Lactobacillus plantarum LP-G7 and application thereof

Abstract

The invention discloses lactobacillus plantarum LP-G7 and application thereof, and the lactobacillus plantarum LP-G7 provided by the invention is acid-resistant and cholate-resistant, has acid production, bacteriostasis and adhesion capabilities, and can improve the growth performance of meat ducks, reduce the deposition of fat in serum, abdomen and liver, improve lipid metabolism and immunocompetence and improve the meat quality of the meat ducks. The method can be widely applied to meat duck breeding, and favorable support is provided for high-quality development of meat duck breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics, and particularly relates to a Lactobacillus plantarum LP-G7 and application thereof in meat ducks. Background Art

[0002] The modern livestock and poultry industry pursues rapid growth, high meat yield, and low feed costs to maximize economic returns. In recent years, with the continued global demand for poultry products, the rapid development of the poultry industry has sparked widespread attention to farming efficiency and nutritional optimization. In particular, the composition of the diet and feed additives directly impact poultry production performance, slaughter performance, and meat quality, thereby affecting the economic benefits of the farming industry. Poultry is highly susceptible to fat deposition under intensive farming conditions. Fat deposits in poultry primarily consist of subcutaneous fat, intramuscular fat, and abdominal fat. Abdominal fat is a major byproduct of slaughter and is often considered a waste product. Excessive body fat deposition not only wastes feed resources but also reduces carcass yield and alters meat quality, compromising the economic efficiency and nutritional value of the product. Studies have shown that probiotics have multiple beneficial effects on the host, including the ability to produce antimicrobial substances, modulate the immune system, and interfere with pathogen colonization. Probiotics also possess multiple functions, including antimicrobial, anti-inflammatory, and antioxidant properties. Screening probiotic strains is an essential component of probiotic research and is directly related to its application. Numerous studies have demonstrated that beneficial bacteria isolated from host organisms exert excellent probiotic effects. In this context, based on the principles of microbial adaptability and specificity to their environment, a strain of lactic acid bacteria was isolated from the cecal contents of broiler ducks. The strain is acid- and bile-tolerant, and possesses acid-producing, antibacterial, and adhesion capabilities. This strain improves duck growth performance, reduces fat deposition in the serum, abdomen, and liver, enhances lipid metabolism and immunity, and improves meat quality, providing favorable support for the high-quality development of broiler duck farming. Summary of the Invention

[0003] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0004] To achieve the above objectives, the present invention discloses the following technical contents: The first objective of the present invention is to provide a strain of Lactobacillus plantarum (Lactiplantibacillus plantarum) LP-G7, which was screened from the cecal contents of meat ducks. The strain is acid- and bile-tolerant, and exhibits acid production, antibacterial, and adhesion capabilities. Lactobacillus plantarum LP-G7 is deposited with the China General Microbiological Culture Collection under the CGMCC No. 34430, dated May 6, 2025. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0005] The physiological and biochemical properties of Lactobacillus plantarum LP-G7 are as follows: it is rod-shaped, Gram-positive, facultative anaerobic, can reach the growth plateau phase after culturing at 37°C for 12 hours, has a certain tolerance to gastric acid and bile salts, can lower the environmental pH by producing acid, and inhibit the growth of pathogenic microorganisms.

[0006] A second objective of the present invention is to provide the use of Lactobacillus plantarum LP-G7 in meat duck farming. This study primarily examines the effects of Lactobacillus plantarum LP-G7 on the growth performance, abdominal fat percentage, liver lipid deposition, serum lipid metabolism indicators, serum immune indicators, and meat quality of Cherry Valley ducks. Experimental results show that Lactobacillus plantarum LP-G7 can reduce abdominal fat percentage, improve liver lipid deposition, enhance serum lipid metabolism, and improve meat quality in ducks.

[0007] The present invention isolates and screens Lactobacillus plantarum LP-G7 from the intestines of meat ducks, obtaining acid-resistant and bile-resistant Lactobacillus plantarum LP-G7, which has acid-producing, antibacterial and adhesion capabilities. Compared with the prior art, the present invention's Lactobacillus plantarum LP-G7 also has the following unexpected technical effects: The Lactobacillus plantarum LP-G7 of the present invention significantly increases the average daily weight gain of meat ducks, reduces the average daily feed intake and feed-to-weight ratio, and improves the growth performance of meat ducks; significantly reduces the abdominal fat rate and liver fat deposition of meat ducks, significantly reduces the total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) contents in the serum of meat ducks, and increases the high-density lipoprotein cholesterol (HDL-C) in the serum; significantly increases the immunoglobulin M (IgM) and immunoglobulin G (IgG) contents in the serum; significantly reduces the storage loss of breast muscle and leg muscle of meat ducks, and reduces the yellowness (b*), gelatinization and chewiness of the breast muscle.

[0008] In summary, the acid-resistant and bile-resistant Lactobacillus plantarum LP-G7 provided by the present invention, which has acid-producing, antibacterial and adhesion capabilities, can also improve the growth performance of meat ducks, reduce fat deposition in serum, abdomen and liver, improve lipid metabolism and immunity, and improve meat quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Electrophoresis test results of PCR amplification products of some strains; Figure 2 Effects of Lactobacillus plantarum LP-G7 on lipid deposition in the liver of broiler ducks; Group C: control group, fed with a basic diet; Group T: experimental group, fed with a basic diet + Lactobacillus plantarum LP-G7 probiotic preparation (≥1×10 9 cfu / g). DETAILED DESCRIPTION

[0010] The present invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art. In addition, the embodiments should be understood to be illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments, without departing from the spirit and scope of the present invention, also fall within the scope of protection of the present invention. The raw materials and reagents used in the present invention are all commercially available. The experimental methods in the following examples, unless otherwise specified, are conventional methods. Example

[0011] A strain of Lactobacillus plantarum LP-G7 that is acid-resistant, bile-resistant, and has acid-producing, antibacterial, and adhesion capabilities (1) Isolation and purification of strains Weigh 1 g of cecal contents and dissolve it in 10 mL of sterile PBS solution. Vortex thoroughly to mix. Use the serial dilution method to dilute the sample serially by taking 1 mL of the suspension and adding it to 9 mL of sterile PBS solution. Take 1 mL of each gradient, centrifuge and discard the supernatant, add 1 mL of MRS liquid medium containing 0.15% bile salts and pH 4, incubate at 37°C for 1 h, centrifuge and discard the supernatant, add 1 mL of sterile PBS and resuspend. The treated samples of different gradients were respectively pipetted into different bacterial culture dishes, poured into MRS solid medium, gently shaken, and transferred to a 37°C incubator for incubation for 24 h after solidification. All single colonies in the culture medium were picked into 1 mL of MRS broth medium, numbered, and placed in an incubator at 37°C for incubation for 12 h. They were cultured separately in MRS solid medium using the three-zone plate streak method. The culture and purification were repeated for three generations until the colonies on the plate were similar in morphology, size, and color. After plate streak isolation and culture, a total of 201 colonies were isolated and purified from the cecal contents of meat ducks for subsequent initial screening of strains.

[0012] (2) Initial screening of lactic acid bacteria All isolated strains were inoculated into MRS broth medium and cultured in a 37°C incubator for 24 h. Then, the bacterial suspension cultured to the stable growth phase was inoculated into MRS broth medium with pH = 2, 3, and 4 and bile salt concentrations of 0.15%, 0.3%, and 0.6% at a rate of 2%. At the same time, the same bacterial suspension was inoculated into the same medium without pH adjustment and bile salt addition as a control. The culture was cultured at 37°C for 12 h, and the OD of the strain was measured using a microplate reader. 600 The survival rate was determined by performing three replicates for each strain. The top 20 strains ranked by acid and bile resistance and their acid and bile resistance are shown in Tables 1 and 2. Table 1 Acid resistance of strains

[0013]

[0014] (3) Molecular biological identification of lactic acid bacteria Take 1 mL of culture medium and extract bacterial genomic DNA using the 16S rDNA universal primers: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3', PCR amplification was performed.

[0015] After amplification, perform 2% agarose gel electrophoresis to observe whether the target band appears. Then send it to Suzhou Jinweizhi Biotechnology Co., Ltd. for first-generation sequencing. Perform BLAST sequence comparison on the obtained sequence in the GenBank database (see Lactobacillus plantarum LP-G7 sequence). Figure 1 The figures are the electrophoresis results of PCR amplification products of some strains. The DNA marker sizes are 5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp. After electrophoresis detection of the amplified products, bands at 1500 bp were found in the amplified fragments of 13 strains (G7, G8, G22, G26, G59, G63, G64, G65, G94, G101, Q75, Q80, and Q84).

[0016] The PCR amplification products of these 13 strains were sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the GenBank database on NCBI by BLAST sequence comparison. The sequencing results of G64, G65, and G94 were Escherichia coli, and the remaining sequence comparison results are shown in Table 3.

[0017]

[0018] Lactobacillus plantarum LP-G7 sequence: (5) Rescreening of lactic acid bacteria A single colony of the strain was inoculated into MRS broth and cultured in a 37°C incubator for 24 hours. A 2% portion of the culture medium that had reached the stable growth phase was then inoculated into MRS broth and cultured in a 37°C incubator for 24 hours. The culture was centrifuged at 4500 rpm for 10 minutes at 4°C. The supernatant was collected and the pH was measured using a pH meter. The acid production capacity of the different strains was compared based on the pH. A single colony of the strain was inoculated into MRS broth and cultured at 37°C for 12 hours. 10 μL of the culture medium was then aspirated onto a blank drug susceptibility paper disc. This was repeated 10 times. After the drug susceptibility paper disc dried, the disc was placed in a sealed centrifuge tube according to the strain and stored at 4°C until ready for use. The activated Escherichia coli k88 and Staphylococcus aureus were inoculated into LB liquid culture medium at 2% and cultured at 37°C for 12 h. After that, 100 μL of the cultured Escherichia coli k88 and Staphylococcus aureus were respectively taken and spread on LB solid culture medium. The prepared drug-sensitive paper pieces were divided and placed on the coated culture medium. They were placed in a 37°C incubator and cultured for a certain period of time. The size of the inhibition zone was observed and the diameter of the inhibition zone was measured. The antibacterial ability of different strains was compared based on the diameter of the inhibition zone. After culturing the isolated lactic acid bacteria for 24 h, they were centrifuged at 4500 r / min and 4°C for 10 min. The supernatant was discarded, the bacteria were collected, washed twice with sterile PBS buffer, and resuspended in PBS buffer again. The OD of the sample was measured. 600 The absorbance of the bacterial suspension was adjusted to 0.25 ± 0.05 by adding sterile PBS buffer, and the initial absorbance was recorded. 4 mL of bacterial suspension was transferred to a 5 mL centrifuge tube and allowed to stand at room temperature for 20 h. The supernatant was aspirated and the OD value of the supernatant was measured. 600 The autoagglutination rate was calculated by culturing the isolated lactic acid bacteria for 24 h, centrifuging at 3000 r / min and 4°C for 15 min, discarding the supernatant, collecting the bacteria, washing them twice with sterile PBS buffer, and resuspending them in PBS buffer. The OD value of the sample was measured. 600 Add 1 mL of xylene to 3 mL of bacterial suspension, let it stand at room temperature for 10 minutes, then shake it on a vortex for 2 minutes, and finally let it stand at room temperature for 15 minutes. After the solution is separated, aspirate the lower aqueous phase and measure the OD value of the aqueous phase. 600 The surface hydrophobicity was calculated using the values obtained from the HPLC. The acid production, antibacterial, and adhesion abilities of lactic acid bacteria are shown in Table 4. The pH of the bacterial cultures of G7 and G8 was significantly lower than that of the other strains, while their acid production was significantly higher. The diameter of the inhibition zone of G7 against Escherichia coli K88 and Staphylococcus aureus was significantly higher than that of the other strains. The hydrophobicity of G7 and G8 was significantly higher than that of the other strains, and the autoagglutination rates of G7, G63, and Q84 were significantly higher than those of the other strains.

[0019]

[0020] Example 2 Application of Lactobacillus plantarum LP-G7 in meat duck production Preparation of probiotics: The frozen Lactobacillus plantarum LP-G7 was inoculated into MRS broth medium and cultured at 37°C for 24 hours. The culture was subcultured twice. The activated strain was inoculated into MRS broth medium and cultured at 37°C. The bacterial solution was centrifuged, freeze-dried, and crushed to prepare freeze-dried powder of Lactobacillus plantarum LP-G7. The number of viable bacteria in the freeze-dried powder of lactic acid bacteria reached 1x10 9 cfu / g. The MRS broth medium formula is as follows: peptone: 10 g / L, beef powder: 10 g / L, yeast powder: 5 g / L, glucose: 20 g / L, magnesium sulfate: 0.1 g / L, sodium acetate: 5 g / L, sodium citrate: 2 g / L, potassium dihydrogen phosphate: 2 g / L, manganese sulfate: 0.05 g / L, and Tween 80: 1 g / L.

[0021] Seventy-two one-day-old Cherry Valley ducks of similar weight and good health were randomly divided into two groups, with six replicates in each group and six ducks in each replicate. The experimental period was 42 days. The groups were group C and group T. Group C was a control group fed with a basic diet; group T was an experimental group fed with a basic diet plus a Lactobacillus plantarum LP-G7 probiotic preparation (≥1×10 9 cfu / g).

[0022] (1) Effects of Lactobacillus plantarum LP-G7 on the production performance of broiler ducks Fasting body weights of broiler ducks were measured at 08:30 on the first and 42nd day before feeding (initial body weight (IW) and final body weight (FW). Feed intake was recorded daily for replicate groups. Weight gain and feed intake were corrected for any Cherry Valley duck deaths. Average daily gain (ADG), average daily feed intake (ADFI), and feed-to-gain ratio (F / G) were then calculated. As shown in Table 5, Lactobacillus plantarum LP-G7 significantly increased ADG and final body weight, reduced ADFI and F / G, and improved the production performance of broiler ducks.

[0023]

[0024] (2) Effects of Lactobacillus plantarum LP-G7 on abdominal fat rate, liver lipid deposition and serum lipid metabolism in ducks On day 42 of the experiment, at 08:30 AM before feeding, one Cherry Valley duck was selected from each replicate. 2 ml of blood was collected through the wing vein. After standing at room temperature for 30 minutes, the blood was centrifuged at 4°C and 4000 rpm for 10 minutes. Serum was aspirated and dispensed into 1.5 ml centrifuge tubes for analysis of serum biochemical parameters. The ducks were then slaughtered. After fasting for 12 hours, the ducks were deprived of food and water. Eviscerated weight and abdominal fat weight were measured, and abdominal fat percentage was calculated. As shown in Table 6, Lactobacillus plantarum LP-G7 significantly reduced serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels in the ducks, increased serum high-density lipoprotein cholesterol (HDL-C), and significantly reduced abdominal fat percentage. Livers were collected, fixed in 4% paraformaldehyde, and stored at 4°C. Oil Red O staining was performed, and sections were examined under an upright / inverted fluorescence microscope for lipid deposition in the liver. As shown in the figure, Lactobacillus plantarum LP-G7 significantly reduced lipid deposition in the liver.

[0025]

[0026] (3) Effects of Lactobacillus plantarum LP-G7 on serum immune indicators of meat ducks On the 42nd day of the experiment, before feeding at 08:30, one Cherry Valley duck was selected from each replicate, and 2 ml of blood was collected through the wing vein. After standing at room temperature for 30 min, the blood was centrifuged at 4°C and 4000 r / min for 10 min. After centrifugation, the serum was aspirated and divided into 1.5 ml centrifuge tubes. Serum immune indicators were measured. As shown in Table 7, Lactobacillus plantarum LP-G7 significantly increased the levels of immunoglobulin M (IgM) and immunoglobulin G (IgG) in the serum.

[0027] Table 7 Effects of Lactobacillus plantarum LP-G7 on serum immune parameters of meat ducks

[0028] (4) Effect of Lactobacillus plantarum LP-G7 on duck meat quality After slaughter, breast and leg muscles of the ducks were harvested and measured for pH, water-binding capacity, shear force, color difference, TBARS, and TPA. As shown in Table 8, Lactobacillus plantarum LP-G7 significantly reduced storage loss in breast and leg muscles of the ducks, as well as the yellowness (b*), stickiness, and chewiness of the breast muscles.

[0029]

[0030]

[0031] Conclusion: In summary, the acid-resistant and bile-resistant Lactobacillus plantarum LP-G7 provided by the present invention, which has acid-producing, antibacterial and adhesion capabilities, can improve the growth performance of meat ducks, reduce fat deposition in serum, abdomen and liver, improve lipid metabolism and immunity, and improve meat quality.

[0032] It should be noted that the above embodiments are only used to illustrate the technical solutions 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 preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A strain of Lactiplantibacillus plantarum characterized by: Lactobacillus plantarum LP-G7 is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO.: 34430.

2. A microecological preparation containing the Lactobacillus plantarum according to claim 1.

3. Use of the Lactobacillus plantarum of claim 1, whose deposit number is CGMCC NO.: 34430, in improving the growth performance of meat ducks.

4. Use of the Lactobacillus plantarum of claim 1, with the deposit number CGMCC NO.: 34430, in reducing abdominal fat rate, improving liver lipid deposition, and enhancing serum lipid metabolism in ducks.

5. Use of the Lactobacillus plantarum of claim 1, whose deposit number is CGMCC NO.: 34430, in improving the quality of duck meat.

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