Application of slezkia dsm 29692 in pig feeding

Abstract

The application discloses Slackia a new application of sp.D-G6, and particularly relates to Slackia application of sp.D-G6 bacterial powder in pig breeding, and belongs to the field of agricultural animal breeding and microbial technology. The bacterial strain has been preserved in the Guangdong Microbial Culture Collection Center on May 8, 2019, and the preservation number is GDMCC NO: 60661. Research shows that the bacterial powder has no obvious adverse effect on pigs, has good feeding safety, can promote pig feeding and weight gain, improve average daily feed intake and average daily weight gain, and improve growth performance. Further, the bacterial powder can regulate the microbial composition of the ileum of growing pigs, increase the abundance of Firmicutes and Lactobacillus, Lactobacillus and reduce the abundance of Proteobacteria and / or Escherichia-Shigella, Escherichia-Shigella and has application prospect of being developed into a new green feed additive.

Description

Technical Field

[0001] This invention belongs to the field of agricultural animal breeding and microbial technology, and relates to... Slackia The application of strain D-G6 in pig farming, specifically involving Slackia Application of sp. D-G6 strain in promoting feed intake, growth and regulating gut microbiota in pigs. Background Technology

[0002] With the development of modern pig farming towards large-scale, intensive, and antibiotic-reduced / alternative practices, the development of safe, green, and efficient feed additives has become a crucial need in pig farming. The gut is a vital organ for pig digestion, absorption, and immune defense, and the gut microbiota plays a vital role in nutrient digestion, intestinal barrier maintenance, and immune regulation. Existing research indicates that gut microbiota homeostasis is closely related to pig health and growth performance; when gut microbiota is disrupted, it often leads to digestive and absorptive disorders, intestinal barrier damage, and enhanced inflammatory responses, resulting in decreased feed intake, stunted growth, and reduced farming efficiency. Therefore, regulating the gut microbiota, maintaining gut health, improving feed intake, and promoting weight gain through the addition of functional microorganisms has become an important strategy for improving pig production performance. Existing research shows that probiotic supplementation can improve pig gut health, enhance nutrient digestibility and utilization, and improve growth performance under certain conditions.

[0003] Our research group has successfully screened vomitoxin-degrading bacteria from the gut microbiota of chickens. Slackia sp. D-G6. This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on May 8, 2019, with accession number GDMCC NO: 60661. After decades of research, our research group has discovered that this strain has multiple functions, including degrading deoxynivalenol (DON), metabolizing soy isoflavones into equol, and promoting growth in chickens and pigs. Based on the strain's ability to efficiently degrade DON into deepoxy-deoxynivalenol (DOM-1), our research group applied for a patent in 2019 for its DON degradation (patent number: ZL201910493701.6); based on the strain's ability to convert soybean isoflavones into equol, our research group applied for a patent in 2022 for its application in metabolizing daidzein to S-equol (patent application number: 202210340260.8); based on the strain's effect on improving broiler growth performance, our research group applied for a patent in 2024 for its application in improving broiler growth performance (patent application number: 202410439651.4).

[0004] Slackiasp. D-G6 shows great promise, and its live bacterial preparations are expected to be used as microbial powders in the livestock industry. Currently, there are relatively few functional microbial powder products in the livestock industry that aim to improve intestinal health, promote feed intake, and increase weight gain in pigs. Since intestinal microecological homeostasis is closely related to pig nutrient digestion, health status, and growth performance, developing novel, safe, and efficient microbial powders is of great significance for promoting healthy pig farming. Summary of the Invention

[0005] The purpose of this invention is to provide a product containing Slackia The application of sp. D-G6 microbial powder in pig farming aims to meet the demand for safe, green, and efficient microbial feed additives in pig production.

[0006] To achieve the above objectives, the following technical solution is adopted:

[0007] This invention provides a product containing Slackia Application of sp. D-G6 bacterial powder in pig farming.

[0008] In some implementations, the application is an application that promotes feed intake and weight gain in growing pigs.

[0009] In some implementations, promoting feed intake and weight gain in growing pigs includes increasing final weight, average daily feed intake, and / or average daily weight gain.

[0010] In some embodiments, the application is an application for regulating the composition of the ileum microbiota in growing pigs.

[0011] In some embodiments, the regulation of the ileum microbial composition in growing pigs includes increasing the levels of Firmicutes and / or Lactobacillus. Lactobacillus The abundance of ).

[0012] In some embodiments, the regulation of the ileum microbial composition in growing pigs further includes reducing Proteobacteria and / or Escherichia coli-Shigella flora. Escherichia-Shigella The abundance of ).

[0013] In some embodiments, the bacterial powder has no significant adverse effects on growing pigs.

[0014] In some implementations, the absence of significant adverse effects is manifested as no significant abnormalities in the major organ indices and no obvious pathological damage observed in the liver and kidney tissues.

[0015] In some embodiments, the bacterial powder is added to the growing pig diet at 0.2% of the basal diet mass.

[0016] In some embodiments, the bacterial powder is freeze-dried bacterial powder.

[0017] The Slackia sp. D-G6 has the accession number GDMCC NO: 60661.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) Slackia sp. D-G6 bacterial powder has no significant adverse effects on growing pigs and has good feeding safety; (2) The bacterial powder can promote feed intake and weight gain in growing pigs, and increase final weight, average daily feed intake and average daily weight gain; (3) The bacterial powder can regulate the composition of ileum microorganisms in growing pigs, increase the abundance of beneficial bacteria such as Firmicutes and Lactobacillus, and reduce the abundance of potentially harmful bacteria such as Proteobacteria and Escherichia coli-Shigella. Attached Figure Description

[0019] Figure 1 for Slackia sp. Results of morphology and viable count detection of D-G6 lyophilized powder; Figure a shows Slackia morphology diagram of sp. D-G6 lyophilized powder, Figure b is... Slackia viable cell counts of sp. D-G6 lyophilized powder on day 1 and day 30 after lyophilization and storage at room temperature.

[0020] Figure 2 for Slackia The effect of adding D-G6 bacterial powder on the growth performance of pigs; Figure a shows the final weight on days 1, 15 and 30 of the experiment; Figure b shows the average daily weight gain on days 1-15 and 1-30 of the experiment; Figure c shows the average daily feed intake on days 1-15 and 1-30 of the experiment.

[0021] Figure 3 for Slackia The effect of adding sp. D-G6 bacterial powder on the organ index of pigs.

[0022] Figure 4 for Slackia Effects of D-G6 bacterial powder supplementation on pig liver tissue morphology; Figure a shows the control group (fed on a normal diet), and Figure b shows... Slackia sp. D-G6 supplementary group. Scale bar = 50 µm.

[0023] Figure 5 for Slackia Effects of D-G6 bacterial powder supplementation on pig kidney tissue morphology; Figure a shows the control group (fed on a normal diet), and Figure b shows... Slackia sp. D-G6 supplementary group. Scale bar = 50 µm.

[0024] Figure 6 for Slackia Effects of D-G6 bacterial powder supplementation on the gut microbiota composition of pigs; Figure a shows the phylum-level microbiota composition analysis, Figure b shows the genus-level microbiota composition analysis, and Figure c shows the main differentially expressed bacterial groups at the phylum and genus levels in the control group and... Slackia Differences in microbial abundance among sp. D-G6 bacterial powder addition groups. Figure 7 for Slackia LEfSe analysis results on the effect of sp. D-G6 bacterial powder addition on ileal microbial composition. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0026] The culture medium used in this experiment:

[0027] 1. WCA solid medium: Weigh 3.3 g WCA liquid medium powder and 1.5 g agar powder, add pure water and stir until completely dissolved, then bring the volume to 100 mL. Autoclave at 121℃ for 15 min and set aside.

[0028] 2. WCA liquid culture medium: Weigh 33 g of WCA liquid culture medium powder, add water and stir until completely dissolved, then bring the volume to 1 L, autoclave at 121℃ for 15 min and set aside.

[0029] 3. Lyophilization protectants: Trehalose, lactose, chitosan oligosaccharide, monosodium glutamate, skim milk powder and cysteine ​​hydrochloride were dissolved in pure water and brought to a final volume of 1 L. The mixture was then mixed and set aside.

[0030] Example 1 Slackia Preparation of sp. D-G6 bacterial powder

[0031] 1. Slackia sp. D-G6 activation

[0032] The D-G6 strain, stored at -80℃, was removed and inoculated onto WCA agar plates and cultured anaerobically at 37℃ for 3 days. After the culture was completed, when colonies that were visible to the naked eye, uniform in morphology, and free from contamination formed on the surface of the plate, 1 mL of sterile water was added to rinse the surface of the plate and the resulting bacterial suspension was collected in sterile EP tubes for later use.

[0033] 2. Slackia sp. D-G6 scale-up culture

[0034] (1) Preparation of seed culture: The collected bacterial suspension was added to 100 mL of WCA liquid medium at a concentration of 1%, and cultured at 37℃ for 3 days. After the culture was completed, the bacterial suspension was inoculated into 1 L of WCA liquid medium at the same ratio and cultured at 37℃ for 3 days. The same method was used to expand the culture stepwise, and the process was repeated 5 times to finally obtain 5 L of fermentation seed culture;

[0035] (2) Slackia sp. D-G6 fermentation: 5 L of fermentation seed liquid was inoculated into a 500 L fermenter and incubated at 37℃ for 3 days.

[0036] 3. Slackia Preparation of sp. D-G6 lyophilized powder

[0037] After fermentation, the fermentation broth was centrifuged using a tubular centrifuge (20,000 rpm) to collect the mycelial sludge. The obtained mycelial sludge was resuspended at a ratio of 1 kg of mycelial sludge to 5 kg of freeze-drying protectant, mixed thoroughly, and then placed into freeze-drying trays. The thickness of the mixture should not exceed 1 / 2 of the tray. The samples were pre-frozen to below -40°C using a programmed cooling process and held at this temperature to ensure complete freezing. After pre-freezing, the samples were quickly transferred to a freeze dryer for primary drying under vacuum conditions. The vacuum degree was controlled at 100-200 mTorr, and the shelf temperature was controlled at -25 to -15°C. Once the sample temperature stabilized and no visible ice crystals were visible in the material, the secondary drying stage began. During the secondary drying, the shelf temperature was gradually increased to approximately 20°C and held at this temperature. After freeze-drying, the freeze-dried material was promptly pulverized, then vacuum-packed and stored at 4°C.

[0038] 4. Slackia sp. D-G6 lyophilized powder activity detection

[0039] Dissolve 0.1g of freshly prepared freeze-dried bacterial powder and bacterial powder stored at room temperature for 1 month in 1 mL of sterile water, then serially dilute with sterile water by 10%. 6 The sample was spread onto WCA solid medium and the colony count was determined. Results are as follows: Figure 1 As shown, the viable count of the lyophilized bacterial powder after reconstitution is approximately 1.5 × 10⁻⁶. 10 CFU / g, viable bacterial count after one month of storage at room temperature is approximately 5×10⁻⁶. 9 CFU / g.

[0040] Example 2 Slackia Application of sp. D-G6 bacterial powder in pig farming

[0041] Seventy-two healthy castrated crossbred boars with an initial weight of 35.56 ± 1.71 kg were randomly assigned to two groups, with six pens in each group and three boars per pen. The experiment included two treatments: a normal diet feeding group (CON group) and a... Slackia The D-G6 group was supplemented with inoculum powder. The basal diet was prepared according to Table 1, and the D-G6 group was supplemented with inoculum powder at 0.2% of the diet mass. Feed and water were freely available during the experiment, which lasted for 30 days.

[0042] Table 1. Dietary composition and nutrient levels (%) Dietary composition content Nutritional level content wheat flour 30.00 Moisture 10.64 Sprayed corn husks 20.00 Crude protein (CP) 14.99 Soybean meal (46%) 6.90 Crude fat (EE) 3.18 <![CDATA[Compound premixed feed for piglets 1 > 2.50 Crude fiber (CF) 3.14 <![CDATA[Pig Trace Element Premix 2 > 0.20 Coarse ash (Ash) 4.59 Type I dicalcium phosphate 1.11 Calcium (Ca) 0.60 Soybean oil (Grade 1) 0.76 Total phosphorus (P-total) 0.55 stone powder 0.74 Sodium chloride 0.57 Sodium chloride 0.35 1 The compound premixed feed for piglets provides the following per kilogram of feed: VA 2.0 KIU, VD3 2.1 KIU, VE 45 IU, VK3 2.0 mg, VB1 3.0 mg, VB2 5.0 mg, VB6 1.8 mg, VB12 0.03 mg, choline chloride 1 g, niacin 25.0 mg, VB5 15.0 mg, biotin 0.08 mg, and folic acid 1.0 mg. 2 The micronutrient premixed feed for piglets provides the following per kilogram of feed: Mn 20.0 mg, Zn 80.0 mg, Fe 80 mg, Cu 6.0 mg, I 0.15 mg, Se 0.25 mg.

[0043] 1. The effect of the bacterial powder on pig feed intake and weight gain.

[0044] After the experiment began, the amount of feed given was recorded daily, and feed intake was calculated every 5 days. Pigs were weighed on days 1, 15, and 30 of the experiment. At the end of the experiment, final weight, average daily weight gain, and average daily feed intake were calculated. Results are as follows: Figure 2 As shown, at 15 days, the final weight of the D-G6 group was 52.07 ± 0.52 kg, and that of the CON group was 50.28 ± 1.03 kg, representing a 3.6% increase compared to the CON group; at 30 days, the final weight of the D-G6 group was 68.50 ± 1.09 kg, and that of the CON group was 67.22 ± 0.69 kg, representing a 2% increase.

[0045] In terms of average daily weight gain, the D-G6 group had an average daily weight gain of 1.10 ± 0.03 kg from 1 to 15 days, which was higher than that of the CON group (0.98 ± 0.07 kg), representing a 12.2% increase; the D-G6 group had an average daily weight gain of 1.10 ± 0.03 kg from 1 to 30 days, which was higher than that of the CON group (1.05 ± 0.02 kg), representing a 4.8% increase.

[0046] Regarding average daily feed intake, the D-G6 group's average daily feed intake from days 1 to 15 was 2.01 ± 0.06 kg, higher than the CON group's 1.82 ± 0.13 kg, representing a 10.4% increase; the average daily feed intake from days 1 to 30 was 2.43 ± 0.10 kg, higher than the CON group's 2.30 ± 0.07 kg, representing a 5.7% increase.

[0047] 2. The effect of the bacterial powder on the safety of pigs.

[0048] The heart, liver, spleen, kidneys, and stomach of the experimental pigs were weighed, and organ indices were calculated. The results are as follows: Figure 3 As shown, there were no significant differences in organ indices between the CON group and the D-G6 group.

[0049] Further examination of liver and kidney tissue sections and HE staining yielded the following results: Figure 4 and 5 As shown, the liver tissue structure of the CON group and the D-G6 group was intact, the liver lobule outline was clear, the hepatocytes were arranged normally, and no obvious inflammatory cell infiltration or degeneration and necrosis were observed. At the same time, the kidney tissue structure of the CON group and the D-G6 group was intact, the glomeruli were normal in shape and clear in structure, the renal tubules were arranged regularly, the epithelial cell structure was intact, and the cells were arranged relatively tightly. No obvious tissue damage or inflammatory reaction was observed.

[0050] The above results indicate that the bacterial powder has no significant adverse effects on growing pigs and has good feeding safety.

[0051] 3. The effect of the bacterial powder on the intestinal microbiota of pigs.

[0052] Ileal contents samples were collected from growing pigs for 16S rDNA sequencing analysis. Results are as follows: Figure 6 As shown, at the phylum level, the ileum microbiota of growing pigs mainly consists of Firmicutes, Proteobacteria, and Fusobacteriota, with Firmicutes being the dominant phylum. Compared with the CON group, the relative abundance of Firmicutes was significantly increased in the D-G6 group, while the relative abundance of Proteobacteria was significantly decreased. This indicates that the bacterial powder can regulate the composition of the ileum microbiota in growing pigs and increase the abundance of Firmicutes.

[0053] At the genus level, the ileum microbiota of growing pigs is mainly composed of Lactobacillus (Lactobacillus genus). Lactobacillus ), Fusobacterium genus ( Fusobacterium ) and Streptococcus spp. Streptococcus The D-G6 group consisted of bacteria such as *Lactobacillus*, with *Lactobacillus* being the dominant genus. Compared to the CON group, the relative abundance of *Lactobacillus* was significantly increased in the D-G6 group, while the abundance of *Escherichia coli*-Shigella group was significantly lower. Escherichia-ShigellaThe relative abundance was significantly reduced. This indicates that the bacterial powder can regulate the composition of the ileum microbiota in growing pigs, increase the abundance of beneficial bacteria, and reduce the abundance of potentially detrimental bacteria.

[0054] Furthermore, such as Figure 7 As shown, LEfSe analysis revealed significant differences in gut microbiota between the D-G6 and CON groups. (Addition) Slackia Following administration of sp. D-G6 bacterial powder, the differentially abundant microbiota in the ileum of growing pigs mainly included Firmicutes, Bacilli, Lactobacillales, Lactobacillaceae, and Lactobacillus. Lactobacillus Unclassified Lactobacillus species (s__Unassigned_ Lactobacillus ) and Lactobacillus reuteri (s__ Lactobacillus_reuteri (e.g., Lactobacilliales, Lactobacilliaceae, Lactobacillus, and Lactobacillus reuteri are typical probiotic groups, while Firmicutes and Bacillus are higher-level taxa related to intestinal homeostasis and the enrichment of beneficial bacteria.) The above results indicate that... Slackia sp. D-G6 can promote the enrichment of beneficial lactobacillus-related bacteria, thereby improving the ileal microecological structure of growing pigs and maintaining intestinal health.

[0055] 4. Statistical Analysis

[0056] All experimental data are expressed as mean ± standard deviation and analyzed using IBM SPSS Statistics 27.0 software. Independent samples t-tests were used for comparisons between two groups. In the results, P < 0.05 indicates a significant difference. P < 0.01 indicates a highly significant difference. P ≥ 0.05 indicates no significant difference; "*" in the attached figure indicates P < 0.05, "**" indicates P <0.01, "***" indicates P < 0.001, “ns” indicates that the difference is not significant.

Claims

1. Application of Shrekella D-G6 bacterial powder in improving pig growth performance and regulating pig intestinal flora.

2. The application according to claim 1, characterized in that, The application described is designed to promote feed intake and weight gain in pigs.

3. The application according to claim 2, characterized in that, The promotion of feed intake and weight gain in growing pigs includes increasing final weight, average daily feed intake, and / or average daily weight gain.

4. The application according to claim 1, characterized in that, The application described is for regulating the composition of the ileum microbiota in growing pigs.

5. The application according to claim 4, characterized in that, The bacterial powder can regulate the composition of the ileum microbiota in growing pigs, including increasing the levels of Firmicutes and / or Lactobacillus. Lactobacillus The abundance of ).

6. The application according to claim 4, characterized in that, The bacterial powder can regulate the composition of the ileum microbiota in growing pigs, including reducing Proteobacteria and / or Escherichia coli-Shigella flora. Escherichia- Shigella The abundance of ).

7. The application according to any one of claims 1-6, characterized in that, The bacterial powder is added to the pig's daily diet at 0.2% of the basal diet weight.

8. The application according to any one of claims 1-7, characterized in that, The bacterial powder is freeze-dried bacterial powder.

9. The application according to any one of claims 1-8, characterized in that, The bacterial strains contained in the bacterial powder are Slackia sp. D-G6, with accession number GDMCC NO: 60661.

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