Heat-resistant pediococcus acidilactici and application thereof in pellet feed production

Abstract

The invention provides heat-resistant pediococcus acidilactici and application of the heat-resistant pediococcus acidilactici in pellet feed production. The heat-resistant pediococcus acidilactici is named as pediococcus acidilactici YJ and is preserved in the China Center for Type Culture Collection on May 19, 2025, and the preservation number of the heat-resistant pediococcus acidilactici is CCTCC (China Center for Type Culture Collection) NO: M 20251096. The strain YJ has heat-resistant and extrusion-resistant functions, and can be added into a feed production process before particle forming to obtain a feed product with high viable count and uniform addition, so that the growth and intestinal development of chicks are promoted, intestinal injury is repaired, and the production performance of the chicks is improved. The strain YJ can tolerate the high temperature of 90 DEG C, and can be directly added in a mixing stage, so that the strain YJ can be uniformly added in powdery, hard pellet feed and expanded pellet feed, meanwhile, the magnitude order of viable count can reach 107 cfu / g feed, and meanwhile, the variable coefficient of mixing uniformity is less than 5.0%.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of feed probiotics, in particular to a heat-resistant Pediococcus acidilactici and its application in the production of pellet feed. BACKGROUND

[0002] Pediococcus acidilactici is an important probiotic additive in the feed field, which mainly plays an important role by improving animal intestinal health, enhancing immunity, improving production performance and reducing antibiotic use. It is widely used in livestock and poultry (such as pigs and chickens), aquatic and ruminant feed, and its specific functions include inhibiting pathogenic bacteria (such as Escherichia coli and Salmonella), enhancing intestinal antioxidant capacity, improving intestinal morphological structure, improving feed utilization, and can be applied to silage fermentation to improve preservation quality and palatability, etc.

[0003] For example, the patent application CN114958649A discloses a Pediococcus acidilactici and its application in degrading ochratoxin A, the preservation number of the bacteria NJB421 is CGMCC NO.28836, which has the characteristics of stable growth performance, high acid-producing capacity, heat resistance and good antibacterial property. As a feed additive, it can be fed to laying hens to reduce the breakage rate and cholesterol content of egg yolk, improve the weight and shell thickness of eggs, and has the effects of improving feed utilization and growth performance. Although the above-mentioned patent application discloses that the Pediococcus acidilactici NJB421 has good tolerance at 40℃-46℃, and after 30 minutes of action in this temperature range, the number of viable bacteria in the bacterial solution has no difference with that at 37℃ for 30 minutes, which can still reach 10 7 CFU / mL; but it does not disclose that it still has good heat resistance at 70℃-90℃. Moreover, the existing Pediococcus acidilactici is difficult to achieve heat resistance at 70℃-90℃.

[0004] It is a very difficult task to add probiotics to feed, and the core goal is to maintain the activity and stability of probiotics in the final feed product. The temperature of the pelleting process in feed processing is usually high temperature of 70-90℃, which can kill most bacteria. Since most probiotics, especially Pediococcus acidilactici, are sensitive to high temperature, high pressure and moisture, the survival rate of probiotics without coating treatment after the pelleting process is usually less than 0.1‰; therefore, before or during the pelleting process, the probiotics must be coated to ensure the activity and stability of the probiotics in the feed. However, coating the probiotics will increase the processing cost of the feed. In order to reduce the cost, the probiotics without coating treatment are usually added in the drinking water or the finished feed after pelleting, which is easy to contaminate the drinking water pipeline or the feed grading. In short, the existing feed lactobacillus probiotics, especially Pediococcus acidilactici, have the problem of being unable to simultaneously consider low-cost addition and high-viable count during feed processing, which restricts its large-scale industrial application. Heat-resistant Pediococcus acidilactici suitable for pellet feed production is urgently needed. SUMMARY

[0005] Therefore, one object of the present application is to provide a heat-resistant Pediococcus acidilactici.

[0006] A second object of the present application is to provide a bacterial agent for preventing and repairing intestinal damage of laying hens.

[0007] A third object of the present application is to provide the application of the above-mentioned heat-resistant Pediococcus acidilactici or bacterial agent in the production of laying hens, so as to achieve the purpose of preventing and repairing intestinal damage of laying hens.

[0008] A fourth object of the present application is to provide the application of the above-mentioned heat-resistant Pediococcus acidilactici or bacterial agent in the production of pellet feed, so as to realize the low-cost, high-activity and high-uniformity production of probiotic feed.

[0009] A fifth object of the present application is to provide a feed containing the above-mentioned heat-resistant Pediococcus acidilactici or bacterial agent, which can promote the growth, intestinal development and repair of intestinal damage of chicks, and improve the production performance of chicks.

[0010] In order to achieve the above-mentioned objects, the technical solutions provided by the present application are as follows: The first aspect of the present application provides a heat-resistant Pediococcus acidilactici, which is named YJ. Pediococcus acidilactici YJ, which was preserved in China Center for Type Culture Collection on May 19, 2025, and the preservation number is CCTCC NO: M 20251096.

[0011] The second aspect of the present application provides a bacterial agent, which comprises the above-mentioned heat-resistant Pediococcus acidilactici. The bacterial agent can be a solid bacterial agent or a liquid bacterial agent. Preferably, the bacterial agent is a solid bacterial agent, and the concentration of the bacterial strain YJ in the solid bacterial agent can be (1-10) x 1011 cfu / g, such as 1 x 10 11 cfu / g, 2 x 10 11 cfu / g, 3 x 10 11 cfu / g, 4 x 10 11 cfu / g, 5 x 10 11 cfu / g, 6 x 10 11 cfu / g, 7 x 10 11 cfu / g, 8 x 10 11 cfu / g, 9 x 10 11 cfu / g, 1 x 10 12 cfu / g, etc.

[0012] The third aspect of the present application provides the use of the above-mentioned heat-resistant Pediococcus acidilactici (Pediococcus acidilactici YJ) or bacterial agent in the production of laying hens. Pediococcus acidilactici The fourth aspect of the present application provides the use of the above-mentioned heat-resistant Pediococcus acidilactici (Pediococcus acidilactici YJ) or bacterial agent in the production of granular feed.

[0013] Further, the use in the production of laying hens includes prevention and repair of intestinal damage of laying hens.

[0014] The fourth aspect of the present application provides the use of the above-mentioned heat-resistant Pediococcus acidilactici (Pediococcus acidilactici YJ) or bacterial agent in the production of granular feed. Pediococcus acidilactici The fourth aspect of the present application provides the use of the above-mentioned heat-resistant Pediococcus acidilactici (Pediococcus acidilactici YJ) or bacterial agent in the production of granular feed.

[0015] Further, the use in the production of feed includes adding by any of the following ways: 1) solid bacterial agent added in the mixing section of feed processing; 2) post-spraying process.

[0016] Further, the use in the production of feed includes: first, adding Pediococcus acidilactici (Pediococcus acidilactici YJ) bacterial agent with a concentration of (1-10) x 10 11 cfu / g to granular basic feed at a mass ratio of 1:1000, and stirring uniformly with a mixer; and then granulating with a granulator. Pediococcus acidilactici The fifth aspect of the present application provides a feed comprising the above-mentioned heat-resistant Pediococcus acidilactici (Pediococcus acidilactici YJ), and the concentration of the heat-resistant Pediococcus acidilactici (Pediococcus acidilactici YJ) in the feed is (1-10) x 10 7 cfu / g.

[0017] Pediococcus acidilactici Further, the feed comprises granular basic feed and heat-resistant Pediococcus acidilactici (Pediococcus acidilactici YJ) solid bacterial agent with a concentration of (1-10) x 10 11 cfu / g, and the mass ratio of the bacterial agent to the granular basic feed is 1:1000. Pediococcus acidilactici

[0018] Further, the feed comprises granular basic feed and heat-resistant Pediococcus acidilactici (Pediococcus acidilactici YJ) solid bacterial agent with a concentration of (1-10) x 10 11 cfu / g, and the mass ratio of the bacterial agent to the granular basic feed is 1:1000. Pediococcus acidilactici

[0019] ​Compared with the prior art, the heat-resistant Pediococcus lactis provided by the present invention ( Pediococcus acidilactici YJ has heat-resistant and compression-resistant properties, allowing it to be added to the feed production process before pelleting to obtain feed products with high live bacteria counts and uniform addition. This promotes chick growth, intestinal development, repairs intestinal damage, and improves chick production performance. The above-mentioned heat-resistant Pediococcus lactis (… Pediococcus acidilactici YJ can withstand high temperatures of 90℃ and can be added directly during the mixing stage, making it a heat-resistant type of Pediococcus lactis. Pediococcus acidilactici YJ can be uniformly added to powdered, hard pellet, and extruded pellet feeds, with the number of live bacteria reaching the order of 10. 7 The cfu / g feed content was measured, while the coefficient of variation for mixing uniformity was less than 5.0%. Attached Figure Description

[0020] Figure 1 The *Pediococcus lactis* provided in Example 1 of this invention ( Pediococcus acidilactici Colony status of YJ (left) and Gram staining (right, with scale bar at 20 μm); Figure 2 The *Pediococcus lactis* provided in Example 1 of this invention (… Pediococcus acidilactici Growth curve of YJ; Figure 3 The *Pediococcus lactis* provided in Example 1 of this invention (… Pediococcus acidilactici The evolutionary tree of YJ; Figure 4 The *Pediococcus lactis* provided in Example 1 of this invention (… Pediococcus acidilactici ) Schematic diagram of the heat resistance test results of YJ.

[0021] Nucleotide Sequence Listing SEQ ID No. 1 is Pediococcus lactis ( Pediococcus acidilactici YJ's 16S rDNA sequence. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0023] Unless otherwise specified, all terms used in this invention are commonly used in the relevant field. The technical means employed in the embodiments, such as preparation processes and testing methods, are conventional methods well-known to those skilled in the art. All reagents and products used are commercially available. The source, trade name, and, where necessary, the components of any reagents are indicated upon their first appearance.

[0024] The culture media used in the following examples are: MRS medium: 1L of basic formulation contains 20 g glucose, 10 g peptone, 8 g beef extract, 4 g yeast extract, 2 g diammonium citrate, 2 g K2HPO4·3H2O, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·H2O, 1 mL Tween 80, 15 g agar (solid), and pH adjusted to 6.2-6.4.

[0025] Example 1: Heat-resistant Pediococcus lactis ( Pediococcus acidilactici Acquisition and identification of YJ Enrichment and screening of strains On May 18, 2024, bacterial strains were obtained from the ileum of Hy-Line Brown chicks in the laboratory. Specifically, fresh ileal mucosal tissue from healthy Hy-Line Brown chicks was aseptically cut into small segments and homogenized with sterile physiological saline. The homogenate was then serially diluted to 10⁻⁻⁻⁶. 4 ~10⁻ 6 Dilute each solution by 0.1 mL and spread onto MRS selective medium. Incubate anaerobically at 35°C for 48 hours. Pick round, milky-white suspected colonies with neat edges and inoculate them onto MRS liquid medium for purification. Repeat streaking three times to obtain single colonies. Figure 1 As shown (left).

[0026] Identification of strains (1) Microbiological characteristics like Figure 1 As shown on the right, the microbiological characteristics of the isolated strain are typical Gram-positive staining, medium-sized cells, milky-white colonies on the culture medium, smooth and moist surface, and raised appearance. This strain was inoculated into MRS medium and cultured statically at 37°C under anaerobic conditions; the culture solution was collected every 4 hours, and the absorbance (OD) at 600 nm was measured using a spectrophotometer. Its growth curve was plotted as shown below. Figure 2 As shown.

[0027] (2) Molecular biological identification The genomes of the selected strains were extracted and gene sequences were determined. The assembled 16S rDNA sequences were compared with relevant sequences in the NCBI GenBank database for similarity. Multiple alignment was performed using the Blast program to determine the species of the strains. A phylogenetic tree of the strains was constructed using the neighbor-joining method with Mega X software (MEGA 5.0). Figure 3 As shown in SEQ ID No. 1, the 16S rDNA sequence of this strain is 926 bp in length. Pediococcus acidilacticiThe 16S rDNA sequence showed 99% homology. Therefore, based on the combined microbiological characteristics and molecular biological identification results, this strain belongs to *Pediococcus lactis* (Lactococcus). Pediococcus acidilactici YJ, which is named YJ (hereinafter referred to as "strain YJ").

[0028] (3) Heat resistance test of strain YJ The YJ strain was inoculated into MRS medium and first incubated statically at 37°C under anaerobic conditions for 24-28 h to obtain a concentration of 6.93 × 10⁻⁶. 9 A bacterial suspension of CFU / g was prepared; 1 mL of this suspension was added to a glass test tube, and the test tube containing the suspension was placed in a 90℃ constant temperature water bath. The viable bacterial count was determined using the plate count method after 1, 2, 3, 4, and 5 minutes in the 90℃ constant temperature water bath. A total of 15 glass test tubes were used in this experiment. Three test tubes were removed at each time point (e.g., 1 minute) to calculate the viable bacterial count and survival rate. The results are shown below. Figure 4 As shown.

[0029] from Figure 4 It can be seen that the initial viable count of strain YJ (CCTCC NO: M 20251096) was 6.93 × 10⁻⁶. 9 The cfu / g count reached 3.33 × 10⁻⁶ cells / g after being kept at 90℃ for 5 minutes. 6 With a cfu / g concentration and a survival rate of 0.48‰, strain YJ exhibits good tolerance.

[0030] Therefore, based on the above identification results of strain YJ, strain YJ is classified and named... Pediococcus acidilactici (Pediococcus lactis), name Pediococcus acidilactici YJ was deposited on May 19, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCCNO: M 20251096.

[0031] Example 2: Application of strain YJ in feed processing The highest temperature in feed processing is usually below 90°C, and the processing time at this temperature is also relatively short. For example, in the pelleting section, the temperature is 70°C-85°C, and the time is generally no more than 10 seconds. Therefore, the strain YJ provided in Example 1 of this invention can theoretically withstand the high temperature of the pelleting section and maintain high activity.

[0032] This embodiment provides a solid microbial agent with a concentration of (4.93±0.56)×10 11Strain YJ of 1.0 x 10 cfu / g. The low-temperature preserved strain was inoculated into MRS liquid medium and cultured at 30-37°C for 18-24 hours to activate and recover. The seed liquid was inoculated into the fermenter at 5-10%, and the optimized MRS medium was used to control the pH to 6.0-6.5 and the culture was carried out at 35°C for 8-12 hours. When the OD600 reached 0.8-1.0 (log phase), centrifugation was carried out at 4000-5000 r / min for 15-20 minutes to obtain the bacterial slurry, which was washed 2-3 times; plate counting was performed to ensure that the viable bacteria were ≥10 11 cfu / g. The bacterial liquid was spray dried at an inlet temperature of 180-200°C and an outlet temperature of 80-90°C to obtain a powder, which was sealed and stored in the dark.

[0033] The present embodiment also provides a feed comprising a granular base feed and the above-mentioned solid bacterial agent, and the mass ratio of the solid bacterial agent to the granular base feed is 1:1000.

[0034] First, the concentration of (4.93±0.56) x 10 11 CFU / g of Pediococcus acidilactici (P. Pediococcus acidilactici ) YJ bacterial agent was added to the granular base feed at a mass ratio of 1:1000, and the mixture was stirred uniformly using a mixer; then granulation was performed using a granulator.

[0035] The present embodiment also provides a processing method of the above-mentioned feed, which comprises: adding 1 g of the above-mentioned solid bacterial agent to 1 kg of the granular base feed, mixing in a mixer for 2 min, steam conditioning at 85°C for 1 min using a conditioner, and then performing a granulation process, wherein the ring die diameter of the granulation is 3.0 mm, and the granulated feed is dried at 60°C for 20 min after molding, so that the viable bacterial count of the granulated feed is 6.55 x 10 7 cfu / g of feed. The viable bacterial count and survival rate of each section in the feed processing process were detected, and the results are shown in Table 1.

[0036] Table 1: Viable bacterial count and survival rate in the feed processing process Item Initial After mixing After conditioning After granulation After drying Viable count cfu / g (4.93 ± 0.56) x 10 8 ]] (3.43 ± 0.48) x 10 8 ]] (3.14 ± 0.61) x 10 8 ]] (9.10 ± 0.88) x 10 7 ]] (6.55 ± 0.72) x 10 7 ]] Survival rate % 100 69.57±7.72 63.69±6.66 18.46±2.05 13.29±0.96 Mixing homogeneity (coefficient of variation) % -- 4.47±0.35 4.48±0.51 4.01±0.45 4.45±0.54 As can be seen from Table 1, Pediococcus acidilactici can be uniformly added to the feed, and after mixing, conditioning, granulation and drying, it still maintains a high viable bacterial count and survival rate (>13%), is uniformly mixed, and the coefficient of variation is <5.0%.

[0037] Therefore, the heat-resistant Pediococcus acidilactici provided by the present application can maintain activity under high temperature conditions, solving the problem that the number of live bacteria of the Pediococcus acidilactici is greatly reduced in the feed processing process, finally leading to unstable Pediococcus acidilactici feeding function and limited probiotic effect; the heat-resistant Pediococcus acidilactici YJ can be used to prepare liquid and solid Pediococcus acidilactici products. The solid product can be added in the mixing section of the feed processing, maintaining the uniform mixing and maintaining the high number of live bacteria in the feed product. The liquid product can be added by spraying process after the feed pellet is formed, and the high number of live bacteria in the feed product can also be maintained.

[0038] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the specific embodiments of the present application can be modified or some technical features can be replaced by equivalent ones; without departing from the spirit of the technical solutions of the present application, they should be covered in the technical solution range of the present application.

Claims

1. A heat tolerant Pediococcus acidilactici strain, characterized in that, designated Pediococcus acidilactici YJ, deposited on May 19, 2025 at China Center for Type Culture Collection, with the accession number CCTCC NO: M 20251096.

2. A bacterial agent comprising the heat-tolerant Pediococcus acidilactici of claim 1.

3. The inoculant according to claim 2, characterized in that It is a solid bacterial agent, wherein the concentration of the bacterial strain YJ is (1-10) x 10 11 cfu / g.

4. Use of the heat-tolerant Pediococcus acidilactici of claim 1 or the bacterial agent of claim 2 or 3 in the production of laying hens.

5. Use according to claim 4, characterized in that, Including prevention and repair of intestinal damage in laying hens.

6. Use of the heat-tolerant Pediococcus acidilactici of claim 1 or the bacterial agent of claim 2 or 3 in the production of pelleted feed.

7. Use according to claim 4, characterized in that, Including addition by either of: 1) solid bacterial agent addition in the mixing section of the feed processing; 2) post-coating process.

8. Use according to claim 7, characterized in that, Including: First, the heat-resistant Pediococcus acidilactici inoculum with a concentration of (1-10)×10 11 The heat-resistant Pediococcus acidilactici inoculum with a concentration of (1-10)×10 11 The heat-resistant Pediococcus acidilactici inoculum with a concentration of (1-10)×10 11 The heat-resistant Pediococcus acidilactici inoculum with a concentration of (1-10)×10 11 The heat-resistant Pediococcus acidilactici inoculum with a concentration of (1-10)×10 9. A feed, characterized in that, The feed includes the heat-resistant Pediococcus acidilactici of claim 1, and the number of viable cells of the heat-resistant Pediococcus acidilactici in the feed is (1-10) x 10 7 cfu / g.

10. The feed according to claim 9, characterized in that, It comprises a granular basal feed and a solid bacterial agent of Pediococcus thermophilus with a concentration of (1-10) x 10 11 cfu / g, and the mass ratio of the bacterial agent and the granular basal feed is 1:1000.

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