTAAGTCTGGT

[0043] GTAAAAGGCAGTGGCTCAACCATTGTATGCATTGGAAACTGGTAGACTTG

[0044] AGTGCAGGAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGA

[0045] TATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGCCTGTAACTGAC

[0046] ACTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGT

[0047] CCACGCCGTAAACGATGAGTGCTAGATGTAGGGAGCTATAAGTTCTCTGT

[0048] ATCGCAGCTAACGCAATAAGCACTCCGCCTGGGGGAGTACGACCGCAAGG

[0049] TTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTG

[0050] GTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATACTCGT

[0051] GCTATTCCTAGAGATAGGAAGTTCCTTCGGGACACGGGATACAGGTGGTG

[0052] CATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC

[0053] GAGCGCAACCCCTATTGTTAGTTGCCATCATTAAGTTGGGCACTCTAACG

[0054] AGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCA

[0055] TGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGA

[0056] GTCGCGAGACAGTGATGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCG

[0057] GATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGC

[0058] GGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCC

[0059] GTCACACCACGG

[0060] 2. Growth curve of Lactococcus lactis

[0061] After activating Lactococcus lactis ZLL111, it was inoculated into MRS liquid medium (available from Beijing Aoboxing) at inoculation amounts of 0.5% (v / v), 1% (v / v), 2% (v / v), and 3% (v / v) respectively, and statically cultured at 37 °C for 48 h. The OD value of the bacterial liquid from 0 to 48 h was measured using a fully automatic growth curve analyzer Bioscreen C (Shanghai Weizai Technology Co., Ltd.), and the growth curve was plotted. As 600nm value, draw the growth curve. AsFigure 1 As shown, when the inoculation amount of Lactococcus lactis is 0.5%, the growth is optimal.

[0062] Example 2 Determination of the stability of bacteriocin produced by Lactococcus lactis

[0063] After the strain was activated, it was inoculated into MRS broth at an inoculation amount of 1%, cultured at 37 °C for 24 h, centrifuged at 6000 r / min for 15 min after the culture, and the supernatant was filtered through a 0.22 μm filter membrane and stored at 4 °C for later use.

[0064] (1) Exclusion of organic acids

[0065] The pH of the supernatant was adjusted to pH 4.0 using 1 mol / L NaOH and HCl solutions. The blank control group was the untreated supernatant. Using Salmonella as the indicator bacterium for the antibacterial test, the diameter of the antibacterial zone was measured to evaluate the effect of organic acids on the antibacterial effect of bacteriocin.

[0066] Table 2 Exclusion test of organic acids (mm)

[0067] Salmonella Staphylococcus aureus Control group 23.5 21.5 pH = 4.0 22.5 22.0

[0068] (2) Exclusion of hydrogen peroxide

[0069] The pH of the bacterial supernatant was adjusted to 7.0 using 1 mol / L NaOH and HCl solutions, 10 mg / mL catalase was added, and after incubation in a 37 °C water bath for 2 h, the pH was adjusted back to the original pH 4.1 for the antibacterial test. The supernatant without catalase treatment was used as the blank control.

[0070] Table 3 Catalase treatment test (mm)

[0071] Salmonella Staphylococcus aureus Control group 23.50 21.50 Catalase treatment 19.50 18.0

[0072] (3) Determination of protease stability

[0073] Take 4 portions of 1 mL supernatant, and add 10 mg / mL trypsin, papain, pepsin, and 50 μL proteinase K respectively. Adjust the pH of the solution to their respective optimal values (trypsin: 7.4; papain: 6.5; pepsin: 4.5; proteinase K: 7.5). After incubating the solution in a 37 °C water bath for 2 h, adjust it back to the original pH value for the antibacterial test. The untreated supernatant was used as the blank control.

[0074] (4) Determination of thermal stability

[0075] The supernatant was treated in a water bath at 40°C, 60°C, 80°C, and 100°C, respectively. It was treated in a water bath at 100°C for 30 minutes, and the remaining temperature gradients were treated in a water bath for 4 hours. The supernatant at room temperature was used as a blank control for the antibacterial test.

[0076] (5) Determination of pH stability

[0077] Take 4 portions of 1 mL of the concentrated supernatant of the isolated bacteria, and adjust the pH value of the supernatant to 3.0, 4.0, 5.0, and 6.0 with 1 mol / L NaOH and HCl solutions, respectively. After standing at room temperature for 1 h, restore it to the initial pH with NaOH and HCl solutions. Determine the antibacterial activity of bacteriocin under different pH treatments, using the untreated supernatant as a blank control.

[0078] The results of the stability detection of bacteriocin produced by the strain are shown in Table 2, Table 3, and Figure 2. The results show that after treatment at pH 3.0 - 6.0 and temperature treatment (40°C, 60°C, 80°C, and 100°C), its antibacterial activity did not decrease significantly; when adding 4 kinds of proteases, compared with the control group, it was more sensitive to proteinase K, but the antibacterial diameter was still above 18 mm. In addition, after the supernatant was treated at pH 4.1 and with catalase, the antibacterial diameter was still above 19.5 mm, indicating that the antibacterial substance in the supernatant was bacteriocin. The bacteriocin had strong acid and high temperature resistance activities, and the bacteriocin had a good antibacterial effect on Salmonella.

[0079] Example 3 Construction of a heterologous expression engineering bacterium of bacteriocin

[0080] Obtain the complete genome sequence of Lactococcus lactis, align its gene sequence using NCBI blast, and then through amino acid sequence alignment, design primers guided by the bacteriocin sequence, and construct a heterologous expression strain that produces bacteriocin through PCR cloning, purification, and insertion into an expression vector.

[0081] (1) Inoculate Lactococcus lactis into MRS liquid medium and incubate it statically at 37 °C for 18 h. Harvest the bacteria by centrifugation, extract the total DNA using the operation instructions of the DNA extraction kit, and store it at -20 °C for later use. Design relevant primers using the software Snapgene 6.0.2 and synthesize them by a biological company: Purify the PCR amplification product according to the PCR product purification kit (F: 5'-TCACCGTTAATTAACCCGGGGATCCATCTTTCAACAATTTTGTAG-3'; R: 5'-GTCAAGGAGAAAAAACCCCCGGATCCATGCTTAGTTTAAAACTTGCG-3'). Ligate the linear plasmid fragment pAM1 and the gene fragment using the one-step method and transfer them into Escherichia coli DH5α for cloning. Pick a single colony into 500 μL of LB liquid medium (containing ampicillin antibiotic), and culture it at 37 °C and 220 rpm in a shaker for 1 h. Perform colony PCR, and then send the single colonies with positive colony PCR identification to a biological company for sequencing.

[0082] Table 4 Ligation system

[0083] Linking component Volume (4 μL) Linearized plasmid 0.5 μL Target fragment 1.5 μL 2× Recombinase 2 μL

[0084] The reaction temperature of the recombinase (2×MultiF Seamless Assembly Mix) is 50 °C, the reaction time is 45 min. After the reaction, centrifugation is required for Escherichia coli transformation.

[0085] (2) Transfer it into yeast and verify its function

[0086] Yeast cell transformation: (1) Inoculate YPG30 yeast cells into 10 mL of YPD medium and culture them overnight at 30 °C and 220 rpm; (2) Dilute the bacterial solution 10-fold and culture it at 30 °C and 220 rpm for about 5 h until the logarithmic growth phase is reached; (3) Centrifuge at 800×g for 2 min, discard the supernatant, add 3 mL of ddH2O and resuspend by pipetting, centrifuge at 800 g for 2 min, and discard the supernatant: Preheat 2 mg / mL ssDNA in a 95 °C metal bath for 5 min and then place it on ice for pre-cooling; (4) Let it stand at room temperature for 30 min, perform heat shock in a 42 °C water bath for 15 min, and then place it in an ice bath for 5 min. Centrifuge at 800×g for 2 min, discard the supernatant, add 200 μL of ddH2O to resuspend the cells, and transfer them to an SD medium (without Ura3) plate and culture at 30 °C for 3 d.

[0087] Verification of yeast function: Transfer the constructed engineering bacteria to 5 mL of YPR medium and culture them at 30 °C and 220 rpm for 16 h. After 16 h, take 1 mL of YPR medium and transfer it to 3 mL of YPG medium and culture at 30 °C and 220 rpm for 4 h.

[0088] (3)Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0089] Centrifuge the YPG bacterial solution at 12,000 rpm for 1 min and discard the supernatant. Use a protein extraction kit to extract the proteins from the sterilized supernatant, and then identify the extracted proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results are as Figure 3 shown. The molecular weight of the bacteriocin is 97 kDa, and protein bands appear after induction with galactose.

[0090] (4)Antibacterial test of engineered bacteria

[0091] Centrifuge the engineered bacterial solution at 6,000×g at 4°C for 30 min, and aspirate the supernatant for the Oxford cup method test to identify its antibacterial effect. The results show that the engineered bacteria have good antibacterial effects against 4 harmful bacteria, among which the antibacterial effect against Salmonella is the best, and its antibacterial diameter reaches 27.5 mm.

[0092] Table 5 Antibacterial zone diameters of engineered bacteria against harmful bacteria (mm)

[0093] Escherichia coli Staphylococcus aureus Salmonella Shigella flexneri Yeast YPG30 0 0 0 0 Empty vector transformed bacteria (pAM1 plasmid transformed into yeast) 0 0 0 0 (YKT30) LL111 engineered bacteria 23.00 24.50 27.50 22.25

[0094] Comparison between Example 4 and existing recombinant bacteriocins

[0095] Among the currently disclosed bacteriocins, bacteriocin strain AI06 (ABC transporter, permease and substrate binding protein, ACCESSION: CP009472.1 REGION: 1866450..1868441, protein_id="AIS04298.1", hereinafter referred to as the control bacteriocin, whose nucleotide sequence is shown in SEQ ID No. 8) is relatively close to the sequence of the present invention. According to its sequence, the present invention synthesizes its sequence by sequence synthesis method and verifies it by sequencing. Then, according to the method of Example 3 above, construct an engineered bacterium for heterologous expression of bacteriocin and perform induced expression. Centrifuge the YPG bacterial solution at 12,000 rpm for 1 min and discard the supernatant. Use a protein extraction kit to extract the proteins from the sterilized supernatant, and then identify the extracted proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that the molecular weight of the bacteriocin is 94 kDa, and protein bands appear after induction with galactose.

[0096] Comparison of bacteriocin antibacterial activity and stability

[0097] According to the method of Example 2, the stabilities of the bacteriocin of the present invention and the control bacteriocin were measured respectively, and the results are as follows:

[0098] The nisin engineering bacteria and ZLL111 engineering bacteria were selected. After being treated with proteinase K (pH = 4.0) in a simulated porcine gastric juice environment, the antibacterial activities of different bacteriocins were measured. Six replicates were set in each group, and the average antibacterial diameter was calculated.

[0099] Table 6 Comparison of the antibacterial effect stability of different bacteriocins against Salmonella (mm)

[0100] Before treatment After treatment Stability (%) Engineered bacteria of strain AI06 23.50±0.25 19.25±0.33 81.91 Engineered bacteria of ZLL111 27.5±0.35 26.0±0.40 94.54 P value 0.056 0.008 0.002

[0101] The above results show that after the bacteriocin of the present invention is treated in a simulated gastric juice environment, its antibacterial activity is still above 26 mm and there is no significant decrease. The antibacterial activities of the control bacteriocins are all lower than that of the present invention. In addition, the stability of the supernatant of the present invention before and after being treated in a simulated gastric juice environment is as high as 94.54%, while the stability of the control group before and after treatment is only 81.91%, which is significantly lower than that of the bacteriocin engineering bacteria of the present invention by 12 percentage points. Generally speaking, both the bacteriocin of the present invention and the control bacteriocin have excellent antibacterial properties, but the bacteriocin of the present invention is significantly superior to the control bacteriocin in terms of the stability in a simulated gastric juice environment.

Claims

1. Lactococcus lactis ZLL111, with the deposit number of CGMCC No. 31943.

2. A bacterial agent containing the Lactococcus lactis described in claim 1.

3. A bacteriocin, with the amino acid sequence being: the amino acid sequence shown in SEQ ID No.2; or, a sequence with the same function formed by substituting, deleting or adding one or several amino acids to the sequence shown in SEQ ID No.

2.

4. A gene encoding the bacteriocin described in claim 3.

5. A vector containing the encoding gene described in claim 4.

6. An engineered bacterium transformed with the bacteriocin described in claim 3.

7. A product containing the Lactococcus lactis described in claim 1, the bacterial agent described in claim 2, the bacteriocin described in claim 3 or the engineered bacterium described in claim 6.

8. The product according to claim 7, characterized in that, The product is a feed additive, feed, food additive, food, preservative, medicine or health product.

9. The application of the Lactococcus lactis described in claim 1, the bacterial agent described in claim 2, the bacteriocin described in claim 3 or the engineered bacterium described in claim 6 in the preparation of antibacterial products.

10. A method for preparing a bacteriocin, which is obtained by culturing the Lactococcus lactis of claim 1 or the engineered bacterium of claim 6.

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