Copper-loaded duck-origin lactobacillus plantarum with high antibacterial peptide yield and application of copper-loaded duck-origin lactobacillus plantarum

Abstract

The invention provides copper-loaded duck-origin lactobacillus plantarum with high antibacterial peptide yield and application of the copper-loaded duck-origin lactobacillus plantarum. The preservation number of the provided copper-loaded duck-origin lactobacillus plantarum is CGMCC (China General Microbiological Culture Collection Center) NO.32835. The domesticated and bred duck-origin lactobacillus plantarum with high antibacterial peptide yield can tolerate high-concentration copper ions, the domesticated and bred lactobacillus plantarum can stably grow, and the contents of antibacterial peptide, extracellular protein, lipase, amylase, lactic acid, acetic acid and the like in the domesticated and bred lactobacillus plantarum strain are increased; and the amino acid composition is changed. The strain can convert inorganic copper into organic copper, the utilization rate of copper is increased, and growth and development of organisms are promoted. Moreover, small peptides and extracellular proteins in metabiotics generated after fermentation have the effect of effectively inhibiting harmful bacteria such as riemerella anatipestifer, escherichia coli, salmonella and staphylococcus of poultry, and are beneficial to eliminating intestinal inflammatory factors, maintaining intestinal health and improving the feed utilization rate.

Description

Technical Field

[0001] This invention belongs to the field of poultry microecological preparation production technology, specifically relating to a copper-loaded duck-derived Lactobacillus plantarum with high antimicrobial peptide production and its application. Background Technology

[0002] Lactobacillus plantarum ( Lactobacillus plantarum Lactobacillus plantarum is a probiotic widely used in medicine, food, and animal husbandry. Many studies have shown that it exerts beneficial effects on the host by maintaining intestinal flora balance, reducing intestinal inflammatory factors, promoting nutrient digestion and absorption, and improving intestinal microbial metabolism. Because of its functions, including inhibiting harmful bacteria, maintaining intestinal health, and promoting growth and development, Lactobacillus plantarum is used as a probiotic preparation in poultry production.

[0003] Copper is an essential trace element for animals, serving as a necessary component and cofactor for numerous enzymes and playing a positive role in a series of life activities, including growth and development. Copper-based additives used in poultry feed are categorized based on their copper source: inorganic copper, organic copper, and nano-copper. Compared to inorganic copper, animals tolerate organic copper better, it has a higher safety profile, is more easily absorbed, and exerts better biological effects. Furthermore, copper deficiency in poultry leads to copper deficiency syndrome, a metabolic nutritional disorder. Long-term excessive copper intake not only reduces utilization but also causes copper poisoning, resulting in resource waste and environmental pollution. Therefore, establishing a method that can inhibit the growth of harmful bacteria in poultry, promote growth and development, maintain intestinal health, improve copper utilization, and reduce copper toxicity and emissions is of great significance to poultry farming and ecological maintenance, and has broad market prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a multifunctional *Lactobacillus plantarum* strain. This domesticated and selected high-copper-load *Lactobacillus plantarum* strain can enhance poultry's ability to inhibit harmful bacteria, maintain intestinal health, promote growth and development, and improve feed utilization, while also meeting the poultry's copper requirements. When used as a feed additive, it can effectively improve the absorption rate of organic copper by farmed poultry, thereby reducing the amount of inorganic copper added to the feed, reducing emissions to the environment, and protecting the ecological environment.

[0005] This invention first provides a duck-derived Lactobacillus plantarum with a high copper loading ( Lactobacillus plantarum The specimen was deposited on November 28, 2024, at the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32835.

[0006] The duck-derived plant lactobacillus with high yield of antibacterial peptide and copper provided by the application can be used as a feed additive; The duck-derived plant lactobacillus with high yield of antibacterial peptide and copper provided by the application can also be used for preparing an antibacterial preparation. The duck-derived plant lactobacillus with high yield of antibacterial peptide and copper provided by the application can also be used for preparing an antibacterial preparation.

[0007] The duck-derived plant lactobacillus with high yield of antibacterial peptide and copper provided by the application can also be used for preparing an antibacterial preparation.

[0008] Compared with the prior art, the application has the following advantages: 1. A duck-derived plant lactobacillus resistant to high-concentration copper ions is obtained.

[0009] 2. The obtained strain is domesticated and selected from duck-derived plant lactobacillus (preserved in the China General Microbiological Culture Collection Center, located at No. 1, Yikuang Road, Beijing, China, on February 21, 2023, with a preservation number of CGMCC No. 26622), can more efficiently inhibit common harmful bacteria of ducks such as duck Riemerella anatipestifer, Salmonella and Escherichia coli, and is facultative anaerobic.

[0010] 3. The obtained strain can convert inorganic copper into organic copper, and poultry has higher tolerance and is more easily absorbed, and can promote the growth and development of the body, improve the feed conversion rate and improve the intestinal health problem of poultry.

[0011] 4. Compared with the starting strain, the content of antibacterial peptides, amino acids, small-molecule organic acids, amylases and lipases prepared by the strain of the application is obviously improved. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Fig. 1 is a comparison diagram of the morphology of duck-derived plant lactobacillus before and after domestication and selection, A is a scanning electron microscope graph of duck-derived plant lactobacillus before domestication and selection, and B is a scanning electron microscope graph of duck-derived plant lactobacillus after domestication and selection; Figure 2 Fig. 2 is a comparison diagram of the growth of duck-derived plant lactobacillus before and after domestication and selection; Figure 3 Fig. 3 is a copper content change curve graph of duck-derived plant lactobacillus after domestication and selection; Figure 4 Fig. 4 is an effect graph of duck-derived plant lactobacillus after domestication and selection on inhibiting Riemerella anatipestifer; Figure 5 Fig. 5 is an effect graph of duck-derived plant lactobacillus after domestication and selection on inhibiting Escherichia coli; Figure 6: The effect diagram of duck-derived plant lactobacillus domesticated and selected to inhibit staphylococcus aureus; Figure 7 : The effect diagram of duck-derived plant lactobacillus domesticated and selected to inhibit salmonella; Figure 8 : The effect diagram of high-load copper duck-derived plant lactobacillus postbiotic on staphylococcus aureus and salmonella; E is a scanning electron microscope diagram of staphylococcus aureus, e is a scanning electron microscope diagram of staphylococcus aureus added with postbiotic, F is a scanning electron microscope diagram of salmonella, and f is a scanning electron microscope diagram of salmonella added with postbiotic; Figure 9 : The flow cytometry diagram of staphylococcus aureus and salmonella when the addition amount of high-load copper duck-derived plant lactobacillus postbiotic is 1:4 Figure 10 : The secondary mass spectrum diagram of AVGLLLATGQAANA sequence; Figure 11 : The 3D structure diagram of AVGLLLATGQAANA sequence. DETAILED DESCRIPTION

[0013] The duck-derived plant lactobacillus provided by the application is fermented and cultured in a culture medium rich in copper ions (Cu 2+ ), and is obtained through intensified domestication and culture screening. The duck-derived plant lactobacillus obtained through selection and breeding can greatly enhance the tolerance to load copper ions (Cu 2+ ), and has good adaptability and carrying capacity to high-concentration Cu 2+ ions. The domesticated bacteria have the characteristics of loading copper, which lays a good foundation for the production of high-load copper duck-derived plant lactobacillus feed additives. In addition, the bacteria have the function of inhibiting duck Riemerella anatipestifer, escherichia coli, staphylococcus aureus and salmonella harmful bacteria, and the produced postbiotic has stronger antibacterial property; at the same time, after multiple generations of adversity domestication, the strain morphology and characteristics change, the activity is stronger, the produced metabolites (postbiotic) are more abundant, and the effect of promoting the growth and development of poultry and intestinal health is better.

[0014] The high-yield antibacterial peptide copper (Cu 2+ ) duck-derived plant lactobacillus domesticated and selected by the application can convert inorganic copper into organic copper, so that more organic copper is loaded in the duck-derived plant lactobacillus bacteria. When the concentration of copper ions in the culture medium is 600.0 mg / L, the content of copper ions in the bacteria can reach 48.23 μg / L.

[0015] The application will be described in detail below in combination with specific embodiments and drawings.

[0016] Example 1: High-yield antibacterial peptide copper ion duck-derived plant lactobacillus domestication and selection The duck-derived plant lactobacillus is fermented and cultured in a culture medium rich in copper ions (Cu(Lactobacillus plantarum) (Deposited on February 21, 2023, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, accession number CGMCC No. 26622) based on the method of gradually increasing the copper ion concentration (Cu) in the culture medium. 2+ The strain was domesticated and selected using a concentration method. After several generations of continuous culture, Lactobacillus plantarum that could tolerate high concentrations of copper sulfate (600 mg / L) was screened out. On November 28, 2024, it was deposited at the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the accession number CGMCC No. 32835.

[0017] Compare the cell morphology, strain number, and copper enrichment effect of duck-derived Lactobacillus plantarum before and after domestication and selection.

[0018] 1) Changes in strain structure from Figure 1 It can be seen that, under the same culture time, microscopic observation after Gram staining revealed that unloaded copper ions (Cu) were present. 2+ The morphology of duck-derived *Lactobacillus plantarum* is basically short rod-shaped or rod-shaped; while the morphology of *Lactobacillus plantarum* highly loaded with copper ions (Cu) is... 2+ The bacteria of *Lactobacillus plantarum* derived from ducks are long rod-shaped, occurring singly, in pairs, or in long chains. Scanning electron microscopy images at the same magnification show that the surface of *Lactobacillus plantarum* derived from ducks is smooth and highly loaded with copper ions (Cu). 2+ The duck-derived Lactobacillus plantarum showed spherical protrusions, attached substances, and elongated bacterial cells.

[0019] 2) Changes in the number of strains from Figure 2 It can be seen that the domesticated and selected duck-derived Lactobacillus plantarum enters the exponential growth phase earlier than the strain before domestication. There is no significant difference in the growth of duck-derived Lactobacillus plantarum before and after domestication and selection, indicating that the domesticated and selected high-load copper duck-derived Lactobacillus plantarum grows well.

[0020] Analysis of the effect of copper loading on duck-derived Lactobacillus plantarum Table 1: Analysis of Copper Loading in Duck-Derived Lactobacillus Plantarum with Different Copper Addition Levels

[0021] As shown in Table 1, the organic copper content of the domesticated and selected high-copper-loaded duck-derived Lactobacillus plantarum was 48.23 ug / L when the copper content was 600 mg / L, which was 131.87% higher than that before domestication and selection, proving that the domesticated strain has a double copper-loading effect.

[0022] Example 2: Comparison of the antibacterial effects of duck-derived Lactobacillus plantarum before and after domestication The starting strain of *Lactobacillus plantarum* derived from ducks (CGMCC No. 26622) and the high-load copper-bearing *Lactobacillus plantarum* derived from ducks selected in Example 1 (CGMCC No. 32835) were used to test the inhibition of the growth of harmful duck bacteria (Escherichia coli, Riemerella lissima, and Staphylococcus aureus). The two strains of *Lactobacillus plantarum* were cultured in MRS broth for 24 hours and the supernatant (metabolites) was collected.

[0023] 1) Antibacterial curve The supernatant was mixed with broth containing harmful bacteria in a ratio of 1:4, and the mixture was placed in a microbial growth analyzer (ELISA reader) to determine the growth curve over 24 hours.

[0024] Table 2: Effects of domesticated and selected duck-derived *Lactobacillus plantarum* on inhibiting harmful bacteria.

[0025] Figure 4 , Figure 5 , Figure 6 , Figure 7 Antibacterial test results showed that a ratio of 1:4 with harmful bacteria significantly inhibited the growth of harmful bacteria within 24 hours. The domesticated and selected high-load duck-derived *Lactobacillus plantarum* exhibited significant inhibitory effects against *Escherichia coli*, *Rimerella*, *Staphylococcus aureus*, and *Salmonella*, indicating that the selected strain retained the ability of duck-derived *Lactobacillus plantarum* to inhibit harmful bacteria, and its effect was even better than the original strain.

[0026] 2) Effects on the morphology and apoptosis rate of harmful bacteria Take the above supernatant and mix it with broth culture medium containing Salmonella and Staphylococcus aureus in a certain ratio (supernatant: harmful bacteria ratio is 1:4), and incubate at 37°C for 18 h. The resulting bacterial culture is processed according to requirements and used for scanning electron microscopy and flow cytometry.

[0027] Depend on Figure 8 Scanning electron microscopy revealed that the high-load copper duck-derived plant lactobacillus metabiotic, bred through domestication and selection, could disrupt the structure of Salmonella. The addition of the metabiotic caused significant rupture of the bacterial cells, leading to cell death, and also had a certain impact on the morphology of Staphylococcus aureus.

[0028] Table 3: Effects of High-Loaded Copper-Bacillus plantarum Postbiotic on Apoptosis Rate of Harmful Bacteria

[0029] From Table 3 and Figure 9The results show that the high-yield antibacterial peptide copper-loaded duck plant lactobacillus probiotics can improve the apoptosis rate of staphylococcus aureus and salmonella, which indicates that the domesticated antibacterial effect is enhanced.

[0030] Example 3: Analysis of physicochemical properties of duck plant lactobacillus before and after domestication and breeding By analyzing the enzyme activity, small molecule organic acid, amino acid and small peptide produced by high copper-loaded duck plant lactobacillus and duck plant lactobacillus before domestication, the physicochemical properties of duck plant lactobacillus after domestication and breeding are further determined.

[0031] 1) Effect of domestication and breeding on enzyme activity, small molecule organic acid and small peptide produced by duck plant lactobacillus fermentation Table 4: Change table of enzyme activity and small peptide content of high copper-loaded plant lactobacillus

[0032] As shown in Table 4, the fermentation broth of duck plant lactobacillus CGMCC NO.32835 strain after domestication and breeding has significantly increased alpha-amylase and lipase compared with before domestication (P<0.01), and small peptides have also increased by 3.29%. It is shown that duck plant lactobacillus after domestication and breeding has significantly increased the ability to produce amylase and lipase and small peptides. P

[0033] 2) Effect of domestication and breeding on small molecule organic acid produced by duck plant lactobacillus fermentation Table 5: Table of organic acid content produced by duck plant lactobacillus before and after domestication and breeding

[0034] As shown in Table 5, the organic acid content in the probiotics of duck plant lactobacillus before and after domestication and breeding has changed significantly. After domestication and breeding, the content is 22.168 mg / ml, which is 31.70% higher than that before domestication and breeding (16.8326 mg / ml). It is shown that the content of lactic acid and acetic acid in the strain after domestication and breeding has significantly increased (P<0.05). p<

[0035] 3) Effect of domestication and breeding on amino acid produced by duck plant lactobacillus fermentation Table 6: Table of amino acid content produced by duck plant lactobacillus before and after domestication and breeding

[0036] Note: ∑ (mg / 100g) represents the total free amino acid content in the fermentation broth of the strain, expressed in units of mg / 100g.

[0037] ​​As shown in Table 6, compared with before domestication and selection, the total amino acid content of duck-derived Lactobacillus plantarum fermentation broth after domestication is 470.41 mg / 100 g, and the total amino acid content before domestication is 461.79 mg / 100 g, which increases by 18.67%, and there is a significant increase (P<0.05), wherein the content of a plurality of amino acids is significantly increased, and a plurality of essential amino acids required by poultry bodies, such as threonine (THR), methionine (MET), lysine (LYS), and arginine (ARG), have an increasing trend. P <0.05), wherein the content of a plurality of amino acids is significantly increased, and a plurality of essential amino acids required by poultry bodies, such as threonine (THR), methionine (MET), lysine (LYS), and arginine (ARG), have an increasing trend.

[0038] Effect of domestication and selection on small peptides produced by duck-derived Lactobacillus plantarum fermentation The small peptides in the fermentation product of the selected duck-derived Lactobacillus plantarum are subjected to antibacterial screening and purification, and a small peptide is obtained, which sequence is specific to duck-derived Lactobacillus plantarum with high copper loading, and the amino acid sequence is AVGLLLATGQAANA (SEQ ID NO: 1).

[0039] The amino acid sequence secondary mass spectrum and 3D structure are shown in Figure 8 、 Figure 9 The structural formula is shown in Figure 10 .

[0040] Antibacterial activity of small peptides on Escherichia coli and Staphylococcus aureus After artificial synthesis of the small peptide with the sequence SEQ ID NO: 1, the duck-derived Lactobacillus plantarum fermentation broth before domestication and selection is diluted so that the total amount of the liquid is consistent with the initial fermentation broth, and the fermentation broth without adding SEQ ID NO: 1 and the fermentation broth adding SEQ ID NO: 1 are taken to carry out growth inhibition test on Escherichia coli and Staphylococcus aureus, so as to verify the antibacterial activity of the small peptide on Escherichia coli and Staphylococcus aureus.

[0041] Table 7: Inhibition rate table of small peptides on Escherichia coli and Staphylococcus aureus

[0042] As shown in Table 7, the small peptide has obvious antibacterial activity on Escherichia coli and also has inhibition ability on Staphylococcus aureus.

[0043] In summary, the small peptide with the sequence SEQ ID NO: 1 has obvious antibacterial activity on Escherichia coli and also has inhibition ability on Staphylococcus aureus. 2+) concentration method, domestication of high-yield antibacterial peptide copper-loaded duck-derived plant lactobacillus obtained by selection and breeding not only retains the probiotic advantages of plant lactobacillus, but also produces small peptides and extracellular proteins with improved ability to inhibit harmful bacteria; adjust the intestinal flora and promote intestinal health. And improve the ability of the bacteria to convert inorganic copper into organic copper, improve the utilization rate of copper in the body, reduce the excretion of copper, reduce the pollution of copper to the environment, increase the content of small molecule organic acid, which is beneficial to eliminate intestinal inflammatory factors and maintain intestinal health; The increase of enzyme and amino acid and other components can improve the feed conversion rate of poultry, promote the growth and development of the body, and improve the quality of poultry products.

Claims

1. A copper-loaded duck-derived *Lactobacillus plantarum* strain that produces high levels of antimicrobial peptides, characterized in that... The preservation number of the duck-derived Lactobacillus plantarum is CGMCC No. 32835.

2. The use of *Lactobacillus plantarum* as described in claim 1 in the preparation of products resistant to *Riemerella anatipestifer*, *Escherichia coli*, *Salmonella*, and *Staphylococcus*.

3. The application of Lactobacillus plantarum as a feed additive as described in claim 1.

4. An antibacterial product, characterized in that, The antibacterial product contains Lactobacillus plantarum as described in claim 1.

5. An antimicrobial peptide, characterized in that, The antimicrobial peptide is isolated and prepared from the metagener of Lactobacillus plantarum as described in claim 1.

6. The antimicrobial peptide according to claim 5, characterized in that, The amino acid sequence of the antimicrobial peptide is SEQ ID NO:

1.

7. A type of duck feed, characterized in that, The duck feed contains the *Lactobacillus plantarum* as described in claim 1.

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