Pediococcus acidilactici and application thereof
By screening and identifying Pediococcus lactis AK-RS1 in the duck intestine, the problem of the scarcity of duck-derived lactic acid bacteria resources has been solved, and the effects of improving feed utilization and immunity and improving the microecology of duck farming have been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-09
- Publication Date
- 2026-04-07
AI Technical Summary
Existing technologies have very few duck-derived lactic acid bacteria strains available for screening and isolation. There is a lack of strains that combine good tolerance, adhesion ability, and safety, leading to frequent bacterial diseases and low feed utilization efficiency in duck farming.
Pediococcus acidilactici AK-RS1 was screened from the intestines of healthy ducks and identified as CCTCC NO: M2025755. It has good acid resistance, alkali resistance, bile salt resistance and adhesion ability. It was prepared into a fermentation culture medium for the preparation of antibacterial agents and feed additives.
It improves feed conversion rate, promotes animal growth, enhances immunity, improves intestinal microecological balance, and provides a safe and efficient microecological preparation reference, suitable for duck farming.
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Figure CN121801727A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, specifically relating to a type of Pediococcus lactis and its applications. Background Technology
[0002] With the continuous improvement of people's living standards, the demand and requirements for the breeding industry are gradually increasing, leading to the rapid development of the duck farming industry. However, compared with the mature model of chicken farming, duck farming technology is relatively lagging behind, resulting in problems such as scattered and unregulated farming, low efficiency, and frequent bacterial diseases. Currently, with the large-scale development of duck farming, the demand for antibiotic-free farming and green feed additives is becoming increasingly urgent. Therefore, people are actively exploring feed additives that can replace antibiotics. Lactic acid bacteria, as a safe and efficient probiotic, possesses characteristics such as acid resistance, bile salt resistance, and alkali resistance. It can colonize the animal intestines and exert beneficial effects, thus being widely used in the treatment and prevention of digestive tract diseases in humans and animals. At the same time, lactic acid bacteria have strong protease, lipase, and cellulase activities, which can improve feed absorption and utilization efficiency; and can stimulate the development of immune organs, enhancing the body's immunity and resistance.
[0003] Currently, research on the screening and application of lactic acid bacteria specific to the duck gut is still relatively limited, especially lacking strains with good tolerance, adhesion ability, and safety. Therefore, isolating high-efficiency probiotic lactic acid bacteria from the duck gut is of great significance for developing microecological preparations suitable for duck farming. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to overcome the deficiency of the limited resources for screening and isolating duck-derived lactic acid bacteria strains in the prior art, and to provide a strain of Pediococcus lactis and its application.
[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: This invention discloses a type of Pediococcus acidilactici AK-RS1, wherein the Pediococcus acidilactici is Pediococcus acidilactici AK-RS1 with preservation number: CCTCC NO: M2025755.
[0006] The present invention also discloses a culture based on the above-mentioned Pediococcus lactis, wherein the culture of Pediococcus lactis AK-RS1 is a fermentation culture broth.
[0007] The preparation method of the above culture includes the following steps: the *Pediococcus lactis* AK-RS1 is inoculated into MRS medium, shaken and cultured for 20-24 h, and the bacterial cells are removed to obtain the culture of *Pediococcus lactis* AK-RS1.
[0008] The above-mentioned Pediococcus lactis AK-RS1 and its culture can be used to prepare antibacterial agents.
[0009] Furthermore, the *Pediococcus lactis* AK-RS1 prepared by this invention exhibits excellent adhesion to the poultry intestine. Therefore, the *Pediococcus lactis* AK-RS1 and its culture disclosed in this invention can also be used to prepare feed additives, thereby achieving the goals of improving feed conversion rate, promoting animal growth, enhancing immunity, and improving intestinal microecological balance.
[0010] Compared with existing technologies, this invention has the following beneficial effects: This invention screens duck-derived bacterial strains with probiotic effects from the intestines of healthy ducks. Through continuous screening from a large number of strains, including isolation, purification and identification, tolerance tests, growth performance tests, acid and alkali production capacity tests, and animal safety tests, strains with good acid, alkali, bile salt, and trypsin resistance were selected. These strains exhibit an adhesion rate of 14.6% to duck intestinal epithelial cells, enabling them to colonize the intestines and exert probiotic effects. Furthermore, these strains are sensitive to most antibiotics, have high safety, and are suitable for use as feed additives in duck farming. This provides a better reference for expanding the duck-derived probiotic resource library and its application in microecological preparations.
[0011] Preservation of biological materials Pediococcus acidilactici AK-RS1 was deposited on April 11, 2025 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2025755, located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description
[0012] Figure 1 Characteristic diagram of MRS agar culture of Pediococcus lactis AK-RS1.
[0013] Figure 2 Image showing the Gram staining results of Pediococcus lactis AK-RS1 under a microscope.
[0014] Figure 3 The bar chart shows the thermostable strain of Pediococcus lactis AK-RS1.
[0015] Figure 4 Phylogenetic tree of Pediococcus lactis AK-RS1 16S rRNA.
[0016] Figure 5 This is a 24-hour growth curve of Pediococcus lactis AK-RS1.
[0017] Figure 6 This is a 24-hour pH change curve for Pediococcus lactis AK-RS1.
[0018] Figure 7 This is a diagram of the hemolysis experiment of Pediococcus lactis AK-RS1.
[0019] Figure 8 A bar chart of acid-resistant Pediococcus lactis AK-RS1.
[0020] Figure 9 Bar chart for alkali-resistant Pediococcus lactis AK-RS1.
[0021] Figure 10 Bar chart showing trypsin resistance of Pediococcus lactis AK-RS1.
[0022] Figure 11 A bar chart of bile salt resistant Pediococcus lactis AK-RS1. Detailed Implementation
[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.
[0024] The *Pediococcus lactis* provided by this invention is classified and named AK-RS1. Its accession number is CCTCC NO: M 2025755. The depositary institution is the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit date is April 11, 2025.
[0025] Example 1 1. Isolation, culture and purification of bacterial strains In a clean bench, a segment of mucosal intestinal tissue from a healthy Wuhe duck was scraped using the back of a sterile scalpel. The tissue was placed in a sterile test tube containing physiological saline, shaken well, and immediately diluted serially with physiological saline at a dilution gradient of 10. -4 10 -5 10 -6 100 drops of the diluted solution were added to MRS-CaCO3 agar medium and spread onto plates. The plates were then incubated overnight at 37°C under aerobic conditions. Plates with good growth and sparse colonies were selected for further screening.
[0026] The colony selection criteria were as follows: colonies that were growing well, white, round, with a raised surface, moist, and neat edges, conforming to the morphology of lactic acid bacteria colonies, and with a large calcium-soluble zone. Colonies meeting these criteria were picked up with an inoculation loop and streaked into MRS-CaCO3 agar medium for incubation, repeating this cycle at least 5 times.
[0027] This allowed for the initial screening of several pure bacterial strains, which were named AK-RS1. The colonies on MRS agar medium are shown as follows: Figure 1 As shown.
[0028] 2. Staining and microscopic examination of smears Single purified colonies were picked and smeared, Gram-stained, and observed under an oil immersion microscope. AK-RS1 cells were found to be purple, spherical or oval, arranged in pairs or chains, and were Gram-positive. Results are shown below. Figure 2 .
[0029] 3. Biochemical tests Fermentation tests were conducted on glucose, lactose, maltose, and sucrose, as well as in peptone water, glucose phosphate peptone water, and Simon's citrate tests. The test bacteria were inoculated into peptone water, glucose phosphate peptone water, Simon's citrate, and sugar fermentation tubes (glucose, lactose, maltose, and sucrose) using an inoculation loop or needle, and incubated at 37°C for 24-48 h. The results are shown in Table 1, indicating that AK-RS1 can decompose glucose, maltose, sucrose, and lactose. (See Table 1 for details.)
[0030] Table 1 shows the results of the AK-RS1 biochemical test.
[0031] 4. PCR identification: PCR amplification was performed using universal primers 27F and 1492R for the bacterial 16S rRNA gene.
[0032] (1) Nucleic acid extraction: A single colony of the bacterium was picked up with an inoculation loop and inoculated into MRS liquid medium and cultured at 37°C and 120 rpm for 24 h with shaking. Nucleic acid was then extracted using the Ezup column-type bacterial genomic DNA extraction kit from Shanghai Sangon Biotech Co., Ltd.
[0033] (2) PCR amplification and sequencing analysis of 16S rRNA: The PCR reaction system was 50 μL: PCR Master Mix 25 μL, upper primer 2 μL, lower primer 2 μL, DNA template 5 μL, and sterile water 16 μL. PCR reaction conditions: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 10 min. The PCR products were detected by agarose gel electrophoresis and then sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing, and the sequence is shown.
[0034]
[0035] Sequencing results were compared and analyzed in the NCBI nucleic acid database. The 16S rRNA gene of AK-RS1 strain and *Pediococcus lactis* strain ABRIIFBI-55 (GenBank accession number: ON391079.1) showed 100% homology, confirming the tested bacterium as *Pediococcus lactis*, and a phylogenetic tree was constructed. During evolution, independent strains such as... Figure 4 The evolutionary branch shown is a new species of *Pediococcus lactis*, and the results are shown in Table 2.
[0036] Table 2 shows the results of the comparative analysis in the nucleic acid database.
[0037] 5. Resistance to acid, alkali, bile salts, and trypsin inhibitors. Add 100 μL of AK-RS1 bacterial suspension to 10 mL of MRS medium and incubate at 37°C with shaking at 120 rpm for 24 h. Collect bacterial cells by centrifugation at 8000 r / min for 10 min. Resuspend the bacterial cells in MRS medium with pH values of 2, 3, 4, 8, and 9, bile salt concentrations of 0.1%, 0.2%, and 0.3%, and trypsin concentrations of 1%, 1.2%, and 1.4%, respectively. Incubate at 37°C with shaking for 2 hours, then plate samples for counting. See Table 3 for details. Figure 9 , Figure 10 , Figure 11 .
[0038] Table 3 Results of acid resistance, bile salt resistance, and pancreatic enzyme resistance tests
[0039] 6. High temperature resistance test 100 μL of AK-RS1 bacterial suspension was added to 10 mL of MRS medium and cultured at 37°C with shaking at 120 rpm for 24 h. Subsequently, 200 μL of AK-RS1 bacterial suspension was incubated at 40°C, 50°C, and 60°C for 20 min each. The results showed that the survival rates of each group were 93%, 64%, and 30%, respectively. (See details...) Figure 3 This indicates that the bacterium has good heat resistance.
[0040] 7. Analysis of growth characteristics and lactic acid / base production capacity The AK-RS1 strain was inoculated into MRS medium and cultured at 37°C with shaking at 120 rpm. Samples were taken every 2 hours, using sterile MRS medium as a control. The OD value and pH value at 600 nm were measured, and curves were plotted with culture time on the x-axis and OD value and pH value on the y-axis, respectively. See Figure 6 , Figure 7As shown in the figure, AK-RS1 bacteria enter the logarithmic growth phase at 4-12 hours and begin to increase rapidly, then enter the plateau phase at around 18 hours with little change in concentration. AK-RS1 has a good acid-producing capacity; the pH drops to 3.88 at 24 hours (acid production ends), and the pH drops rapidly to 4.0 between 2-14 hours, indicating that its metabolic process can regulate the acidic environment of the intestine.
[0041] 8. Drug sensitivity test 100 μL of AK-RS1 bacterial suspension was added to 10 mL of MRS medium and incubated at 37℃ with shaking at 120 rpm for 24 h. The bacterial suspension was then evenly spread onto MRS agar plates using a cotton swab, and drug sensitivity test strips were attached to the plates. The plates were incubated at 37℃ for 24 h. The results showed that AK-RS1 was sensitive to E, MI, FFC, IPM, C, CC, CZ, PIP, and PEN; moderately sensitive to AMP, TET, AZI, DO, CXM, CPZ, and CTR; and resistant to CIP, NOR, VAN, MY, XO, LEV, CAZ, SXT, PB, CN, S, AMK, and GEN. These results indicate that AK-RS1 is relatively sensitive to common antibiotics and has good safety.
[0042] Table 4 Results of drug resistance test for strain AK-LY1
[0043] Note: S = Sensitive; R = Tolerant; I = Moderately Sensitive 9. Hemolysis test Streaking of fresh AK-RS1 bacterial colony onto sheep blood agar plates and incubating at 37°C for 24 hours. Hours later, the results were as follows Figure 8 This indicates that AK-RS1 is a gamma hemolytic agent and has good safety.
[0044] 10. Cell adhesion The experimental cells were duck intestinal epithelial cells. 200 μL of bacteria were inoculated into a 24-well plate containing duck intestinal epithelial cells and incubated for 1 h. Cell counts were then performed on the plate. The AK-RS1 cell adhesion rate was 14.6%, indicating that it has good adhesion ability in the intestine and can exert a probiotic function.
[0045] Table 5 AK-RS1 cell adhesion rate
[0046] In this invention, a high-performance lactic acid bacteria strain AK-RS1 was isolated from the cecum of ducks and identified as a type of Pediococcus lactis.
[0047] Experiments showed that the isolated AK-RS1 strain exhibited good resistance to acid, alkali, bile salts, and trypsin.
[0048] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.
[0049] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A type of Pediococcus acidilactici AK-SF1, characterized in that, The *Pediococcus lactis* mentioned is *Pediococcus lactis* AK-RS1 with the preservation number: CCTCC NO: M2025755.
2. A culture based on *Pediococcus lactis* AK-RS1 as described in claim 1, characterized in that, The culture of *Pediococcus lactis* AK-RS1 is a fermentation broth.
3. A method for preparing a culture of *Pediococcus lactis* according to claim 2, characterized in that, The procedure includes the following steps: inoculating the *Pediococcus lactis* AK-RS1 into a medium containing MRS, shaking and culturing for 20-24 h, removing the bacterial cells to obtain a culture of *Pediococcus lactis* AK-RS1.
4. The use of the *Pediococcus lactis* AK-RS1 as described in claim 1 or the culture as described in claim 2 in the preparation of antibacterial agents.
5. A drug, characterized in that, The active ingredient of the drug comprises the *Pediococcus lactis* AK-RS1 of claim 1 or the culture of claim 2.
6. A feed additive, characterized in that, The active ingredient of the feed additive comprises the *Pediococcus lactis* AK-RS1 as described in claim 1 or the culture as described in claim 2.