Lactococcus lactis CX212, bacteriocin Nisin Z and applications thereof

Abstract

The present invention proposes a Lactococcus lactis CX212, a bacteriocin Nisin Z and an application thereof, which belongs to the field of beneficial microorganisms in aquatic products and their application, and can solve the problem that there is currently no probiotic with significant antagonistic effect against common aquatic pathogens such as Aeromonas salmonicida. The Lactococcus lactis CX212 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on December 6, 2024, with a deposit number of CGMCC No.32948, and its Latin name is Lactococcus lactis Lactococcus lactis CX212, isolated from the intestines of the Manila clam, is a probiotic that can significantly inhibit Aeromonas salmonicida. The probiotic was developed by mining the gene cluster related to the synthesis of bacteriocin Nisin Z from the genome of Lactococcus lactis CX212 and isolating bacteriocin Nisin Z from CX212 metabolites. Lactococcus lactis CX212 and bacteriocin Nisin Z are used in bactericidal compositions, antibacterial agents, or feed additives.

Description

Technical Field

[0001] The invention belongs to the field of beneficial aquatic microorganisms and applications thereof, and in particular relates to Lactococcus lactis CX212, bacteriocin Nisin Z and applications thereof. Background Art

[0002] Aeromonas salmonicida is a major bacterial pathogen that causes severe boils in rainbow trout and other fish, resulting in extremely high mortality rates. Currently, antibiotics are widely banned in aquaculture, and exploring alternatives to antibiotics is a key approach to achieving healthy aquaculture.

[0003] Beneficial bacteria such as lactic acid bacteria are one of the important ways to replace antibiotics. Factors such as the number of live probiotics and the colonization effect affect the use of probiotics. As postbiotics, probiotic metabolites have the characteristics of higher efficiency, stability, and targeting. Among them, bacteriocins are a class of polypeptides or protein substances with antibacterial biological activity produced during bacterial metabolism. They are diverse in type, have unique antibacterial mechanisms, and are not prone to drug resistance. Therefore, the application of bacteriocins in the aquaculture field is of great significance for precise disease prevention and the discovery of new materials to replace antibiotics. However, there are currently no effective probiotics for common aquatic pathogens such as Aeromonas salmonicida. Summary of the Invention

[0004] The present invention aims to solve the technical problem that there is currently no probiotic with significant antagonistic effect against common aquatic pathogens such as Aeromonas salmonicida, and proposes Lactococcus lactis CX212, bacteriocin Nisin Z and applications thereof.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A Lactococcus lactis CX212, which was deposited in the General Microbiology Center of the China Culture Collection Administration on December 6, 2024, with a deposit number of CGMCC No. 32948 and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its Latin name is Lactococcus lactis.

[0007] In one embodiment, the Lactococcus lactis CX212 is isolated from the intestinal tract of Ruditapes philippinarum, and the complete 16S rDNA sequence of the Lactococcus lactis CX212 is shown as SEQ ID NO.1.

[0008] The present invention also provides a feed additive, which includes the Lactococcus lactis CX212 described in the above embodiment.

[0009] In one embodiment, the amount of Lactococcus lactis CX212 added to the feed additive is such that the number of viable bacteria in the feed is greater than or equal to 10 8CFU / g;

[0010] The number of live bacteria in the feed is the number of live bacteria of the probiotic component contained in a unit mass of the feed detected by a plate coating method.

[0011] The present invention also provides a bacteriocin Nisin Z, which is extracted from Lactococcus lactis CX212. The amino acid sequence of the bacteriocin Nisin Z is:

[0012] MSTKDFNLDLVSVSKKNSGASPRITSISLCTPGCKTGAVMGCNMKTATCNCSIHVSK.

[0013] In one embodiment, the nucleotide sequence of the bacteriocin Nisin Z is shown as SEQ ID NO. 2-3.

[0014] The present invention also provides a feed additive, which includes purified bacteriocin Nisin Z, wherein the purified Nisin Z is added to the feed additive at a ratio of 0.5-3.0 g / kg.

[0015] The present invention also provides a bactericidal composition or bacteriostatic preparation, which comprises Lactococcus lactis CX212; or, the bactericidal composition or bacteriostatic preparation comprises bacteriocin Nisin Z.

[0016] The present invention further proposes a use of Lactococcus lactis CX212 or bacteriocin Nisin Z in inhibiting aquatic pathogens for non-disease treatment purposes, wherein the aquatic pathogens include Aeromonas salmonicida, Escherichia coli, Edwardsiella and Aeromonas hydrophila.

[0017] The present invention further proposes the use of a feed additive in improving the intestinal flora structure of aquatic animals for non-disease treatment purposes, wherein the aquatic animals include rainbow trout.

[0018] Compared with the prior art, the advantages and positive effects of the present invention are:

[0019] 1. The present invention proposes a probiotic strain, Lactococcus lactis CX212, which is isolated from the intestine of the Manila clam and can significantly inhibit Aeromonas salmonicida. The present invention discloses a probiotic strain, Lactococcus lactis CX212, which can significantly inhibit Aeromonas salmonicida. The present invention discloses a probiotic strain, Lactococcus lactis CX212, which is isolated from the intestine of the Manila clam ...

[0020] 2. The present invention proposes a bacteriocin, Nisin Z, which can significantly inhibit the growth of Aeromonas salmonicida by killing its outer membrane, rupturing the cytoplasmic membrane, and causing the cytoplasm to flow out of the cell and die.

[0021] 3. The present invention proposes that by feeding rainbow trout with Lactococcus lactis CX212 or bacteriocin Nisin Z as a feed additive or a dietary supplement, the growth of juvenile rainbow trout can be promoted, the intestinal flora structure can be improved, and the resistance to Aeromonas salmonicida can be enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the antibacterial properties of Lactococcus lactis CX212 provided in Example 1 of the present invention;

[0023] Figure 2 The prediction graph for bacteriocin production by Lactococcus lactis CX212 provided in Example 1 of the present invention;

[0024] Figure 3 This is a graph showing the antibacterial properties of the bacteriocin Nisin Z provided in Example 1 of the present invention;

[0025] Figure 4 This is an electron microscopic observation of the antibacterial properties of the bacteriocin Nisin Z provided in Example 1;

[0026] Figure 5 This is a graph showing weight changes of rainbow trout provided in Example 2 of the present invention;

[0027] Figure 6 This is a schematic diagram of the specific growth rate of rainbow trout provided in Example 2 of the present invention;

[0028] Figure 7 The alpha diversity graph (Shannon index) of the rainbow trout intestinal flora provided in Example 2 of the present invention;

[0029] Figure 8 The alpha diversity graph (Simpson index) of the rainbow trout intestinal flora provided in Example 2 of the present invention;

[0030] Figure 9 The α-diversity graph (Ace index) of the rainbow trout intestinal flora provided in Example 2 of the present invention;

[0031] Figure 10 The alpha diversity map (Chao1 index) of the rainbow trout intestinal flora provided in Example 2 of the present invention;

[0032] Figure 11 A horizontal stacked histogram of the intestinal flora of rainbow trout provided in Example 2 of the present invention;

[0033] Figure 12A horizontal stacked histogram of the rainbow trout intestinal flora provided in Example 2 of the present invention;

[0034] Figure 13 The functional prediction of the intestinal microbial community provided in Example 2 of the present invention;

[0035] Figure 14 This is a weight change graph provided in Example 3 of the present invention;

[0036] Figure 15 A schematic diagram of a specific growth rate provided in Example 3 of the present invention;

[0037] Figure 16 The mortality rate of the challenge provided in Example 3 of the present invention;

[0038] Figure 17 A horizontal stacked histogram of the rainbow trout intestinal flora provided in Example 3 of the present invention;

[0039] Figure 18 A horizontal stacked histogram of the rainbow trout intestinal flora provided in Example 3 of the present invention;

[0040] Figure 19 The predicted pathway related to amino acids in the rainbow trout intestinal flora provided in Example 3 of the present invention (10d);

[0041] Figure 20 This is the predicted pathway (20d) related to amino acids in the rainbow trout intestinal flora provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0042] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0043] An embodiment of the present invention provides a Lactococcus lactis CX212, which was deposited in the General Microbiology Center of the China Culture Collection Administration on December 6, 2024, with a deposit number of CGMCC No. 32948 and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its Latin name is Lactococcus lactis.

[0044] In a specific embodiment, Lactococcus lactis CX212 is isolated from the intestinal tract of Ruditapes philippinarum, and the complete 16S rDNA sequence of Lactococcus lactis CX212 is shown as SEQ ID NO.1.

[0045] An embodiment of the present invention provides a feed additive, which includes the Lactococcus lactis CX212 described in the above embodiment.

[0046] In a specific embodiment, the amount of Lactococcus lactis CX212 added to the feed additive is such that the number of viable bacteria in the feed is greater than or equal to 10 8 CFU / g;

[0047] The number of live bacteria in the feed is the number of live bacteria of the probiotic component contained in a unit mass of the feed detected by a plate coating method.

[0048] The present invention provides a bacteriocin Nisin Z, which is extracted from Lactococcus lactis CX212. The amino acid sequence of the bacteriocin Nisin Z is:

[0049] MSTKDFNLDLVSVSKKNSGASPRITSISLCTPGCKTGAVMGCNMKTATCNCSIHVSK.

[0050] In a specific embodiment, the nucleotide sequence of the bacteriocin Nisin Z is shown as SEQ ID NO.2-3.

[0051] It should be noted that the full nucleotide sequence of Nisin Z is 20,111 bp long. Due to its length, the XML version of the sequence listing is split into two sequences, as shown in SEQ ID NOs. 2 and 3. SEQ ID NO. 2 is 10,673 bp long, and SEQ ID NO. 3 is 9,438 bp long. The concatenation of the two sequences gives the above-mentioned Nisin Z nucleotide sequence, totaling 20,111 bp.

[0052] An embodiment of the present invention provides a feed additive, which includes purified bacteriocin Nisin Z, wherein the purified Nisin Z is added to the feed additive at a ratio of 0.5-3.0 g / kg.

[0053] An embodiment of the present invention further provides a bactericidal composition or bacteriostatic preparation, which includes Lactococcus lactis CX212; or, the bactericidal composition or bacteriostatic preparation includes bacteriocin Nisin Z.

[0054] The present invention also provides an embodiment of the use of Lactococcus lactis CX212 or bacteriocin Nisin Z in inhibiting aquatic pathogens for non-disease treatment purposes. The aquatic pathogens include but are not limited to Aeromonas salmonicida, Escherichia coli, Edwardsiella and Aeromonas hydrophila.

[0055] The embodiment of the present invention also provides a use of a feed additive in improving the intestinal flora structure of aquatic animals for non-disease treatment purposes, wherein the aquatic animals include but are not limited to rainbow trout.

[0056] In order to more clearly and in detail introduce the Lactococcus lactis CX212, bacteriocin Nisin Z and applications thereof provided by the embodiments of the present invention, they will be described below in conjunction with specific examples.

[0057] Example 1

[0058] This embodiment provides a method for screening, identifying, and discovering and purifying bacteriocins of Lactococcus lactis, specifically:

[0059] In this example, a lactic acid bacteria strain was isolated from the intestinal tract of the philippine clam, identified as Lactococcus lactis by 16S rRNA and designated CX212. The complete 16S rDNA sequence is shown in SEQ ID NO. 1. In vitro antibacterial experiments using the Oxford cup method revealed that CX212 exhibited significant antibacterial effects against common aquatic pathogens such as Aeromonas salmonicida, Escherichia coli, Edwardsiella pneumoniae, and Aeromonas hydrophila. The results are shown in Table 1. Figure 1 As shown, Figure 1 A in the middle is Aeromonas salmonicida, Figure 1 B is Escherichia coli, Figure 1 C in the middle is Edwardsiella tarda, Figure 1 D in the middle is Aeromonas hydrophila.

[0060] (1) Discovery of Lactococcus lactis bacteriocins

[0061] The whole genome sequencing of Lactococcus lactis CX212 was carried out, and the potential bacteriocin gene clusters in the sequence were mined by combining the secondary metabolite database antiSMASH and the bacteriocin online software BAGEL4 to predict the types of bacteriocins that CX212 may produce. The results showed that CX212 can produce bacteriocin Nisin Z ( Figure 2 ).

[0062] (2) Nisin Z purification

[0063] (2-1) Vector construction

[0064] Nisin Z was heterologously expressed using direct cloning: Geneious Prime software was used to construct a P15A promoter-chloramphenicol resistance gene-Nisin Z synthetic gene cluster vector. Specific primers were designed to amplify the P15A promoter-chloramphenicol resistance gene within the P15A plasmid. The entire genome of Lactococcus lactis strain CX212 was digested with AflIII (400 μL of digestion system) to excise the Nisin Z synthetic gene cluster. The P15A promoter-chloramphenicol resistance gene portion was amplified using the designed specific primers. The amplified product was ligated to the digested genome using T4 ligase (20 μL of ligation system). The ligation product was desalted for 40 min-1 h and then electroporated into competent Escherichia coli cells on ice. Positive colonies were screened and verified by restriction digestion. Successfully transformed cells were kept as stocks.

[0065] (2-2) Expanding culture and breaking bacteria

[0066] Select a single colony containing the recombinant plasmid and inoculate it into 10 mL of LB liquid medium (with chloramphenicol) and culture it at 37°C overnight with shaking; inoculate 10 mL of the bacterial solution into 1000 mL of LB liquid medium and expand the culture at 37°C until the OD 600 =0.5-0.6, cool to 16°C, add IPTG for induction for more than 20 hours; centrifuge at 4°C, 6000 rpm for 5 minutes to collect the cells, pour out the supernatant, collect the cells into a 50 ml centrifuge tube, disrupt the cells by ultrasound, rinse with Lysis buffer and shake until there are no particles; centrifuge at 4°C, 12000 rpm for 1 hour, collect the supernatant;

[0067] (2-3) Recombinant protein purification

[0068] The nickel column was washed 2-3 times with clean water and then once with Lysis buffer. The supernatant was eluted with a chromatography column at 4°C using 10mM, 25mM, 50mM, 100mM, and 500mM eluents. The eluate was collected in an ultrafiltration centrifuge tube, concentrated by centrifugation, and desalted using NaCl-containing Tris-HCl buffer and high-concentration Tris-HCl buffer. The protein sample was retained for final analysis.

[0069] (3) Antibacterial properties of Nisin Z

[0070] The Oxford cup method was used to verify the antibacterial effect of Nisin Z on Aeromonas salmonicida. At the same time, after Nisin Z was co-cultured with Aeromonas salmonicida, samples were collected at 6h, 12h, and 24h of the co-culture experiment, and electron microscopy was performed to observe its specific bactericidal mechanism. It was found that Nisin Z killed the outer membrane of Aeromonas salmonicida, rupturing the cytoplasmic membrane, causing the cytoplasm to flow out and the cell to die, thereby significantly inhibiting its growth. The experimental results are as follows Figure 3 and Figure 4 As shown. Among them, Figure 3 L is the low-concentration group of 0.2 g Nisin Z per kg feed, M is the medium-concentration group of 1.0 g Nisin Z per kg feed, and N is the high-concentration group (H group) of 3.0 g Nisin Z per kg feed. Figure 4 The figures in the figure show electron micrographs of Nisin Z-damaged salmonicidal Aeromonas at different times, among which, Figure 4 Figures AB in the middle are the control group, i.e., electron micrographs of Aeromonas salmonicida without the addition of Nisin Z; Figure 4 Middle CD are electron microscopic images of Nisin Z damaging Aeromonas salmonicida when they were co-cultured for 6 hours; Figure 4 Middle EF is the electron microscopic image of Nisin Z damaging Aeromonas salmonicida when Nisin Z and Aeromonas salmonicida were co-cultured for 12 hours; Figure 4 GH in the middle are electron microscopic images of Nisin Z damaging Aeromonas salmonicida when they were co-cultured for 24 hours.

[0071] Example 2

[0072] This example studies the effect of feeding Lactococcus lactis CX212 on the growth and intestinal flora of rainbow trout, specifically:

[0073] The rainbow trout feed composition includes: crude protein 45%, crude fat 20%, crude fiber 3%, crude ash 9%, moisture 8.5%, total phosphorus 2.0%, calcium 0.8%, and amino acids 3.3%. The experiment designed four treatment groups, namely:

[0074] 10 per gram of feed 7 low concentration group of cfu CX212 (group A);

[0075] 10 per gram of feed 8 cfu CX212 medium concentration group (group B);

[0076] 10 per gram of feed 9 high-concentration group of cfu CX212 (group C);

[0077] The control group (CK group) was not supplemented with CX212.

[0078] The experiment was conducted at the Integrated Aquaculture Center of Qingdao Agricultural University using a static aquaculture system. Three replicates were performed in each of the three groups: a low-concentration group (A), a medium-concentration group (B), a high-concentration group (C), and a control group (CK). Twelve aquaculture tanks, each containing 300 L of water, were used, with 30 rainbow trout per tank weighing 37.57 ± 1.01 g. The average aquaculture water temperature was 15.5 ± 0.5°C, the dissolved oxygen level was 6.9 ± 0.7 mg / L, and the pH was 7.0 ± 0.5. Fish were fed daily at 9:00 AM and 4:00 PM, at a feed rate of 2% of their body weight. The experimental period lasted 30 days, with fish samples collected on days 7, 14, and 21 for analysis of growth and changes in the intestinal microbiota.

[0079] The experimental results are as follows:

[0080] At the beginning and end of the experiment, the average weight of the fry was tested to analyze the growth of rainbow trout. Figure 5-6 The intestine was collected for 16S rRNA gene sequencing, and intestinal microbial diversity, intestinal flora differences and functional prediction analysis were performed. The analysis results are shown as follows: Figure 7-13 shown.

[0081] Result analysis:

[0082] (1) From Figure 5-Figure 6 The experimental results shown here show that the final body weight (FW) and specific growth rate (SGR) of the feed groups supplemented with probiotics at all three concentrations were significantly higher than those of the control feed group (CK group), indicating that the probiotics used in this example can promote the growth of rainbow trout. Furthermore, throughout the entire experimental process of this example, no fry mortality occurred in any of the experimental groups, and the survival rate before challenge was 100%.

[0083] (2) Figure 7-12 Feeding the CX212 strain showed no significant change in community diversity after 10 days. However, after 20 days, the low-concentration group (A) significantly reduced community diversity, the medium-concentration group (B) significantly reduced community diversity and increased community richness, and the high-concentration group (C) significantly increased both diversity and richness. Specifically, at the phylum level, Firmicutes generally increased, while Bacteroidetes and Proteobacteria decreased. At the genus level, Mycoplasma generally increased and Chryseobacterium generally decreased across all treatment groups. The abundance of the potentially pathogenic Chryseobacterium and Comamonas decreased, while the abundance of some potentially beneficial bacteria, Lactobacilli, increased.

[0084] (3) Figure 13It was shown that feeding the CX212 strain could increase the abundance of multiple amino acid metabolic pathways (cysteine, methionine, alanine, aspartic acid, glutamate, glycine, serine and threonine, etc.), glyoxylate and dicarboxylic acid metabolism, and oxidative phosphorylation.

[0085] Example 3

[0086] This example investigates the effects of Nisin Z preparation on the growth and disease resistance of rainbow trout by feeding them. Specifically:

[0087] The experimental design includes four treatment groups, as follows:

[0088] a low-concentration group (L group) of 0.2 g Nisin Z per kilogram of feed;

[0089] a medium concentration group (M group) of 1.0 g Nisin Z per kilogram of feed;

[0090] a high-concentration group (H group) receiving 3.0 g of Nisin Z per kilogram of feed;

[0091] The control group (CK group) was not supplemented with Nisin Z. The in vivo feeding experiment method was the same as that in Example 2.

[0092] The experimental results are as follows:

[0093] At the beginning and end of the experiment, the average weight of the fry was tested to analyze the growth and disease resistance of rainbow trout. The test results are as follows: Figure 14-15 .

[0094] The intestine was collected for 16SrRNA gene sequencing, and intestinal microbial diversity, intestinal flora differences and functional prediction analysis were performed. The analysis results are shown in Table 1. Figures 16-20 shown.

[0095] Table 1 Alpha diversity of rainbow trout intestinal flora

[0096]

[0097] Result analysis:

[0098] (1) By Figure 14-16 As shown, the specific growth rate (SGR) and weight gain (WG) of the Nisin Z treatment groups at all concentrations were significantly higher than those of the control group (CK). Within seven days of infection with Aeromonas salmonicida, the survival rate of rainbow trout in all groups was significantly reduced. The survival rate of rainbow trout in the CK group was the lowest of all groups, falling below 60% five days after infection. The survival rates of the other treatment groups, particularly the M and H groups, exceeded 70% after five days, significantly higher than those in the CK group (P < 0.05).

[0099] (2) By Figure 17-18The intestinal α-diversity index of rainbow trout treated with Nisin Z showed a decreasing trend on the 10th day and an increasing trend on the 20th day. At the phylum level, feeding Nisin Z increased the abundance of Firmicutes and decreased the abundance of Proteobacteria and Fusobacteria. At the genus level, the abundance of Mycoplasma increased and Proteobacteria decreased. Furthermore, the relative abundance of potential pathogens such as Acinetobacter, Streptococcus, and Shewanella decreased.

[0100] (3) By Figure 19-20 It can be seen that the Nisin Z-treated group significantly increased the abundance of peptidase-related functions, pentose and glucuronic acid conversion, and multiple amino acid biosynthesis (phenylalanine, tyrosine, tryptophan and lysine).

[0101] The above description is merely an example of the present invention and does not limit the present invention in any other form. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes for application in other fields. However, any simple modification, equivalent change and modification of the above embodiment made according to the technical essence of the present invention without departing from the technical solution content of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A use of bacteriocin Nisin Z in inhibiting aquatic pathogens for non-disease treatment purposes, characterized in that: The aquatic pathogen is Aeromonas salmonicida, and the amino acid sequence of the bacteriocin Nisin Z is: MSTKDFNLDLVSVSKKNSGASPRITSISLCTPGCKTGAVMGCNMKTATCNCSIHVSK.

2. The use of the bacteriocin Nisin Z according to claim 1 in inhibiting aquatic pathogens for non-disease treatment purposes, characterized in that: The bacteriocin Nisin Z is extracted from Lactococcus lactis CX212, which was deposited in the General Microbiology Center of China Culture Collection Administration on December 6, 2024, with a deposit number of CGMCC No.32948. Its Latin name is Lactococcus lactis .

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