Pig intestine source clostridium butyricum cell-free supernatant and application thereof

Abstract

The invention discloses a pig intestine source clostridium butyricum cell-free supernatant and application thereof, and belongs to the technical field of microorganisms. The preparation method of the cell-free supernate comprises the following steps: co-culturing clostridium butyricum and escherichia coli K88, centrifuging fermentation liquor to remove thalli, sequentially filtering the fermentation liquor through a 0.22 mu m ceramic membrane and a 3kDa ultrafiltration membrane, collecting components of which the molecular weight is less than 3kDa, and performing freeze drying and resuspension to obtain the cell-free supernate. The supernate is rich in various active small molecule peptide fragments. The invention further discloses application of the supernate in preparation of a medicament for relieving diarrhea of weaned piglets or a feed additive for improving growth performance of the weaned piglets. Test results show that the cell-free supernate can significantly improve the growth performance of weaned piglets, effectively reduce the diarrhea rate, optimize the intestinal flora structure and increase the abundance of beneficial bacteria, and has significant beneficial effects.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a cell-free supernatant of Clostridium butyricum from pigs and its application. Background Technology

[0002] my country is the world's largest producer and consumer of pigs. The weaning stage is a crucial phase in pig farming, directly determining later growth potential. Intestinal mucosal barrier damage caused by weaning stress is a major factor affecting piglet development. During early weaning, piglets are subjected to multiple stressors, including nutritional (changing from sow's milk to solid feed), psychological (separation from sow and mixing of piglets from different litters), and environmental (transportation, transfer from farrowing crates to nursery pens, and increased exposure to pathogens). Their incompletely developed gastrointestinal and immune systems make them highly susceptible to weaning stress. Symptoms include decreased appetite, digestive disorders, diarrhea, and slow growth. Diarrhea is the most severe problem caused by weaning stress. The causes of diarrhea in weaned piglets are cross-cutting and diverse, including both intestinal mucosal barrier damage due to weaning stress and microbial imbalance caused by pathogen infection. Therefore, repairing intestinal mucosal barrier damage and regulating intestinal microbial balance are effective strategies to improve weaning stress.

[0003] Clostridium butyricum is a probiotic. As a Gram-positive anaerobic bacillus, it consumes dietary fiber in the intestine and produces short-chain fatty acids, playing an important role in the host, such as regulating intestinal immune homeostasis, improving gastrointestinal barrier function, and reducing inflammation. However, current research on Clostridium butyricum mainly focuses on the production and application of Clostridium butyricum cells or freeze-dried bacterial powder. This patent discloses the innovative effects of Clostridium butyricum cell-free supernatant on the growth and intestinal health of weaned piglets.

[0004] In existing technologies, for example, patent CN118497286A discloses a cell-free supernatant of *Lactobacillus plantarum* with broad-spectrum antibacterial properties and the ability to delay food spoilage, and its application. The cell-free supernatant produced by *Lactobacillus plantarum* DACN996 has broad-spectrum antibacterial activity, exhibiting strong inhibitory capabilities against various foodborne pathogens and spoilage bacteria, making it suitable for use in food and pharmaceutical production to control pathogens and spoilage bacteria. Simultaneously, this fermented lactobacillus can significantly inhibit the growth of *Listeria monocytogenes* in meat products and extend their shelf life. It can be added to meat products as a natural antibacterial agent, effectively replacing antibiotics or chemical antibacterial agents for meat preservation. However, this invention only uses the cell-free supernatant produced by *Lactobacillus plantarum* DACN996, and its effect is no different from directly using *Lactobacillus plantarum* DACN996. It does not identify the components of the supernatant, nor does it provide a specific method for preparing the supernatant. Summary of the Invention

[0005] The purpose of this invention is to provide a cell-free supernatant of Clostridium butyricum from pigs and its application, in order to solve the problems that the existing supernatant preparation methods are not perfect, and the use of supernatant has not been truly developed or verified.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A cell-free supernatant of Clostridium butyricum from pig intestine was prepared by the following steps:

[0008] a. Inoculate *Clostridium butyricum* into enhanced *Clostridium* medium (RCM) and culture at 37°C for 24 hours under strictly anaerobic conditions; b. Transfer the culture to 35 L of industrial medium and continue anaerobic fermentation for 48 hours; c. Add *E. coli* for co-culture; d. Centrifuge to remove bacterial cells, filter through a 0.22 μm ceramic membrane and a 3 kDa ultrafiltration membrane, and collect the cell-free supernatant with a molecular weight <3 kDa; e. Freeze-dry and resuspend to obtain the cell-free supernatant.

[0009] Preferably, step a of the above preparation specifically involves resuscitating the Clostridium butyricum at a concentration of 0.5-2% in enhanced Clostridium tumefaciens medium RCM, placing it in a strictly anaerobic incubator, and culturing it at 37°C for 24 hours.

[0010] Preferably, the industrial culture medium formulation in step b above is as follows: 1% glucose, 2% tryptone, 0.2% yeast extract, 0.1% K₂HPO₄, 0.1% MgSO₄·7H₂O, 0.02% MnSO₄·H₂O, pH 7.0. Ammonia is added to balance the pH after 26 hours of cultivation. After 27 hours of cultivation, 800 ml of 50% glucose solution is added and cultivation continues until 36 hours. During 18-36 hours, samples are taken hourly for microscopic examination. Once the budding rate of Clostridium butyricum reaches 99%, ammonia is added again to balance the pH after 26 hours of cultivation. After 27 hours of cultivation, 800 ml of 50% glucose solution is added.

[0011] Preferably, the above preparation step c specifically involves adding Escherichia coli K88 with an OD of 600, i.e., a volume ratio of 1:40000, and culturing for 48 hours.

[0012] Preferably, step d of the above preparation specifically involves centrifuging the culture medium, discarding the bacterial cell precipitate, filtering with a 0.22 μm ceramic membrane, and then using a 3 kDA ultrafiltration membrane for molecular retention to obtain a small molecule cell-free supernatant with <3 kDA.

[0013] Preferably, the above preparation step e specifically involves placing the supernatant in a freeze dryer, pre-freezing it at -20°C for 1 hour, freeze-drying it for 48 hours, resuspending it at a ratio of 30, freeze-drying the precipitate, and then obtaining a cell-free supernatant of Clostridium butyricum from pigs.

[0014] Preferably, the cell-free supernatant described above contains at least one of the following peptides:

[0015] CBP1:LAAVAIPKFGAVKKDANISADKANAK;

[0016] CBP2: IVQDVSNIKDVIIEVQK;

[0017] CBP3: ICGQIKITPKS;

[0018] CBP4: VTKLPTITL.

[0019] The present invention also provides a medicine for relieving diarrhea in weaned piglets, by adding the above-mentioned cell-free supernatant.

[0020] The present invention also provides a feed additive to improve the growth performance of piglets, which includes the above-mentioned cell-free supernatant, and the additive replaces corn and soybean meal by 10% of the total daily weight of the diet, and adds 1.0%-2.0% soybean oil.

[0021] The beneficial effects of this invention are as follows:

[0022] 1. This invention is the first to truly study the independent role of the supernatant, which no longer works simultaneously with probiotics, and it also identifies the peptide sequence in the postbiotic, providing a framework for the subsequent artificial synthesis of peptides for drug production.

[0023] 2. Innovative preparation process: By co-culturing Clostridium butyricum and pathogenic bacteria (Escherichia coli K88), and using industrial-scale fermentation (35L scale) and 3kDa ultrafiltration technology, high-purity small molecule active ingredients (<3kDa) are obtained, which are suitable for feed additive production.

[0024] 3. Significantly improved growth performance: The final weight of weaned piglets increased by 16.3% (13.88 kg vs. 11.93 kg), and the average daily weight gain increased by 25.6% (151.73 g / d vs. 120.80 g / d).

[0025] 4. Reduced the incidence of diarrhea: The diarrhea rate decreased by 55% (10.32% vs. 22.86%), and the stool score improved by 40.9% (1.23 vs. 2.08) (P<0.05).

[0026] 5. Optimize the gut microbiota structure: increase the abundance of beneficial bacteria (lactic acid bacteria, Coprococcus, Blautia), inhibit pathogenic bacteria (such as Campylobacterota), and restore the balance of the gut microbiota. Attached Figure Description

[0027] Figure 1 This is the total ion current chromatogram of the present invention; the horizontal axis represents time (min), and the vertical axis represents relative abundance.

[0028] Figure 2 This is a comparative graph of serum pro-inflammatory factor TNF-α in this invention; from top to bottom, the graphs show the serum TNF-α content, serum IL18 content, and serum IL10 content, respectively. The vertical axis represents the content of TNF-α, IL18, and IL10, in pg / mL, and the horizontal axis represents the control group (CON), LPS-induced group (LPS), artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant group (CBP), and artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant plus LPS-induced group (CBPLPS); where [*] indicates P<0.05, [**] indicates P<0.01, [***] indicates P<0.001, and [****] indicates P<0.0001;

[0029] Figure 3 This is a comparative diagram of the pro-inflammatory factor TNF-α in the ileum of this invention; from left to right and from top to bottom, the diagrams show the content of TNF-α, IL18, IL10, IL6, IL-1β, and IFN-γ in the ileum, respectively, in pg / mL. The horizontal axis represents the control group (CON), the LPS-induced group (LPS), the group with artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant (CBP), and the group with artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant plus LPS-induced group (CBPLPS); where [*] indicates P<0.05, [**] indicates P<0.01, [***] indicates P<0.001, and [****] indicates P<0.0001.

[0030] Figure 4This is a comparative diagram of the pro-inflammatory factor TNF-α in the colon in this invention. From left to right and from top to bottom, the diagrams show the TNF-α content in the ileum, IL18 content in the colon, IL10 content in the colon, IL6 content in the colon, IL-1β content in the colon, and IFN-γ content in the colon, in pg / mL. The horizontal axis represents the control group (CON), the LPS-induced group (LPS), the group with artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant (CBP), and the group with artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant plus LPS-induced group (CBPLPS). Wherein, [*] indicates P<0.05, [**] indicates P<0.01, [***] indicates P<0.001, and [****] indicates P<0.0001.

[0031] Figure 5 Tissue section images showing the effect of Clostridium butyricum cell-free supernatant on the intestinal morphology of weaned piglets; including control group (CON), LPS-induced group (LPS), artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant group (CBP), and artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant with added LPS-induced group (CBPLPS).

[0032] Figure 6 This invention illustrates the effect of Clostridium butyricum cell-free supernatant on the levels of short-chain fatty acids in the colon of weaned piglets. From left to right and top to bottom, the graph shows the contents of isovaleric acid, isobutyric acid, aceticacid, valeric acid, butyric acid, and propanoic acid, in pg / g. The horizontal axis represents the control group (CON), the LPS-induced group (LPS), the artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant group (CBP), and the artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant plus LPS-induced group (CBPLPS). Wherein, [*] indicates P < 0.05, [**] indicates P < 0.01, [***] indicates P < 0.001, and [****] indicates P < 0.0001.

[0033] Figure 7 To demonstrate the antibacterial effect of the Clostridium butyricum cell-free supernatant of this invention, the following is a breakdown of the contents: lower cup: the original solution of Clostridium butyricum cell-free supernatant (CBP) prepared in this invention; upper left cup: the key peptide solution screened in this invention, which was artificially synthesized and reconstituted; upper right cup: sterile physiological saline as a negative control.

[0034] Figure 8This invention relates to the effect of Clostridium butyricum cell-free supernatant on the composition of the colonic flora in weaned piglets. Rarefaction curves are presented, with the horizontal axis representing the number of sequences sampled, i.e., sequencing depth, and the vertical axis representing the coverage index, reflecting the proportion of species detected in the sample. The control group (CON), the LPS-induced group (LPS), the artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant group (CBP), and the artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant with added LPS-induced group (CBPLPS) are also included.

[0035] Figure 9 This invention presents the Ace index plots for different treatment groups based on the Kruskal-Wallis H test. The Ace index is an important indicator of α-diversity in microbial communities, used to estimate species richness (i.e., the number of different microorganisms) in the community; a higher value indicates greater species richness. The vertical axis represents the Ace index based on amplicon sequence variant (ASV) levels, reflecting the quantitative value of microbial species richness. The horizontal axis represents different groups: control group (CON), LPS-induced group (LPS), artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant group (CBP), and artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant plus LPS-induced group (CBPLPS).

[0036] Figure 10 This invention presents Sobs index plots for different treatment groups based on the Kruskal-Wallis H test. The Sobs index is an important indicator of microbial community α-diversity, used to estimate species richness (i.e., the number of different microorganisms) in the community; a higher value indicates greater species richness. The vertical axis represents the Sobs index based on amplicon sequence variant (ASV) levels, reflecting the quantitative value of microbial species richness. The horizontal axis represents different groups: control group (CON), LPS-induced group (LPS), artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant group (CBP), and artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant plus LPS-induced group (CBPLPS).

[0037] Figure 11This invention presents Shannon index plots for different treatment groups based on the Kruskal-Wallis H test. The Shannon index is an important indicator of α-diversity in microbial communities, used to estimate species richness (i.e., the number of different microorganisms) in the community; a higher value indicates greater species richness. The vertical axis represents the Shannon index based on amplicon sequence variant (ASV) levels, reflecting the quantitative value of microbial species richness. The horizontal axis represents different groups: control group (CON), LPS-induced group (LPS), artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant group (CBP), and artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant plus LPS-induced group (CBPLPS).

[0038] Figure 12 This invention presents the Chao index plots for different treatment groups based on the Kruskal-Wallis H test. The Chao index is an important indicator of α-diversity in microbial communities, used to estimate species richness (i.e., the number of different microorganisms) in the community; a higher value indicates greater species richness. The vertical axis represents the Chao index based on amplicon sequence variant (ASV) levels, reflecting the quantitative value of microbial species richness. The horizontal axis represents different groups, including the control group (CON), the LPS-induced group (LPS), the group with artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant (CBP), and the group with artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant plus LPS-induced group (CBPLPS).

[0039] Figure 13 The principal coordinate analysis (PCoA) results based on the ASV level in this invention are used to display the β diversity of the microbial community. The horizontal axis (PC1) explains 14.51% of the community structure variance, making it the largest principal component affecting community differences; the vertical axis (PC2) explains 11.55% of the community structure variance, making it the second largest principal component; the Kruskal-Wallis H test results of the box plot show: R=0.2626, P=0.001000.

[0040] Figure 14 The results of this invention, based on nonmetric multidimensional scaling (NMDS) analysis at the ASV level, are used to demonstrate the β diversity of the microbial community. The horizontal axis (NMDS1) explains the community structure variance, which is the largest principal component affecting community differences; the vertical axis (NMDS2) explains the community structure variance in another dimension; the stress value is 0.181, R=0.2626, P=0.001000.

[0041] Figure 15This is a community bar chart analysis diagram of the present invention. The height of the bar chart represents the relative abundance ratio of the phylum in the corresponding group. The control group (CON), LPS-induced group (LPS), artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant group (CBP), and artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant added to LPS-induced group (CBPLPS) are included.

[0042] Figure 16 This is a bar chart analysis of the microbial community (genus level) of the present invention. The vertical axis represents the relative abundance at the genus level. The control group (CON), the LPS-induced group (LPS), the artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant group (CBP), and the artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant with added LPS-induced group (CBPLPS) are included.

[0043] Figure 17 This is a bar chart of the LEfSe (Linear discriminant analysis Effect Size) analysis of the present invention, used to identify microbial markers (genus level) with statistical differences between different groups. The horizontal axis is the linear discriminant analysis score, and the vertical axis is the different taxonomic units at the genus level. The control group (CON), the LPS-induced group (LPS), the artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant group (CBP), and the artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant plus LPS-induced group (CBPLPS) are included. Detailed Implementation

[0044] The present invention will be described in more detail below with reference to the embodiments. It should be understood that the implementation of the present invention is not limited to the embodiments below, and any modifications or alterations made to the present invention fall within the protection scope of the present invention; and the methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0045] Example 1:

[0046] Preparation of cell-free supernatant

[0047] Clostridium butyricum ZJU-F1 (Chinese Patent CN201410137978.2), deposited at the China General Microbiological Culture Collection Center (CGMCC No. 8939) of our research group, was revived in ordinary RCM medium at a concentration of 0.5-2% and incubated in a strictly anaerobic incubator at 37°C for 24 h. The culture was then transferred to 35 L of industrial-grade medium at a ratio of 0.1-0.5% and placed in a strictly anaerobic pilot-scale fermenter, incubated at 37°C for 48 h. Then, Escherichia coli K88 with OD=600 was added at a ratio of 1:40000, and the fermentation was continued for 24 h. The culture was centrifuged, the bacterial precipitate was discarded, and the mixture was filtered through a 0.22 μm ceramic membrane, followed by molecular rejection using a 3 kDa ultrafiltration membrane to obtain a cell-free supernatant with low molecular weight (<3 kDa). The supernatant was then pre-frozen at -20°C for 1 day. After 48 hours of freeze-drying, the precipitate was resuspended at a ratio of 30 and freeze-dried to obtain a cell-free supernatant.

[0048] Mass spectrometry was used to quantitatively detect peptide sequences in cell-free supernatants, and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was used to identify the amino acid sequences of the peptides. 100 μL of sample was added to 400 μL of methanol, sonicated in an ice-water bath for 5 minutes, and then centrifuged at 12000 rpm for 10 minutes at 4°C. 400 μL of supernatant was collected and freeze-dried under vacuum for later use. 200 μL of wash buffer was added to dissolve the sample. After desalting, column activation, impurity removal, equilibration, and elution, the final sample volume was 100 μL. The sample was concentrated using a vacuum freeze-centrifuge, and the concentrated sample was reconstituted with mobile phase A before being loaded onto the spectrometer. 200 ng of total peptides from each sample were separated using a liquid chromatography system and then coupled with a mass spectrometer equipped with a nanoliter ion source for data acquisition. Chromatographic separation was performed using a reversed-phase column. The mobile phase was an acetonitrile-water-formic acid system, where mobile phase A was a 0.1% formic acid aqueous solution and mobile phase B was a 0.1% formic acid-acetonitrile solution. After the column was equilibrated with 100% A phase, the sample was directly loaded onto the column via an autosampler and separated by a gradient at a duration of 44 min. The mass spectrometer used DDA PaSEF mode for DDA data acquisition, with a scan range of 100-1700 m / z. During the PASEF MS / MS scan, the collision energy increased linearly with ion mobility, from 20 eV (1 / K0 = 0.6 Vs / cm²) to 59 eV (1 / K0 = 1.6 Vs / cm²). The raw data files were searched in the Uniprot database using FragPipe (V21.1) software, and qualitative analysis was performed after the search was completed.

[0049] Proteins were extracted from cell-free supernatant, and the extracted proteins were quantitatively analyzed by reductive alkylation. The processed samples were then analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) to obtain raw mass spectrometry results. These results were then analyzed using PEAKSStudio 10.6 software, matched against a database, and the identification results were obtained. The total ion chromatogram is shown below. Figure 1 As shown, each peak represents a component, with peak height indicating the ionic intensity of that component and peak width indicating the retention time. Table 1 presents the results of the identified peptides with higher scores.

[0050] Table 1. Results of some identified peptides

[0051]

[0052] Example 2: Proof of Antibacterial Effect

[0053] The peptides with higher scores identified in Table 1 were artificially synthesized, and the most effective peptides were screened for subsequent experiments.

[0054] Table 2 Peptides after initial screening

[0055]

[0056] like Figure 7 As shown, to verify whether the peptides identified in this invention have direct antibacterial activity, this embodiment selected a peptide (CBP1) with a high score in mass spectrometry quantitative detection for artificial chemical synthesis. The Oxford cup method (Agardiffusion test) was used to detect its inhibitory effect on pathogenic Escherichia coli K88, a common pathogen in weaned piglets.

[0057] The specific steps are as follows:

[0058] Escherichia coli K88, Staphylococcus aureus, and Salmonella in the logarithmic growth phase were evenly spread on LB agar plates to form bacterial motility.

[0059] Place three sterile Oxford cups at equal intervals on a plate.

[0060] Add 120 μL of sample to each of the three Oxford cups:

[0061] Lower cup: The cell-free supernatant (CBP) stock solution of Clostridium butyricum prepared in this invention.

[0062] Top left cup: A solution of the key peptides screened in this invention, which has been artificially synthesized and reconstituted.

[0063] Upper right cup: Sterile saline as a negative control.

[0064] Incubate the plates in a 37°C incubator for 16-24 hours, and observe and measure the diameter of the zone of inhibition.

[0065] The test results are as follows Figure 7 As shown, from left to right, the plates are the negative control plate (without antibacterial substance) and the plates from the three replicate test groups. The results are clearly visible:

[0066] A noticeable inhibition zone was formed around the added CBP supernatant stock solution, proving that the supernatant itself has an antibacterial effect.

[0067] The addition of the key peptide forms a large, clear, and transparent antimicrobial zone, exhibiting antimicrobial activity similar to that of CBP.

[0068] No inhibition zone formed around the upper right cup after adding sterile saline.

[0069] Example 3: The regulatory effect of cell-free supernatant on the intestinal mucosal barrier function of weaned piglets

[0070] To investigate the regulatory effect of Clostridium butyricum cell-free supernatant on the intestinal mucosal barrier function of weaned piglets, weaned Jinhua piglets from five litters with an average weight of approximately 9.95 ± 2.03 kg were randomly divided into two groups according to sex, weight, and litter origin, with five replicates per group and five piglets per replicate. The control group (CON) and the group receiving artificially synthesized and reconstituted Clostridium butyricum cell-free supernatant (CBP) were respectively divided into two groups. The experimental period was 21 days. The CBP group received 3 ml of Clostridium butyricum cell-free supernatant daily by gavage, while the CON group received an equal volume of sterile water daily by gavage. All piglets had free access to water during the experiment, but their diet was restricted. On day 21, 18 piglets with similar weights were selected from the CBP group. Nine of these piglets were intraperitoneally injected with 100 μg / kg of LPS, and the other nine were intraperitoneally injected with an equal volume of sterile saline. Slaughter and sampling were performed 6 hours after LPS injection.

[0071] Compared with the CON group, daily gavage administration of Clostridium butyricum cell-free supernatant significantly increased the final weight and average daily weight gain of piglets (P < 0.05); CBP also significantly reduced the diarrhea rate and diarrhea score of piglets (P < 0.05), indicating that CBP can improve the growth performance of weaned piglets and reduce the incidence of diarrhea (Table 3).

[0072] Table 3. Effects of Clostridium butyricum cell-free supernatant on growth performance of weaned piglets.

[0073]

[0074] Patent CN104164377A demonstrates the application effects of Clostridium butyricum preparations. Calculations based on its data show that treatment group 2, compared to its control group:

[0075] The patient's weight increased by 5.23% in the final stage (from 9.37 kg to 9.86 kg).

[0076] The average daily weight gain increased by 25.42% (from 137.12g / d to 171.97g / d).

[0077] The material weight ratio was optimized (reduced) by 20.30% (from 2.02 to 1.61).

[0078] The diarrhea rate decreased by 69.19% (from 14.77% to 4.55%).

[0079] Example 4: Detection of Inflammatory Factors

[0080] like Figures 2-4 As shown, inflammatory factors in serum and intestine were detected using an ELISA kit. Compared with the CON group, the LPS group showed significantly higher levels of pro-inflammatory factors in serum and intestine (P < 0.05), while CBP significantly reduced the LPS-induced levels of TNF-α in serum (P < 0.05), and reduced the levels of TNF-α, IL-18, and IFN-γ in ileum and colon (P < 0.05).

[0081] like Figure 5 As shown, 1 cm sections of the duodenum, jejunum, ileum, and mid-colon were harvested, washed with PBS phosphate-buffered saline, and fixed with 4% paraformaldehyde. After fixation, the tissues underwent trimming, rinsing, dehydration, clearing, paraffin embedding, sectioning, HE staining, and mounting. Sections were prepared for histological observation. The height of five intact villi (VH) and the depth of crypts (CD) were measured in each field of view of the jejunum and ileum, and the villus-crypt ratio (VH / CD) was calculated. H&E staining results of piglet intestinal tissues showed that, compared with the CON group, the LPS group exhibited severe damage to the small intestinal epithelium, and the villus height of the duodenum, jejunum, ileum, and colon was significantly shorter (P < 0.05). Compared with the LPS group, the CBPLPS group showed more intact epithelial morphology and increased villus height of the duodenum, jejunum, ileum, and colon (P < 0.05), indicating improved intestinal morphology and suggesting that CBP can improve LPS-induced intestinal inflammation.

[0082] like Figure 6As shown, the level of short-chain fatty acids (SCFAs) is largely influenced by the symbiotic gut microbiota. Fecal SCFA content was quantitatively detected using GC-MS. After thawing colonic contents at 4℃, 0.2 g of sample was accurately weighed into a 2 mL centrifuge tube, 600 μL of ultrapure water was added, and the mixture was allowed to stand for 30 min. The mixture was then centrifuged at 12,000 rpm for 15 min. 400 μL of the supernatant was collected into a centrifuge tube, and 100 μL of 25% metaphosphate was added. After mixing, the mixture was incubated at 4℃ for 30 min, centrifuged at 12,000 rpm for 15 min, and the supernatant was filtered through a 0.22 μm filter membrane. The contents of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid were determined using gas chromatography-ion mobility spectrometry. The results showed that CBP significantly increased the butyric acid content in the intestines of piglets (P < 0.05).

[0083] like Figures 8-17 As shown, 16S rRNA was used to study the microbial composition of the colon contents of piglets. The sample dilution curve gradually flattened, and the Coverage index reached 99.9%, indicating abundant ASV coverage. The ace, sobs, shannon, and chao indices showed no significant differences in community richness and diversity among the groups. PCoA and NMDS were used to analyze β-diversity based on Bray-Curtis distance. The results showed significant differences in gut microbial β-diversity (P < 0.05), indicating that CBP has a significant regulatory effect on the piglet gut microbiota. Species analysis based on ASV is shown below. Figures 13-14As shown, at the phylum level, the intestinal microbiota of piglets in each group mainly consisted of Bacillota, Bacteroidetes, Spirochaetota, Actinomycetota, Methanobacteriota, and Pseudomonadota. Bacillota was the dominant phylum in all groups, followed by Bacteroidetes. Compared with the CON group, the LPS group showed a decrease in the proportions of Actinomycetes and Fusobacteria (P < 0.05), and an increase in the proportions of Verruicrobiota, Planctomycetota, Campylobacterota, and Elusimicrobia (P < 0.05). Conversely, the CBPLPS group showed a decrease in the proportions of Actinomycetes and Fusobacteria compared to the LPS group (P < 0.05), and a decrease in the proportions of Planctomycetota, Campylobacterota, and Elusimicrobia (P < 0.05). Compared with the CON group, LPS reduced the abundance of beneficial bacteria, such as Prevotellaceae_UCG-003, Ruminococcus, Coprococcus, Corynebacterium, Prevotellaceae_UCG-001, Lactobacillus, and Blautia (P < 0.05). Compared with the LPS group, after CBP administration, the proportion of beneficial bacteria such as Lactobacillus, Coprococcus, and Blautia increased (P < 0.05), suggesting that CBP can alleviate the intestinal flora disorder caused by LPS by increasing the abundance of beneficial bacteria. LEfSe analysis revealed that p-251-o5, WCHB1-41, Lactobacillus, and Corynebacterium had the highest linear discriminant scores (LDA>2.5) in the CON, LPS, CBP, and CBPLPS groups, respectively.

[0084] This invention not only successfully isolated and identified multiple active peptides in the supernatant, but also demonstrated through in vitro antibacterial experiments that the screened key peptides have a direct and potent inhibitory effect on pathogenic Escherichia coli and other intestinal pathogens in piglets. This reveals the key mechanism by which this invention works from a material perspective, and lays a solid scientific foundation for the subsequent development of a new generation of precise and efficient antibacterial drugs or feed additives using artificially synthesized peptides.

[0085] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A cell-free supernatant of Clostridium butyricum of porcine intestinal origin, characterized in that, Prepared by Clostridium butyricum through the following steps: a. Clostridium butyricum is inoculated in reinforced Clostridium medium RCM for culture; b. The culture solution is transferred into industrial medium for further anaerobic fermentation; c. Escherichia coli is added for co-culture; d. The bacteria are removed by centrifugation, and the cell-free supernatant is collected by filtration; e. After freeze-drying, resuspension is obtained, and the cell-free supernatant is obtained.

2. The cell-free supernatant of Clostridium butyricum from pig intestine according to claim 1, characterized in that, The preparation step a is specifically that the Clostridium butyricum is resuscitated in the reinforced Clostridium medium RCM at a concentration of 0.5-2%, and is placed in a strict anaerobic incubator for culture at 37℃ for 24h.

3. The cell-free supernatant of Clostridium butyricum from pig intestine according to claim 1, characterized in that, The industrial medium formula in the preparation step b is: glucose 1%, tryptone 2%, yeast extract 0.2%, K2HPO4 0.1%, MgSO4·7H2O 0.1%, MnSO4·H2O 0.02%, pH value 7.

0.

4. The cell-free supernatant of Clostridium butyricum from pig intestine according to claim 1, characterized in that, The preparation step b is specifically that the culture solution is transferred into 35L industrial medium at a ratio of 0.1-0.5%, and is further anaerobically fermented at 37℃ for 48h.

5. The cell-free supernatant of Clostridium butyricum from pig intestine according to claim 1, characterized in that, The preparation step c is specifically that Escherichia coli K88 with OD=600 is added, i.e. the ratio of dissolution is 1:40000, and co-culture is carried out for 48h.

6. The cell-free supernatant of Clostridium butyricum from pig intestine according to claim 1, characterized in that, The preparation step d is specifically that the culture solution is centrifuged, the bacteria are discarded, and the precipitate is filtered with a 0.22μm ceramic membrane, and then a 3kDA ultrafiltration membrane is used for molecular cutoff to obtain small molecule cell-free supernatant with a molecular weight of 0.5-3kDA.

7. The cell-free supernatant of Clostridium butyricum from pig intestine according to claim 1, characterized in that, The preparation step e is specifically that the supernatant is placed in a freeze-drier, pre-frozen at-20℃ for 1h, and freeze-dried for 48h, and the freeze-dried precipitate is resuspended at a ratio of 30 times to prepare the cell-free supernatant of Clostridium butyricum of pig intestinal origin.

8. The cell-free supernatant of Clostridium butyricum from pig intestine according to claim 1, characterized in that, At least one of the following peptide segments: CBP1: LAAVAIPKFGAVKKDANISADKANAK; CBP2: IVQDVSNIKDVIIEVQK; CBP3: ICGQIKITPKS; CBP4: VTKLPTITL.

9. A medicament for alleviating diarrhea in weaned piglets, characterized by, The cell-free supernatant of claim 1 is added.

10. A feed additive for improving growth performance of piglets, characterized in that, The cell-free supernatant of claim 1 is added, and the additive is used to replace corn and soybean meal at 10% of the total weight of the diet, and 1.0%-2.0% soybean oil is added.

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