Application of pediococcus acidilactici probiotics in improving energy utilization rate of broilers

Abstract

This invention belongs to the field of biotechnology and feed additives, and discloses the application of *Pediococcus lactis* postbiotics in improving the energy utilization rate of broilers. The *Pediococcus lactis* is... Pediococcus acidilactici PC13, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2022426. The core of this invention lies in the application of a metabiotic prepared by fermenting and inactivating the aforementioned *Pediococcus lactis* strain to broiler farming, particularly for broilers fed low-energy diets. Animal experiments have demonstrated that adding this metabiotic to the diet significantly improves the apparent metabolic rate and energy utilization efficiency of broilers, while reducing the feed conversion ratio. Its mechanism of action involves improving jejunal intestinal morphology and regulating serum energy metabolism-related biochemical indicators. This metabiotic product exhibits stable performance, providing a highly efficient and stable novel additive solution for resolving the contradiction between "reducing formulation costs and maintaining growth performance" in broiler farming.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to the application of a postbiotic from *Pediococcus lactis* in improving the energy utilization rate of broilers. Background Technology

[0002] In modern broiler farming, feed costs account for 60%-70% of total production costs, with energy being the core component of these costs. Improving feed energy utilization is key to reducing farming costs and increasing production efficiency. Currently, common methods for improving energy utilization in farming practice mainly include: 1) using high-energy raw materials, such as adding a high proportion of oil, but this significantly increases feed costs, and high-oil diets are prone to oxidative rancidity, affecting palatability and animal health; 2) adding exogenous enzyme preparations (such as amylase, protease, and lipase), but their effects are substrate-specific, limiting their overall energy enhancement effect on complex diets, and they are easily inactivated during feed processing (such as pelleting); 3) adding live probiotics, which indirectly promote nutrient absorption by regulating the intestinal flora. However, live bacteria have high requirements for storage conditions (such as temperature and humidity) and are easily inactivated during feed processing, storage, and passage through the acidic environment of the animal's stomach, leading to unstable effects.

[0003] Metabiotics are mixtures of inactivated microbial cells, metabolic byproducts, and cellular components, including peptidoglycans, extracellular polysaccharides, short-chain fatty acids, and bacteriocins. Compared to live probiotics, metabiotics offer advantages such as high stability, ease of storage and transportation, immunity to stomach acid and bile salts, and good safety, demonstrating significant application potential in livestock and poultry farming. However, current research and applications of metabiotics largely focus on general functions such as enhancing animal immunity, improving gut health, and inhibiting pathogens. While these existing technologies have validated the health benefits of certain probiotics or their metabolites, they lack solutions with clearly defined mechanisms of action specifically targeting the key economic trait of "precisely improving energy utilization."

[0004] Pediococcus lactis, a common lactic acid bacteria used in feed, has been proven to have functions such as acid production and antibacterial activity. However, there are currently no reports on the application of its inactivated form—Pediococcus lactis postbiotic—in specifically improving the energy utilization efficiency of broilers. Especially given the current industry's pursuit of cost reduction and efficiency improvement, and the experimentation with low-energy diets to reduce formulation costs, there is an urgent need for a feed additive that can effectively compensate for the deficiencies of low-energy diets and maintain or even improve the growth performance and energy utilization efficiency of broilers. Therefore, developing a Pediococcus lactis postbiotic product based on a specific high-efficiency strain with a clear function of improving energy utilization efficiency has significant industrial application value and market prospects. Summary of the Invention

[0005] This invention first provides a *Pediococcus lactis* strain, wherein the *Pediococcus lactis* is *Pediococcus lactis* (… Pediococcus acidilactici PC13 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2022426.

[0006] The present invention also provides a metabiotic, which is obtained by fermenting and culturing the above-mentioned *Pediococcus lactis* PC13 and then inactivating it.

[0007] In some embodiments, the metabiotic contains ≥40% inactivated bacterial cells, ≥15% extracellular polysaccharides, ≥8 mg / g of total short-chain fatty acids, and ≥20 U / mg of protease activity.

[0008] The present invention also provides the application of the above-mentioned metagenic agent in the preparation of feed additives for improving the energy utilization of broilers.

[0009] In some embodiments, the amount of the metabiotic added to the feed is 0.5 kg / ton of feed.

[0010] In some embodiments, the application is to add a feed additive containing the metagener to the basal or low-energy diet of broilers.

[0011] In some embodiments, the metabolizable energy of the low-energy diet is less than 3.2 MJ / kg.

[0012] The present invention also provides a feed for improving the energy utilization of broilers, the feed containing a basal diet and the above-mentioned post-biotics.

[0013] In some embodiments, the feed is a low-energy diet, and the amount of post-biotic added is 0.5 kg / ton of feed.

[0014] The present invention also provides the application of the above-mentioned Pediococcus lactis PC13 in the preparation of feed additives for improving the energy utilization of broilers.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: (1) This invention is the first to isolate Pediococcus lactis ( Pediococcus acidilactici )PC13.

[0016] (2) Significantly improves energy utilization: Animal experiments have shown that adding the post-biotic of this invention to broiler diets can increase the apparent energy metabolic rate by more than 2 percentage points, significantly improve energy utilization efficiency, and effectively reduce the feed conversion ratio. When added to low-energy diets, it can restore the body weight and growth performance of broilers at 42 days of age to levels close to those of normal-energy diet groups, thus resolving the contradiction between cost reduction and efficiency improvement.

[0017] (3) Mechanism of action is clear: This metabiotic improves the morphology of the jejunum in broilers (significantly increasing villus height and decreasing crypt depth, thereby increasing the villus-crypt ratio), enhancing the intestinal absorption area and efficiency of nutrients. At the same time, it can regulate serum energy metabolism indicators, significantly increasing serum glucose (GLU) and triglyceride (TG) levels and decreasing free fatty acid (NEFA) levels, indicating that it promotes the body's energy storage and utilization, and alleviates metabolic stress caused by low-energy diets.

[0018] (4) Stable and efficient performance: The metabiotic prepared by the Pediococcus lactis PC13 strain of this invention through a specific process is rich in active substances such as extracellular polysaccharides and short-chain fatty acids. Compared with ordinary Pediococcus lactis, its regulation efficiency on energy absorption in the broiler intestine is significantly improved. The metabiotic form is not dependent on live bacteria, has strong stability, and retains a high activity rate after 6 months of storage at room temperature, which is convenient for industrial production and application.

[0019] Preservation Instructions The lactic acid cocci ( Pediococcus acidilactici PC13, isolated from the intestines of pigs, was deposited at the China Center for Type Culture Collection on April 19, 2022, and classified as: Pediococcus lactis ( Pediococcus acidilactici PC13, accession number: CCTCC NO:M 2022426, address: Wuhan University, Wuhan, Hubei, China. Detailed Implementation

[0020] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0021] Example 1: Preparation of postbiotics from Pediococcus acidilactici Lactic acid bacteria liquid culture medium (MRS): 10 g / L beef extract, 10 g / L peptone, 5 g / L yeast extract, 20 g / L glucose, 3.02 g / L sodium acetate, 1.16 g / L dipotassium hydrogen phosphate, 2 g / L triammonium citrate, 0.05 g / L magnesium sulfate, 0.03 g / L manganese sulfate, 1 ml / L Tween-80, pH 6.2±0.2, sterilized at 121℃ for 15 min.

[0022] Lactic acid bacteria fermentation medium: glucose: 20 g / L, yeast extract: 15 g / L, peptone: 23 g / L, corn steep liquor powder: 5-10 g / L, dipotassium hydrogen phosphate: 2 g / L, calcium carbonate: 5 g / L, magnesium sulfate: 0.2 g / L, manganese sulfate: 0.1 g / L, Tween-80: 1 ml / L, pH: 6.5 ± 0.2, sterilized at 121℃ for 15 min.

[0023] Select the *Pediococcus lactis* strain with accession number CCTCC NO: M 2022426 ( Pediococcus acidilactici PC13 was inoculated into MRS liquid medium and anaerobic cultured at 37°C for 36 hours in an anaerobic workstation to prepare a seed culture. The seed culture of *Pediococcus lactis* was inoculated into lactic acid bacteria fermentation medium at an inoculation rate of 3% and cultured in a 5L fermenter for 24 hours, with the temperature controlled at a constant 37°C, pH at a constant 6.2, and the rotation speed at 150 rpm.

[0024] After fermentation, the bacteria were inactivated by moist heat at 60℃ for 30 minutes. Samples were taken for plate counting to confirm the absence of viable bacteria. The inactivated bacterial solution was then centrifuged at 8000 rpm for 15 minutes. The supernatant was collected, filtered through a 5kDa ultrafiltration membrane, and freeze-dried (-50℃, 0.1MPa) to obtain Pycnococcus lactis post-biotic powder.

[0025] Example 2: Preliminary Experiment of Live Pleurotus ostreatus PC13 and Postbiotic Feeding 2.1 Experimental Design: This experiment adopted a single-factor experimental design. One-day-old healthy AA broilers were randomly divided into three groups, with six replicates per group and eight birds per replicate. The specific groups were as follows: control group, PA live bacteria group, and PA post-biotic group. The control group was fed a basal diet (metabolizable energy 3.2 MJ / kg), while the PA live bacteria group was fed a basal diet supplemented with 0.5 kg / t of live lactic acid bacteria (1×10⁻⁶) prepared as powder by carrier adsorption before inactivation in Example 1. 9 The PA postbiotic group was fed a basal diet supplemented with 0.5 kg / t of *Pediococcus lactis* postbiotic prepared in Example 1.

[0026] 2.2 Measurement Indicators: 1. Growth performance: On the 42nd day of age, the experimental flocks were fasted for 8 hours. They were weighed on an empty stomach in repeated units, and the amount of feed consumed was recorded. The average daily weight gain, average daily feed intake and feed conversion ratio were calculated.

[0027] 2. Energy utilization rate: On days 41-42 of the experiment, the total energy of feed / feces was measured using the "chromium trioxide indicator method" (addition amount 0.3%) (GB / T 26437-2010), and the apparent metabolic rate of energy (%) and energy utilization efficiency (energy deposition / energy intake, %) were calculated. 3. Intestinal morphology: Two birds from each treatment group were slaughtered on day 42 of the experiment, and jejunal tissue sections were prepared (hematoxylin and eosin (HE) staining) to measure villus height (VH) and crypt depth (CD). 4. Serum biochemistry: Serum was collected from the wing veins of the 42-day-old birds and separated to measure glucose (GLU), free fatty acids (NEFA), and triglycerides (TG) (enzymatic method). 5. Storage retention rate: After pelleting, the test feed was stored at room temperature for 30 days, and the residual rate of live bacteria in the live bacteria group was tested.

[0028] 2.3 Statistical Methods: One-way ANOVA was performed using SPSS 26.0 software, and Duncan's method was used for multiple comparisons. P < 0.05 indicated significant differences between groups, and P < 0.01 indicated highly significant differences between groups.

[0029] 2.4 Experimental Results 2.4.1 Storage retention rate: The residual rate of PA live bacteria in the PA live bacteria group feed was 10.9%. High temperature during feed pelleting and long-term storage and water loss will cause the vast majority of live bacteria to become inactive, and the actual effect will be greatly reduced. PC post-biotics do not depend on live bacteria. The active components such as inactivated bacteria, extracellular polysaccharides, and short-chain fatty acids are not affected by processing or room temperature storage conditions, and their activity does not decrease significantly.

[0030] 2.4.2 Growth performance: As shown in Table 1, there was no significant difference in feed intake among the three groups; at the same dosage, PA live bacteria only slightly increased weight gain and decreased feed conversion ratio; the final weight of PA post-biotic group at 42 days of age was 31.24 g higher than that of PA live bacteria group, and the feed conversion ratio was further reduced by 0.01.

[0031] Table 1. Growth performance of broilers at 42 days

[0032] 2.4.3 Energy utilization rate: As shown in Table 2, compared with the control group, the apparent metabolic energy of the PA live bacteria group increased by 0.46%; the PA post-biotic group increased by 1.29%, which is 2.8 times that of the live bacteria group; compared with the PA live bacteria group, the apparent metabolic rate of the PA post-biotic group still increased by 0.83%.

[0033] Table 2. Energy utilization rate of broilers

[0034] 2.4.4 Intestinal morphology: As shown in Table 3, both the PA live bacteria group and the PA postbiotic group significantly increased the height of jejunal villi and decreased the crypt depth; the height of PA postbiotic villi was 28.68 μm higher than that of the PA live bacteria group.

[0035] Table 3. Broiler intestinal morphology (jejunum)

[0036] 2.4.5 Serum energy metabolism biochemical indicators: As shown in Table 4, the PA live bacteria group had a weak regulatory effect on glucose and lipid metabolism; the PA post-biotic group could significantly upregulate blood glucose and triglycerides and downregulate free fatty acid content, and the regulatory strength was significantly better than that of PA live bacteria.

[0037] Table 4. Serum energy metabolism indicators of broilers

[0038] In summary, live bacteria preparations have inherent application defects: PC13 live bacteria are not resistant to the high temperature of feed pelleting and room temperature storage, resulting in the inactivation of a large number of live bacteria and a significant reduction in actual application effects; post-biotics are inactivated complex active products, which do not require the survival of live bacteria, and their processing and storage stability is significantly better than that of live bacteria, making them more suitable for industrial applications. Metabiotics exhibit unexpected synergistic effects: Under the same strain and dosage conditions, metabiotics show significantly greater improvement than live bacteria in all core indicators, including growth performance, apparent energy metabolism rate, intestinal barrier function, and serum glucose and lipid metabolism. Example 3: Broiler chicken feeding trial to verify the effect 3.1 Experimental Design: This experiment adopted a two-factor experimental design. One-day-old healthy AA broilers were randomly divided into 4 groups, with 6 replicates per group and 10 birds per replicate. The specific groups are as follows: control group, PA post-biotic group, low-energy group, and low-energy plus PA post-biotic group. The control group was fed a basal diet (metabolizable energy 3.2 MJ / kg), the PA post-biotic group was fed a basal diet supplemented with 0.5 kg / t of Peptococcus lactis post-biotic prepared in Example 1, the low-energy group was fed a reduced-energy diet (metabolizable energy 3.0 MJ / kg), and the low-energy plus PA post-biotic group was fed a reduced-energy diet supplemented with 0.5 kg / t of Peptococcus lactis post-biotic prepared in Example 1.

[0039] During the trial, all broilers aged 1-14 days were fed the same basal diet; the feed was changed at 15 days of age, and the formal trial began. The trial lasted for 42 days, during which no immunization or medication was administered, and the chickens were weighed at 14 and 42 days of age to analyze their growth performance.

[0040] 3.2 Measurement Indicators: 1. Growth performance: On the 14th and 42nd day of age, the experimental flocks were fasted for 8 hours. They were weighed on an empty stomach in repeated units, and the amount of feed consumed was recorded. The average daily weight gain, average daily feed intake and feed conversion ratio were calculated.

[0041] 2. Energy utilization rate: On days 41-42 of the experiment, the total energy of feed / feces was measured using the "chromium trioxide indicator method" (addition amount 0.3%) (GB / T 26437-2010), and the apparent metabolic rate of energy (%) and energy utilization efficiency (energy deposition / energy intake, %) were calculated. 3. Intestinal morphology: On day 42 of the experiment, 2 broilers were slaughtered from each treatment group to prepare jejunal tissue sections (HE staining), and villus height (VH) and crypt depth (CD) were measured 4. Serum biochemistry: Serum was collected on day 42 of the experiment to measure glucose (GLU), non-esterified fatty acid (NEFA) and triglyceride (TG) by enzymatic method; 3.3 Statistical methods: Two-way analysis of variance (Two-way ANOVA) was performed using SPSS 26.0, with the main effects being "PA (postbiotic)" and "low energy", and the interaction effect (PA*low energy) was analyzed. Significance judgment: P < 0.05 indicates a significant difference, 0.05 < P < 0.1 indicates a trend of difference, and P < 0.01 indicates an extremely significant difference.

[0042] 3.4 Experimental results: 3.4.1 Growth performance: It can be seen from Table 5 that there were no significant differences in BW, ADG, ADFI and FCR of broilers during the early stage of the experiment, that is, at 1-14 days of age (P > 0.05); it can be seen from Table 6 that at 1-42 days of age, compared with normal energy, reducing energy significantly increased ADG, BW and F / G of broilers (P < 0.001); compared with no postbiotic supplementation, postbiotic supplementation significantly reduced ADG, BW and F / G of broilers (P < 0.001); meanwhile, energy and PA had interaction effects on BW, ADFI and F / C of broilers (P < 0.001), specifically, compared with the control group, reducing energy significantly increased F / C and decreased BW and ADFI, and adding postbiotics on the basis of reducing energy significantly decreased F / C and increased BW and ADFI (P < 0.001).

[0043] Table 5 Growth performance of broilers at 1-14 days of age

[0044] Table 6 Growth performance of broilers at 1-42 days of age

[0045] 3.4.2 Energy utilization rate: It can be seen from Table 7 that compared with normal energy, reducing energy significantly decreased the apparent metabolizable energy and energy utilization rate of broilers (P < 0.001); compared with no postbiotic supplementation, postbiotic supplementation significantly increased the apparent metabolizable energy and energy utilization rate of broilers (P < 0.001); meanwhile, energy and PA had interaction effects on the apparent metabolizable energy and energy utilization rate of broilers (P < 0.001), specifically, compared with the control group, reducing energy significantly decreased the apparent metabolizable energy and energy utilization rate of broilers, and adding postbiotics on the basis of reducing energy significantly increased the apparent metabolizable energy and energy utilization rate of broilers (P < 0.05).

[0046] Table 7 Energy digestibility and utilization rate of broiler chickens at 42 days of age

[0047] 3.4.3 Intestinal Morphology: As shown in Table 8, compared with normal energy, reducing energy significantly reduced villus height and villus-crypt ratio in the jejunum of broilers and increased crypt depth (P < 0.001); compared with no added metabiotic, adding metabiotic significantly increased villus height and villus-crypt ratio in broilers (P < 0.001); at the same time, energy and PA had an interactive effect on villus height, crypt depth and villus-crypt ratio in the jejunum of broilers (P < 0.001). Specifically, compared with the control group, reducing energy significantly reduced villus height and villus-crypt ratio in the jejunum of broilers and increased crypt depth, while adding metabiotic on the basis of reducing energy significantly increased villus height and villus-crypt ratio and decreased crypt depth (P < 0.05).

[0048] Table 8. Jejunal intestinal morphology of 42-day-old broiler chickens

[0049] 3.4.4 Serum Biochemistry: As shown in Table 9, compared with normal energy, reducing energy significantly decreased the levels of GLU and TG in broiler serum and increased the level of NEFA (P < 0.001); compared with no added postbiotic, adding postbiotic significantly increased the levels of GLU and TG in broiler serum and significantly decreased the level of NEFA (P < 0.001); at the same time, energy and PA had an interactive effect on broiler serum GLU, NEFA, and TG (P < 0.001). Specifically, compared with the control group, reducing energy significantly decreased the levels of GLU and TG in broiler serum and increased the level of NEFA. At the same time, adding postbiotic on the basis of reduced energy significantly increased the levels of GLU and TG in broiler serum and significantly decreased the level of NEFA (P < 0.001).

[0050] Table 9 Serum biochemistry of broiler chickens at 42 days of age

[0051] In summary, the addition of the present invention's *Pediococcus lactis* postbiotic to low-energy diets can effectively improve broiler growth performance and gut health, significantly increase the efficiency of broiler energy utilization, and effectively alleviate metabolic disorders caused by low-energy diets.

[0052] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Pediococcus lactis, characterized in that, The lactic acid cocci are *Pediococcus lactis* ( Pediococcus acidilactici PC13 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2022426.

2. An epigenetic agent, characterized in that, The metabiotic is obtained by inactivation treatment of Pietrococcus lactis PC13 as described in claim 1 after fermentation culture.

3. The epigenetic agent according to claim 2, characterized in that, The metabiotic contains ≥40% inactivated bacterial cells, ≥15% extracellular polysaccharides, ≥8 mg / g total short-chain fatty acids, and ≥20 U / mg protease activity.

4. The use of the post-genetic agent as described in claim 2 or 3 in the preparation of feed additives for improving the energy utilization of broilers.

5. The application according to claim 4, characterized in that, The amount of the post-biotic added to the feed is 0.5 kg / ton of feed.

6. The application according to claim 4 or 5, characterized in that, The application involves adding the feed additive containing the post-genetic agent to the basal or low-energy diet of broilers.

7. The application according to claim 6, characterized in that, The metabolizable energy of the low-energy diet is less than 3.2 MJ / kg.

8. A feed for improving the energy utilization rate of broilers, characterized in that, The feed contains a basal diet and the post-biotic as described in claim 2 or 3.

9. The feed according to claim 8, characterized in that, The feed is a low-energy diet, and the amount of post-biotic added is 0.5 kg / ton of feed.

10. The use of the *Pediococcus lactis* PC13 of claim 1 in the preparation of a feed additive for improving the energy utilization of broilers.

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