Lactococcus lactis CX212, bacteriocin Nisin Z and application thereof

Abstract

The invention provides lactococcus lactis CX212, bacteriocin Nisin Z and application of the lactococcus lactis CX212 and the bacteriocin Nisin Z, belongs to the field of beneficial microorganisms of aquatic products and application of the beneficial microorganisms, and can solve the problem that at present, no probiotics with remarkable effects of antagonizing common pathogenic bacteria of the aquatic products such as aeromonas salmonicida exists. The lactococcus lactis CX212 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 6, 2024, the preservation number of the lactococcus lactis CX212 is CGMCC No.32948, and the Latin name of the lactococcus lactis CX212 is Lactococcus lactis. According to the lactococcus lactis CX212, probiotics capable of remarkably inhibiting aeromonas salmonicida are separated from intestinal tracts of ruditapes philippinarum, relevant gene clusters are synthesized by excavating bacteriocin Nisin Z from a genome of the lactococcus lactis CX212, and the bacteriocin Nisin Z is separated from metabolites of the lactococcus lactis CX212. The lactococcus lactis CX212 and the bacteriocin Nisin Z are used for a bactericidal composition, a bacteriostatic agent or a feed additive.

Description

Technical Field

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

[0002] Aeromonas salmonicida is an important bacterial pathogen that causes severe furunculosis in rainbow trout and other fish, causing extremely high mortality. Currently, antibiotics are widely banned in aquaculture, and exploring alternatives to antibiotics is an important way to achieve 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 field of aquaculture 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] Aiming at the technical problem that there is no probiotic with significant antagonistic effect on common aquatic pathogens such as Aeromonas salmonicida, the present invention provides Lactococcus lactis CX212, bacteriocin Nisin Z and application thereof.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: A Lactococcus lactis CX212, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on December 6, 2024, with a deposit number of CGMCC No.32948, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Its Latin name is Lactococcus lactis .

[0006] 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.

[0007] The present invention also provides a feed additive, which includes the Lactococcus lactis CX212 described in the above embodiment.

[0008] In one embodiment, the amount of Lactococcus lactis CX212 added to the feed additive is such that the number of live bacteria in the feed is greater than or equal to 10 8 CFU / g; 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.

[0009] 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: MSTKDFNLDLVSVSKKNSGASPRITSISLCTPGCKTGAVMGCNMKTATCNCSIHVSK.

[0010] In one embodiment, the nucleotide sequence of the bacteriocin Nisin Z is shown as SEQ ID NO.2-3.

[0011] 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.

[0012] The present invention also provides a bactericidal composition or an antibacterial preparation, wherein the bactericidal composition or the antibacterial preparation comprises Lactococcus lactis CX212; or the bactericidal composition or the antibacterial preparation comprises bacteriocin Nisin Z.

[0013] The present invention further proposes an application 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.

[0014] 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.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are: 1. The present invention provides a Lactococcus lactis CX212, which is a probiotic isolated from the intestinal tract of the Philippine clam and can significantly inhibit Aeromonas salmonicida. The gene cluster related to the synthesis of bacteriocin Nisin Z is mined from the genome of Lactococcus lactis CX212, and bacteriocin Nisin Z is separated from the metabolites of CX212; 2. The present invention proposes a bacteriocin Nisin Z, which can significantly inhibit the growth of Aeromonas salmonicida by killing the outer membrane of the bacterium, causing the cytoplasmic membrane to rupture, leading to cell death caused by cytoplasm outflow; 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 rainbow trout fry can be promoted, the intestinal flora structure can be improved, and the resistance to Aeromonas salmonicida can be enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the antibacterial properties of Lactococcus lactis CX212 provided in Example 1 of the present invention; Figure 2 The prediction graph of bacteriocin production by Lactococcus lactis CX212 provided in Example 1 of the present invention; Figure 3 This is a graph showing the antibacterial properties of the bacteriocin Nisin Z provided in Example 1 of the present invention; Figure 4 This is an electron microscope observation diagram of the antibacterial properties of the bacteriocin Nisin Z provided in Example 1; Figure 5 This is a graph showing weight changes of rainbow trout provided in Example 2 of the present invention; Figure 6 A schematic diagram of a specific growth rate of rainbow trout provided in Example 2 of the present invention; Figure 7 The alpha diversity graph (Shannon index) of the intestinal flora of rainbow trout provided in Example 2 of the present invention; Figure 8 The alpha diversity graph (Simpson index) of the intestinal flora of rainbow trout provided in Example 2 of the present invention; Fig. 9 The alpha diversity map (Ace index) of the intestinal flora of rainbow trout provided in Example 2 of the present invention; Fig.10 The alpha diversity map of the intestinal flora of rainbow trout (Chao1 index) provided in Example 2 of the present invention; Fig.11 A horizontal stacked bar graph of the intestinal flora of rainbow trout provided in Example 2 of the present invention; Fig.12 A horizontal stacked bar graph of the intestinal flora of rainbow trout provided in Example 2 of the present invention; Fig.13 The functional prediction of the intestinal microbial community provided in Example 2 of the present invention; Fig.14 The weight change diagram provided by Example 3 of the present invention; Fig.15 A schematic diagram of a specific growth rate provided in Example 3 of the present invention; Fig.16 The attack mortality rate provided by Example 3 of the present invention; Fig.17A horizontal stacked bar graph of the intestinal flora of rainbow trout provided in Example 3 of the present invention; Fig.18 A horizontal stacked bar graph of the intestinal flora of rainbow trout provided in Example 3 of the present invention; Fig.19 The prediction pathway related to amino acids in the intestinal flora of rainbow trout provided in Example 3 of the present invention (10d); Fig. 20 This is the predicted pathway (20d) related to amino acids in the intestinal flora of rainbow trout provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] The embodiment of the present invention provides a Lactococcus lactis CX212, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on December 6, 2024, with a deposit number of CGMCC No.32948, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Its Latin name is Lactococcus lactis .

[0019] 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.

[0020] An embodiment of the present invention provides a feed additive, which includes the Lactococcus lactis CX212 as described in the above embodiment.

[0021] In a specific embodiment, the amount of Lactococcus lactis CX212 added to the feed additive is such that the number of live bacteria in the feed is greater than or equal to 10 8 CFU / g; 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.

[0022] The embodiment of the present invention provides a bacteriocin Nisin Z, wherein the bacteriocin Nisin Z is extracted from Lactococcus lactis CX212, and the amino acid sequence of the bacteriocin Nisin Z is: MSTKDFNLDLVSVSKKNSGASPRITSISLCTPGCKTGAVMGCNMKTATCNCSIHVSK.

[0023] In a specific embodiment, the nucleotide sequence of the bacteriocin Nisin Z is shown as SEQ ID NO.2-3.

[0024] It should be noted that the full length of the nucleotide sequence of Nisin Z is 20111 bp. Because the sequence length is too long, it is split into two sequences in the XML version of the sequence table, as shown in SEQ ID NO.2-3. Among them, the sequence length of SEQ ID NO.2 is 10673 bp, and the sequence length of SEQ ID NO.3 is 9438 bp. The two are sequentially connected to form the above-mentioned nucleotide sequence of Nisin Z, totaling 20111 bp.

[0025] 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.

[0026] The embodiment of the present invention further provides a bactericidal composition or an antibacterial preparation, wherein the bactericidal composition or the antibacterial preparation comprises Lactococcus lactis CX212; or the bactericidal composition or the antibacterial preparation comprises bacteriocin Nisin Z.

[0027] The embodiment of the present invention also provides a use of Lactococcus lactis CX212 or bacteriocin Nisin Z in inhibiting aquatic pathogens for non-disease treatment purposes, wherein the aquatic pathogens include but are not limited to Aeromonas salmonicida, Escherichia coli, Edwardsiella and Aeromonas hydrophila.

[0028] 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, and the aquatic animals include but are not limited to rainbow trout.

[0029] In order to more clearly and in detail introduce a 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 embodiments.

[0030] Example 1 This embodiment provides a method for screening, identifying, and discovering and purifying bacteriocins of Lactococcus lactis, specifically: In this example, a lactic acid bacteria strain was isolated from the intestinal tract of the Philippine clam, and was identified as Lactococcus lactis by 16S rRNA. Lactococcus lactis), numbered as CX212, and its 16S rDNA full sequence is shown in SEQ ID NO.1. The Oxford cup method was used to conduct in vitro antibacterial experiments, and it was found that CX212 had significant antibacterial effects on common aquatic pathogens such as Aeromonas salmonicida, Escherichia coli, Edwardsiella and Aeromonas hydrophila. The experimental results are shown in 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 figure represents Vibrio parahaemolyticus.

[0031] (1) Discovery of bacteriocins from Lactococcus lactis The whole genome sequencing of Lactococcus lactis CX212 was performed, 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 ).

[0032] (2) Nisin Z purification (2-1) Vector construction Nisin Z was heterologously expressed using direct cloning: Geneious Prime software was used to construct the P15A promoter-chloramphenicol resistance gene-Nisin Z synthetic gene cluster vector, and specific primers for the P15A promoter-chloramphenicol resistance gene amplification of the P15A plasmid were designed. The whole genome of Lactococcus lactis CX212 strain was digested with AflⅢ enzyme (400 μL of digestion system), and the Nisin Z synthetic gene cluster was digested. At the same time, the P15A promoter-chloramphenicol resistance gene part was amplified using the designed specific primers, and the amplified product was linked to the digested genome with T4 ligase (20 μL of ligation system). After the ligation product was desalted for 40 min to 1 h, it was placed on ice and electroporated into Escherichia coli competent cells, followed by positive colony screening and restriction digestion verification, and the successfully transformed positive bacteria were preserved.

[0033] (2-2) Expansion culture and bacterial disruption 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 OD 600=0.5~0.6, cool to 16℃, add IPTG for induction for more than 20h; centrifuge at 4℃, 6000rpm for 5min to collect the bacteria, pour out the supernatant, collect the bacteria into a 50ml centrifuge tube, break the bacteria with ultrasound, rinse with Lysis buffer and shake until there are no particles; centrifuge at 4℃, 12000rpm for 1h in a centrifuge, collect the supernatant; (2-3) Recombinant protein purification The nickel column was washed 2-3 times with clean water and then washed once with Lysis buffer; the supernatant was eluted with a chromatography column at 4°C with 10mM, 25mM, 50mM, 100mM, and 500mM eluents, the eluents were collected in an ultrafiltration centrifuge tube, concentrated by centrifugation, and desalted with NaCl Tris-HCl buffer and high concentration Tris-HCl buffer, and the protein sample was retained for final analysis.

[0034] (3) Antibacterial properties of Nisin Z 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 of the cell and 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 a low-concentration group of 0.2 g Nisin Z per kg feed, M is a medium-concentration group of 1.0 g Nisin Z per kg feed, and N is a 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 AB in the middle is the control group, i.e., the electron microscopic images of Aeromonas salmonicida without the addition of Nisin Z; Figure 4 CD in the middle are electron microscopic images of Nisin Z damaging Aeromonas salmonicida when Nisin Z and Aeromonas salmonicida 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.

[0035] Example 2 In this example, the effect of feeding Lactococcus lactis CX212 on the growth and intestinal flora of rainbow trout was studied, specifically: The rainbow trout feed composition includes: crude protein 45%, crude fat 20%, crude fiber 3%, crude ash 9%, water 8.5%, total phosphorus 2.0%, calcium 0.8%, amino acid 3.3%. The experiment designed four treatment groups, namely: 10 per gram of feed 7 cfu CX212 low concentration group (Group A); 10 per gram of feed 8 cfu CX212 medium concentration group (group B); 10 per gram of feed 9 cfu CX212 high concentration group (Group C); The control group (CK group) was not supplemented with CX212.

[0036] The experimental process was as follows: the experiment was conducted at the Comprehensive Breeding Center of Qingdao Agricultural University, and the breeding system was still water breeding. The experiment was divided into a low concentration group (group A), a medium concentration group (group B), a high concentration group (group C) and a control group (group CK), with 3 parallels in each group. Twelve breeding barrels were used, with a single barrel of 300L of water, 30 rainbow trout in each barrel, and an initial body weight of 37.57±1.01 g. The average temperature of the breeding water was 15.5±0.5℃, the dissolved oxygen was 6.9±0.7 mg / L, and the pH was 7.0±0.5. Feeding was carried out at 9:00 am and 16:00 pm every day, and the feeding amount was 2% of the fish body weight. The experimental period was 30 days, and the fish were sampled on the 7th, 14th and 21st days to analyze the changes in fish growth and intestinal flora.

[0037] The experimental results are as follows: 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-Figure 13 shown.

[0038] Result analysis: (1) From Figure 5-Figure 6 The experimental results shown in the figure show that the final body weight (FW) and specific growth rate (SGR) of the three concentrations of probiotics added to the feed group were significantly higher than those of the common feed group (CK group), that is, the probiotics of this embodiment applied to the feed can promote the growth of rainbow trout. At the same time, during the entire experimental process of this embodiment, no fry died in each experimental group, and the survival rate before the challenge was 100%.

[0039] (2) Figure 7-Figure 12The results showed that by feeding the CX212 strain, the diversity did not change significantly at 10 days, the low concentration group (A) significantly reduced the community diversity at 20 days, the medium concentration group (B) significantly reduced the community diversity and increased the community richness, and the high concentration group (C) significantly increased the community diversity and community richness. Specifically, at the phylum level, the Firmicutes generally increased, the Bacteroidetes decreased, and the Proteobacteria generally decreased; at the genus level, the Mycoplasma genus in each treatment group generally increased, and the Chryseobacterium genus generally decreased. The abundance of potential pathogenic bacteria Chryseobacterium and Comamonas decreased, while the abundance of some potential beneficial bacteria Lactobacillus increased.

[0040] (3) Fig.13 It was shown that feeding the CX212 strain increased the abundance of various amino acid metabolic pathways (cysteine, methionine, alanine, aspartic acid, glutamate, glycine, serine and threonine, etc.), glyoxylate and dicarboxylic acid metabolism, and oxidative phosphorylation.

[0041] Example 3 This example explores the effects of Nisin Z preparation on the growth and disease resistance of rainbow trout by feeding them, specifically: The experimental design included four treatment groups, as follows: A low-concentration group (L group) with 0.2 g Nisin Z per kg feed; The medium concentration group (M group) was 1.0 g Nisin Z per kg feed; The high-concentration group (H group) received 3.0 g of Nisin Z per kg of feed; 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.

[0042] The experimental results are as follows: 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 .

[0043] 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. Figure 16-Figure 20 shown.

[0044] Table 1 Alpha diversity of intestinal flora of rainbow trout Result analysis: (1) By Figure 14-16It can be seen that the specific growth rate (SGR) and weight gain (WG) of the Nisin Z treatment groups were significantly higher than those of the control group (CK). Within 7 days after 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 among all groups, less than 60% after 5 days of attack. The survival rates of other treatment groups, especially M and H groups, exceeded 70% after 5 days, which was significantly higher than that of the CK group (P<0.05).

[0045] (2) By Figure 17-Figure 18 It can be seen that the intestinal α-diversity index of rainbow trout in the Nisin Z treatment group showed a trend of decreasing on the 10th day and increasing on the 20th day. At the phylum level, feeding Nisin Z increased the abundance of Firmicutes and reduced the abundance of Proteobacteria and Fusobacteria; at the genus level, the abundance of Mycoplasma increased and the abundance of Proteobacteria decreased. At the same time, the relative distribution of potential pathogens such as Acinetobacter, Streptococcus and Shewanella decreased.

[0046] (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 interconversion, and multiple amino acid biosynthesis (phenylalanine, tyrosine, tryptophan, and lysine).

[0047] The above description is only an example of the present invention and is not intended to limit the present invention in other forms. Any technician familiar with the profession may use the technical contents disclosed above to change or modify them into equivalent embodiments with equivalent changes and apply them to other fields. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A Lactococcus lactis CX212, characterized in that The lactococcus lactis was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on December 6, 2024, and its deposit number is CGMCC No.32948. Its Latin name is Lactococcus lactis .

2. The Lactococcus lactis CX212 according to claim 1, characterized in that The Lactococcus lactis CX212 is isolated from the intestinal tract of the Ruditapes philippinarum. The complete 16S rDNA sequence of the Lactococcus lactis CX212 is shown in SEQ ID NO.

1.

3. A feed additive, characterized in that: The feed additive comprises the Lactococcus lactis CX212 as claimed in claim 1.

4. The feed additive according to claim 3, characterized in that The amount of Lactococcus lactis CX212 added in the feed additive is such that the number of live bacteria in the feed is greater than or equal to 10 8 CFU / g; 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.

5. A bacteriocin Nisin Z, characterized in that The bacteriocin Nisin Z is extracted from the Lactococcus lactis CX212 as claimed in claim 1, and the amino acid sequence of the bacteriocin Nisin Z is: MSTKDFNLDLVSVSKKNSGASPRITSISLCTPGCKTGAVMGCNMKTATCNCSIHVSK.

6. The bacteriocin Nisin Z according to claim 5, characterized in that The nucleotide sequence of the bacteriocin Nisin Z is shown in SEQ ID NO.2-3.

7. A feed additive, characterized in that: The feed additive comprises the purified bacteriocin Nisin Z according to claim 5, wherein the purified Nisin Z is added to the feed additive at a ratio of 0.5-3.0 g / kg.

8. A bactericidal composition or bacteriostatic preparation, characterized in that: The bactericidal composition or antibacterial preparation comprises the Lactococcus lactis CX212 according to claim 1; or The bactericidal composition or bacteriostatic preparation comprises the bacteriocin Nisin Z as claimed in claim 5.

9. Use of the Lactococcus lactis CX212 according to claim 1 or the bacteriocin Nisin Z according to claim 5 in inhibiting aquatic pathogens for non-disease treatment purposes, characterized in that: The aquatic pathogens include Aeromonas salmonicida, Escherichia coli, Edwardsiella and Aeromonas hydrophila.

10. Use of the feed additive according to claim 3 or the feed additive according to claim 7 for improving the intestinal flora structure of aquatic animals for non-disease treatment purposes, characterized in that: The aquatic animals include rainbow trout.

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