Enterococcus faecalis, low-temperature probiotic compound bacteria as well as preparation method and application of enterococcus faecalis and low-temperature probiotic compound bacteria

Abstract

The invention relates to the technical field of aquaculture microorganisms, in particular to enterococcus faecalis, low-temperature probiotic compound bacteria as well as a preparation method and application of the enterococcus faecalis and the low-temperature probiotic compound bacteria. The low-temperature probiotics used in the invention can keep good growth ability and functional activity in a low-temperature environment. The low-temperature probiotic compound bacteria are added into the feed for feeding, so that the intestinal health of cultured objects in a low-temperature culture environment can be effectively improved, and the abundance of pathogenic bacteria such as vibrio, salmonella and edwardsiella can be effectively reduced. The probiotic compound bacteria can effectively increase the protein content in the cultured objects, and compared with cultured objects without probiotics, the protein content in fish meat of the cultured objects can be increased by 8.4% when the cultured objects are continuously fed with the probiotic compound bacteria for 40 days.

Description

Technical Field

[0001] This invention relates to the field of aquaculture microbial technology, specifically to a type of Enterococcus faecalis, a low-temperature probiotic compound, its preparation method, and its application. Background Technology

[0002] Rainbow trout and other cold-water fish are widely farmed globally due to their high economic and nutritional value. The protein content of fish meat is one of the key indicators for measuring its nutritional and economic value. In intensive aquaculture, how to safely and efficiently improve fish growth performance and muscle protein deposition is a significant challenge for the industry. Currently, farmers typically achieve this by optimizing feed formulations or using growth promoters. However, high-protein feeds are expensive, and excessive undigested protein can lead to water quality deterioration; while the use of chemical growth promoters and antibiotics poses risks such as drug residues, environmental pollution, and bacterial resistance, and has been restricted or banned in an increasing number of countries and regions.

[0003] Probiotics, as a green and safe feed additive, are increasingly widely used in aquaculture. They promote host health through various means, such as improving gut health and reducing the number of pathogenic microorganisms. However, most commercial probiotic products are developed for ambient temperature aquaculture environments (such as livestock or warm-water fish). Their strains are not compatible with the low-temperature living environment (such as 10-18℃) of cold-water fish in terms of optimal growth temperature and enzyme activity, resulting in poor colonization ability, low functional activity, and unstable application effects in cold-water aquaculture systems.

[0004] Most commercially available aquaculture probiotic products currently use strains of thermophilic microorganisms (20-40℃), lacking true low-temperature adaptability. At low temperatures, these strains exhibit poor growth, slow metabolism, and significantly reduced reproductive and colonization capabilities, resulting in low functional activity and difficulty in forming effective protection in the gut or water, leading to limited application effects. Therefore, developing a specialized probiotic product suitable for low-temperature environments, capable of effectively improving the gut microbiome health of cold-water fish and increasing fish protein content, has significant practical application value and market potential. Summary of the Invention

[0005] To address the lack of efficient probiotic agents specifically designed for cold-water aquaculture systems in existing technologies, this invention screens probiotic strains from the natural environment that can maintain high activity at low temperatures (15°C). The specific method is as follows:

[0006] A strain of Enterococcus faecalis, named Enterococcus sp. SD-R5, with accession number CCTCC NO: M20251593, is deposited at the China Center for Type Culture Collection on July 14, 2025.

[0007] A low-temperature probiotic complex includes Enterococcus sp. SD-R5, with accession number CCTCC NO: M20251593; and Bacillus sp. BS-K1, with accession number CCTCC NO: M20242022.

[0008] A method for preparing a low-temperature probiotic compound involves streaking two bacterial strains, Enterococcus sp. SD-R5 (CCTCC NO: M20251593) and Bacillus sp. BS-K1 (CCTCC NO: M 20242022), onto LB agar plates, culturing them, picking single colonies, inoculating them into bottles containing LB medium, and culturing them on a shaker until OD600 ≥1.0 to obtain seed culture. The seed cultures of the two strains are then mixed in a ratio of 0.5-2:0.5-2 to obtain the probiotic compound agent.

[0009] A low-temperature probiotic compound is used to reduce the abundance of pathogenic bacteria in fish, thereby improving the survival rate of farmed fish.

[0010] A low-temperature probiotic complex is used to improve the intestinal environment and promote fish metabolism and growth.

[0011] The low-temperature probiotics used in this invention can maintain good growth capacity and functional activity at low temperatures (15°C). Adding the low-temperature probiotic complex to feed can effectively improve the intestinal health of farmed animals in low-temperature aquaculture environments and effectively reduce the abundance of pathogens such as Vibrio, Salmonella, and Edwardsiella. The probiotic complex in this invention can effectively increase the protein content in farmed animals. After 40 days of continuous feeding, compared with farmed animals fed feed without probiotics, it can increase the protein content in the fish meat by 8.4%. Attached Figure Description

[0012] Figure 1 Figure showing the growth capacity of Enterococcus sp. SD-R5 strain at 15℃;

[0013] Figure 2 The graph shows the growth capacity of Bacillus sp. BS-K1 strain at 15℃.

[0014] Figure 3 Phylogenetic tree of Enterococcus sp. SD-R5 strain;

[0015] Figure 4 Phylogenetic tree of Bacillus sp. BS-K1 strain;

[0016] Figure 5 A bar chart showing the relative abundance of species at the Genus level in experimental group A and control group B;

[0017] Figure 6 This is a bar chart showing the relative abundance of species at the species level for experimental group A and control group B. Detailed Implementation

[0018] Basic concept: This invention screens low-temperature probiotics from the natural environment, prepares a probiotic compound agent, ferments it to prepare a bacterial powder, applies it to actual aquaculture water, mixes it with feed and feeds it to verify its performance. Specific Implementation

[0019] Culture medium:

[0020] LB medium: 10g peptone; 5g yeast extract; 10g NaCl; pH 7.0 - 7.2; 1000mL distilled water.

[0021] Verification medium: Sodium acetate 5g; (NH4)2SO4 0.047g (10mg / L); KH2PO4 0.5g; Na2HPO4 0.5g; MgSO4·7H2O 0.4g; trace salt solution 2ml; distilled water 1000ml; pH 7.0-7.2, solid medium with 2% agar.

[0022] Trace salt solution: 50 g ethylenediaminetetraacetic acid (EDTA), 2.2 g ZnSO4, 5.5 g CaCl2, 1.57 g CuSO4•5H2O, 5.06 g MnCl2•4H2O, 5 g FeSO4•7H2O, 1.61 g CoCl2•6H2O, 1000 mL distilled water, pH 7.0-7.2.

[0023] Add 2% agar to the solid culture medium.

[0024] Example 1: Screening and preparation of low-temperature probiotics

[0025] 1. Screening of strains:

[0026] Sample enrichment: Take 1 mL of water sample from the biofilter of the recirculating aquaculture system and add it to a 250 mL Erlenmeyer flask containing 100 mL of sterilized pure water. Incubate at 15 °C and 180 r / min for 2 h.

[0027] Coating screening: The enriched bacterial suspension was diluted to 10⁻⁶ using a serial dilution method. -1 - 10 -8The concentration was evenly spread onto LB solid medium plates and incubated at 15°C for 24 hours. After colony formation, different colonies with different morphologies and colors were selected from the LB solid medium for isolation.

[0028] Sequencing identification: Single colonies were streaked onto LB solid medium and incubated in a culture incubator for 24 hours at 15°C. After that, their 16S rDNA sequences were determined to identify the species.

[0029] A strain of Enterococcus faecalis sp. SD-R5 (accession number: CCTCC NO: M20251593) was obtained. Its seed culture was inoculated into validation medium at a 1% inoculum size and cultured at 15℃ and 180 r / min. The OD600 value was measured every 12 h. The results are as follows: Figure 1 As shown, the OD of strain SD-R5 at 24h 600 It can reach 0.323, with an OD of 36h. 600 The highest value was 0.783. Simultaneously, another patented strain, Bacillus sp. BS-K1 (accession number: CCTCC NO: M 20242022), was selected to verify its low-temperature growth performance, and the results are as follows. Figure 2 As shown, in 24hOD 600 It can reach 0.623, with an OD of 36h. 600 The value reached a maximum of 0.853. Both strains exhibited good growth ability at low temperatures.

[0030] 2. Preparation of compound microbial agents

[0031] Two preserved bacterial strains, Enterococcus sp. SD-R5 and Bacillus sp. BS-K1, were streaked onto LB agar plates and incubated at 30°C for 24 hours. Single colonies were picked and inoculated into Erlenmeyer flasks containing LB medium. The flasks were then incubated at 15°C and 180 rpm on a shaker until OD reached [value missing]. 600 ≥ 1.0, used as seed culture. The seed cultures of the two strains were mixed in a 1:1 ratio to obtain a probiotic compound bacterial agent.

[0032] Example 2 Identification of the strain

[0033] 1. Molecular biological identification: Single colonies were selected for colony PCR using primers 27F (5'-agagtttgatcctggctag-3') and 1495R (5'-CTACGGCTACCTTGT TACGA-3'). The products were purified and sequenced. Sequences were retrieved from GenBank using BLAST and compared to identify 16S rDNA sequences with high similarity. A phylogenetic tree was constructed using MEGA 7.0 to find the most homologous strains.

[0034] 2. The results of the 16S rDNA sequencing are as follows:

[0035] Enterococcus sp.SD-R5

[0036]

[0037] Bacillussp.BS-K1:

[0038] GGGGTGCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGAAGTCC

[0039] 3. The results were compared with the 16S rDNA gene sequence of the type strain in the NCBI database for homology. The results are as follows: Figure 3 and Figure 4 As shown, SD-R5 was identified as Enterococcus faecium, which is Gram-positive, and BS-K1 was identified as Bacillus subtilis, which is also Gram-positive.

[0040] Example 3: Investigation into the effect of low-temperature probiotic complex on the protein content of farmed organisms

[0041] 1. Preparation and administration of low-temperature probiotic compound bacteria powder:

[0042] Mix wheat bran (40%) and soybean meal (60%) in a specific ratio, add sterile water at a material-to-water ratio of 1:0.6-0.7, and mix thoroughly. Dispense into shallow trays (1-2 cm thick) and sterilize at 121℃ for 30 minutes. After the substrate carrier cools to room temperature (approximately 37℃), in a clean bench, spray the prepared seed solution at an inoculation rate of 10% (v / w) evenly onto the sterilized carrier and mix thoroughly. Place the inoculated carrier in a constant temperature incubator and incubate at 30℃ for 36 hours. Turn the carrier every 12 hours to ensure ventilation and heat dissipation. After fermentation, place the material in a 45℃ drying oven and dry it in a well-ventilated place until the moisture content is below 10%. Crush the dried lumps using a pulverizer and pass them through a 100-mesh sieve to obtain a uniform inoculum powder. Dispense the inoculum powder into aluminum foil bags, seal them, and store them in a cool, dry place away from light. Use the plate count method to count the viable cells after fermentation, after drying, and in the final product. The final product has a viable count ≥ 1.0 × 10⁻⁶. 9 CFU / g. Add the prepared bacterial powder to the feed at a ratio of 2%, mix well, and then feed.

[0043] 2. The effect of probiotic compound agents on the protein content of livestock.

[0044] The probiotic compound was applied in a rainbow trout recirculating aquaculture system (culture temperature 15-18℃). One hundred rainbow trout were randomly selected from the same culture pond and placed in pond A as the experimental group, while another 100 rainbow trout were placed in pond B as the control group. Experimental group A was fed feed supplemented with probiotic compound powder, while experimental group B was fed normal feed without probiotic compound powder. After 40 days of feeding, fish samples were taken from both groups for analysis of intestinal microbial composition and protein and fat content in the fish meat. The protein detection method followed the National Food Safety Standard: Determination of Protein in Food (GB 5009.5-2016).

[0045] The protein content of the fish meat is shown in Table 1. Two rainbow trout, A and B, were randomly selected from the experimental group's fishpond, and two rainbow trout, C and D, were randomly selected from the control group's fishpond, respectively, and sent for testing. The results showed that the protein content of the fish meat in the experimental group was 23.8 g / 100g, while the protein content of the fish meat in the control group was 21.9 g / 100g. Compared with the control group, the protein content of the fish meat in the experimental group fed with feed supplemented with probiotic compound powder increased by 8.4%.

[0046] Table 1. Fish protein and fat content in the experimental and control groups

[0047]

[0048] Example 4: Effects of Low-Temperature Probiotic Compound Agent on the Intestinal Microbial Composition of Cultured Animals

[0049] The intestines of fish from both the experimental and control groups were sent for sequencing to detect changes and differences in their gut microbiota. Sequencing was performed by Novogene, using metagenomic sequencing analysis. Results are as follows: Figure 5 and Figure 6 As shown, at the Genus level, compared with the control group B, experimental group A, fed with probiotic-added feed, showed a 24.02% decrease in the relative abundance of Salmonella, a 26.22% decrease in Vibrio, and a 7.25% decrease in Edwardsiella. These genera contain a variety of fish pathogens, while Stutzerimonas increased by 42.90%. At the Species level, Vibrio sp. Y159 decreased by 29.32%, Edwardsiella tarda decreased by 8.14%, these are common aquatic pathogens, while Stutzerimonas stutzeri increased by 41.14%.

[0050] Therefore, feeding fish with probiotic-fortified feed can effectively reduce the abundance of pathogens such as Vibrio, Salmonella, and Edwardsiella, thereby reducing the risk of disease and mortality in fish and improving survival rates. Increasing the abundance of beneficial bacteria such as Stutzerimonas helps with nitrogen cycling and detoxification, improves the intestinal environment, and promotes fish metabolism and growth.

[0051] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Enterococcus faecalis, characterized in that: Named Enterococcus faecalis Enterococcus sp.SD-R5, accession number CCTCC NO: M20251593.

2. A low-temperature probiotic complex, characterized in that: Including Enterococcus faecalis Enterococcus sp.SD-R5, with accession number CCTCC NO: M20251593; and strain Bacillus. Bacillu s sp.BS-K1, its accession number is CCTCCNO: M 20242022.

3. A method for preparing the low-temperature probiotic complex as described in claim 2, characterized in that: Two bacterial strains, Enterococcus sp. SD-R5 (CCTCC NO: M20251593) and Bacillus sp. BS-K1 (CCTCC NO: M 20242022), were streaked onto LB agar plates and cultured. Single colonies were picked and inoculated into bottles containing LB medium. The culture was then shaken until OD600 ≥ 1.0, which served as seed culture. The seed cultures of the two strains were mixed at a ratio of 0.5-2:0.5-2 to obtain a probiotic compound bacterial agent.

4. A low-temperature probiotic compound as described in claim 2 is used to reduce the abundance of pathogenic bacteria in fish, thereby improving the survival rate of fish farming.

5. A low-temperature probiotic compound as described in claim 2 is used to improve the intestinal environment and promote fish metabolism and growth.

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