Application of a porcine-derived Bacillus in antioxidant activity

Abstract

This invention discloses the application of a porcine-derived Bacillus in antioxidant activity, wherein the porcine-derived Bacillus is used to prepare antioxidant products. The porcine-derived Bacillus is Bacillus subtilis YB-X-721, classified as Bacillus subtilis, and was deposited at the China General Microbiological Culture Collection Center on November 10, 2023, with accession number CGMCC No. 28942. The fermentation supernatant, bacterial suspension, and cell-free extract of the porcine-derived Bacillus subtilis YB-X-721 of this invention all possess certain DPPH free radical, superoxide anion free radical, and hydroxyl free radical scavenging abilities, as well as a certain reducing ability, and can be used to prepare antioxidant products. Furthermore, when the porcine-derived Bacillus subtilis YB-X-721 of this invention is added to piglet feed as an animal feed additive, it can improve the growth performance of weaned piglets, significantly increasing average daily weight gain, and significantly reducing average daily feed intake, feed conversion ratio, and diarrhea rate.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, this invention relates to the application of porcine Bacillus in antioxidant activity. Background Technology

[0002] The long-term use of antibiotics in feed has led to problems such as bacterial resistance, animal food residues, and environmental pollution, seriously affecting food safety and human health. my country completely banned the use of feed antibiotics in pig farming in 2020, ushering in a new era of antibiotic-free pig farming. After the ban, common pathogenic enterobacteria easily infect pigs, causing diseases such as yellow and white diarrhea, edema disease, gastroenteritis, and septicemia, leading to decreased growth performance and, in severe cases, death, thus reducing the efficiency of pig farming. Furthermore, these diseases are difficult to prevent with vaccines. Therefore, to eliminate the negative impacts of antibiotic-free pig farming, improve pig farming efficiency, and ensure the intestinal health of pigs, there is an urgent need to develop green and safe alternatives with comparable efficacy to antibiotics. Against this backdrop, probiotics, with their advantages of being non-toxic, residue-free, less prone to inducing drug resistance, anti-intestinal infection, and immune-boosting, have become one of the best choices for antibiotic alternatives. Therefore, developing efficient and green antibiotic alternatives that can improve the antioxidant capacity of piglets is a problem that the pig farming industry urgently needs to solve. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0004] To achieve these objectives and other advantages of the present invention, an application of porcine-derived Bacillus subtilis in antioxidant activity is provided. The porcine-derived Bacillus subtilis YB-X-721, classified as Bacillus subtilis, was deposited at the China General Microbiological Culture Collection Center on November 10, 2023, with accession number CGMCC No. 28942.

[0005] Preferably, the porcine-derived Bacillus is used to prepare antioxidant products.

[0006] Preferably, the antioxidant product contains Bacillus subtilis YB-X-721, Bacillus subtilis YB-X-721 fermentation supernatant, Bacillus subtilis YB-X-721 bacterial suspension, or Bacillus subtilis YB-X-721 cell-free extract.

[0007] Preferably, the preparation method of the Bacillus subtilis YB-X-721 fermentation supernatant is as follows: Bacillus subtilis YB-X-721 is inoculated into LB liquid medium at 1-3% (v / v), cultured at 37°C for 24 hours, centrifuged at 8000g for 10 minutes, and the supernatant is collected at 4°C, which is the Bacillus subtilis YB-X-721 fermentation supernatant.

[0008] Preferably, the method for preparing the Bacillus subtilis YB-X-721 bacterial suspension is as follows: Bacillus subtilis YB-X-721 is inoculated into LB liquid medium at 1-3% (v / v), cultured at 37°C for 24 h, centrifuged at 8000g for 10 min, and collected at 4°C. The obtained bacterial cells are washed 2-3 times with sterile water, resuspended in PBS, and the density is adjusted to 1×10⁻⁶. 9 CFU / mL was used to obtain a suspension of Bacillus subtilis YB-X-721.

[0009] Preferably, the preparation method of the cell-free extract of Bacillus subtilis YB-X-721 is as follows: the Bacillus subtilis YB-X-721 bacterial suspension is ultrasonically disrupted under ice bath conditions, centrifuged at 8000g for 10min, and the supernatant is collected to obtain the cell-free extract of Bacillus subtilis YB-X-721.

[0010] Preferably, the specific conditions for ultrasonic fragmentation are: power 240-260W, ultrasonic time 3-10s, interval 5-15s, for a total of 10min.

[0011] Preferably, the 16S rDNA sequence of the porcine Bacillus is shown in SEQ ID NO.1.

[0012] Preferably, the antioxidant product is a microbial agent or an animal feed additive.

[0013] Preferably, the amount of the animal feed additive added is such that the viable count of porcine Bacillus in the feed is 0.5 × 10⁻⁶. 7 ~2×10 7 CFU / g.

[0014] Preferably, the method for preparing the fermentation supernatant includes:

[0015] Step 1: Mix corn gluten powder, quercetin and PBS buffer, place in a sweep pulse sonication device, set the sonication parameters, perform sweep pulse sonication, add konjac glucomannan, and continue sweep pulse sonication to obtain nutrient solution. Add the nutrient solution to LB liquid culture medium, stir evenly, and autoclave at 121℃ for 20 minutes to obtain fermentation medium.

[0016] Step 2: Inoculate Bacillus subtilis YB-X-721 strain at 1-3% (v / v) into fermentation medium, incubate at 37℃ for 24h, centrifuge at 8000g for 10min, collect the supernatant at 4℃, and obtain YB-X-721 fermentation supernatant.

[0017] Step 3: Inoculate Bacillus tequilensis strain YB-2 (preservation number CCTCC No: M 20221531) into fermentation medium at 1-3% (v / v), incubate at 37℃ for 24h, centrifuge at 8000g for 10min, and collect the supernatant at 4℃ to obtain YB-2 fermentation supernatant.

[0018] Step 4: Mix the fermentation supernatant of YB-X-721 and the fermentation supernatant of YB-2 at a volume ratio of 2 to 5:1 to obtain a compound fermentation supernatant.

[0019] Preferably, the mass-to-volume ratio of corn gluten powder, quercetin, konjac glucomannan, and PBS buffer is 5-15g:1-3g:0.5-2g:50-300mL; the nutrient solution is added to LB liquid culture medium at 10-30% (v / v).

[0020] Preferably, the swept-frequency pulse ultrasound processing parameters are: temperature 15-30℃, ultrasound power 400-800W, frequency 22-40kHz, swept-frequency amplitude ±0.5-±2kHz, swept-frequency period 100-500ms, pulse ultrasound time 100-300s, interval time 5-15s, and processing time 5-30min.

[0021] The present invention has at least the following beneficial effects: the fermentation supernatant, bacterial suspension and cell-free extract of the porcine Bacillus subtilis YB-X-721 of the present invention all have certain DPPH free radical, superoxide anion free radical, and hydroxyl free radical scavenging abilities as well as certain reducing abilities, and can be used to prepare antioxidant products; and the porcine Bacillus subtilis YB-X-721 of the present invention, when added to piglet feed as an animal feed additive, can improve the growth performance of weaned piglets, significantly increase the average daily weight gain, and significantly reduce the average daily feed intake, feed conversion ratio and diarrhea rate.

[0022] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0023] Figure 1 This is a comparative diagram of the inhibition zones of porcine-derived Bacillus strains YB-M-32, YB-M-123, YB-X-416, and YB-X-721 of the present invention, wherein: A is Escherichia coli; B is Salmonella; and C is Staphylococcus aureus.

[0024] Figure 2 This is a Gram staining image of the porcine Bacillus YB-X-721 of this invention;

[0025] Figure 3 This is a staining image of the porcine Bacillus YB-X-721 spores from this invention;

[0026] Figure 4 This is a colony morphology diagram of the porcine Bacillus YB-X-721 of the present invention;

[0027] Figure 5 This is the phylogenetic tree of the porcine Bacillus YB-X-721 of this invention;

[0028] Figure 6 This is the growth curve of the porcine Bacillus YB-X-721 of the present invention. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0030] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0031] Example 1

[0032] An application of a porcine-derived Bacillus in antioxidant activity, wherein the porcine-derived Bacillus subtilis YB-X-721, classified as Bacillus subtilis, was deposited at the China General Microbiological Culture Collection Center on November 10, 2023, with accession number CGMCC No. 28942;

[0033] Screening and isolation of porcine Bacillus:

[0034] Fresh feces from healthy, antibiotic-free adult Rongchang pigs were used as samples. An appropriate amount of fecal sample was aseptically weighed, placed in PBS buffer, shaken to mix thoroughly, and incubated in a 90°C water bath for 10 min to kill other non-spore-forming bacteria. Then, the sample was serially diluted with sterile water, and an appropriate amount of the diluted solution was spread on soybean casein agar (TSA) medium and incubated upside down at 37°C for 48 h. The growth of colonies on the plates was observed, and single colonies were streaked and inoculated onto new plates for purification and culture. Finally, four Bacillus strains were isolated and numbered YB-M-32, YB-M-123, YB-X-416, and YB-X-721.

[0035] Screening for antagonistic pathogen characteristics of porcine Bacillus:

[0036] To further obtain Bacillus strains with good antibacterial effects, an inhibitory test was conducted on the candidate bacteria: LB solid medium was sterilized and cooled to approximately 50°C, then poured into sterile petri dishes. After the plates were allowed to air dry naturally, 0.1 mL of indicator bacterial suspension (10 mL / min) was transferred to each dish. 8 The bacterial suspension (CFU / mL) was evenly spread on a plate and allowed to air dry. Wells were then punched in the plate, and 0.1 mL of the test bacterial suspension was added to each well. The plate was allowed to stand until the bacterial suspension had permeated the plate, and then incubated at 37°C for 24 hours. The size of the inhibition zone was measured. The indicator bacteria were Escherichia coli, Staphylococcus aureus, and Salmonella. The inhibition results are shown in Table 1, and the inhibition zone comparison diagram is shown below. Figure 1 As shown (A. Escherichia coli; B. Salmonella; C. Staphylococcus aureus).

[0037] Table 1

[0038]

[0039] One strain of Bacillus, YB-X-721, was screened and found to inhibit Escherichia coli, Staphylococcus aureus, and Salmonella. Morphological, physiological and biochemical, and molecular biological characteristics were identified.

[0040] Morphological identification of Bacillus subtilis YB-X-721 (a porcine-derived Bacillus subtilis):

[0041] In a sterile operating room, the purified Bacillus isolates were streaked onto LB agar plates and incubated at 37°C for 24 hours. The size, shape, gloss, color, and transparency of single colonies were observed and recorded. Gram staining was performed on the isolates. Figure 2 ) and spore staining ( Figure 3 The staining procedure was performed according to the staining kit. Bacterial morphology was observed and recorded under a microscope. The colony morphology of *Bacillus subtilis* strain YB-X-721 (from porcine origin) is shown in Table 2. Figure 4 .

[0042] Table 2

[0043] colonies color Size (mm) form edge protrusion transparent texture YB-X-721 White 3.5 round irregular no no Unsmooth

[0044] Acid and bile salt resistance tests of porcine Bacillus subtilis YB-X-721:

[0045] Bile salt tolerance test:

[0046] The bacterial strain was inoculated into LB liquid medium and cultured for 24 h. Then, the bacterial suspension was inoculated at a ratio of 5% (v / v) into LB liquid medium with different bile salt levels (0.15% and 0.3%) and incubated at 37°C for 4 h. At 0 and 4 h, 100 μL of the bacterial suspension was serially diluted 10-fold, and 10 μL of the diluted suspension was used as the final product. -5 Gradual dilutions were spread onto LB plates and incubated at 37°C for 24 hours. Colony counts were performed, with three replicates for each sample.

[0047] Survival rate (%) = (4h colony count / 0h colony count) × 100%

[0048] Acid resistance test:

[0049] The bacterial strain was inoculated into LB liquid medium and cultured for 24 hours. Then, the bacterial suspension was inoculated at a ratio of 5% (v / v) into LB liquid medium at pH 2 and 3, and incubated at 37°C for 4 hours. At 0 and 4 hours, 100 μL of the bacterial suspension was serially diluted 10-fold. -5 Gradual dilutions were spread onto LB plates and incubated at 37°C for 24 hours. Colony counts were performed, with three replicates for each sample.

[0050] Survival rate (%) = (4h colony count / 0h colony count) × 100%

[0051] The experimental results are shown in Table 3. It can be seen that Bacillus subtilis YB-X-721, a porcine-derived Bacillus, has excellent acid and bile salt resistance.

[0052] Table 3

[0053]

[0054] Physiological and biochemical characteristics of porcine Bacillus subtilis YB-X-721:

[0055] The physiological and biochemical characteristics of strain YB-X-721 were tested according to the steps in the biochemical detection kit, and the results are shown in Table 4. Comparative analysis with the *Manual of Systematic Identification of Common Bacteria* and the *Berge's Manual of Bacterial Identification* preliminarily identified it as a Bacillus genus.

[0056] Table 4

[0057] project result project result catalase + Sorbitol - VP reaction - Seven-leaf spirit - Methyl red reaction - glucose - Starch hydrolysis - Raffinose - Cellobiose - sucrose - maltose - gelatin + Mannitol -

[0058] In the table, + indicates positive; - indicates negative.

[0059] Molecular biological identification of porcine Bacillus subtilis YB-X-721:

[0060] PCR amplification was performed using universal primers 27F and 1492R for bacterial identification of 16S rDNA. The primer sequences are shown in Table 5. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0061] Table 5

[0062]

[0063] The PCR amplification template was activated fresh bacterial culture, and the system was 50 μL. The reaction system is shown in Table 6.

[0064] Table 6

[0065]

[0066]

[0067] The PCR amplification program was as follows: 95℃ for 5 min; 95℃ for 45 s; 55℃ for 45 s; 72℃ for 1 min; repeated for 35 cycles; 72℃ for 8 min. The PCR amplification products were identified by 1% agarose gel electrophoresis and then sequenced by Shanghai Sangon Biotech Co., Ltd., followed by BLAST alignment analysis in GenBank.

[0068] Perform BLAST alignment in GenBank to construct a genetic phylogenetic tree, such as... Figure 5 As shown in the figure. The results showed that YB-X-721 and the reference strain Bacillus subtilis (NCIB 3610) were on the same branch and had a very high degree of phylogenetic relationship. Therefore, YB-X-721 could be identified as Bacillus subtilis and named Bacillus subtilis YB-X-721. The 16S rDNA sequence of the porcine Bacillus subtilis YB-X-721 was obtained by sequencing and is shown in SEQ ID NO.1.

[0069] Biological characteristics analysis of porcine Bacillus subtilis YB-X-721:

[0070] After activation, the bacterial strain was inoculated into 100 mL of LB liquid medium at a 2% (v / v) inoculation rate and cultured with shaking at 37°C. Every 4 hours, 5 mL of bacterial solution was collected for analysis. The bacterial concentration (OD value) was measured using a UV spectrophotometer at 600 nm for a total of 24 hours. The growth curve of *Bacillus subtilis* YB-X-721 (a porcine-derived Bacillus subtilis) was determined... Figure 6 It can be observed that YB-X-721 enters the logarithmic growth phase after 2 hours and the stationary phase after 10 hours.

[0071] Example 2

[0072] Application of a porcine-derived Bacillus in antioxidant activity

[0073] The in vitro antioxidant activity of the porcine-derived Bacillus of the present invention:

[0074] I. Preparation of bacterial culture and in vitro antioxidant detection

[0075] 1. Reagents

[0076] Anhydrous ethanol, potassium ferricyanide, PBS buffer, acetic acid, ferric chloride, ascorbic acid, hydrogen peroxide, ferrous sulfate, 3,5-dinitrosalicylic acid (DNS), pyrogallol, trihydroxymethylaminomethane-hydrochloric acid buffer (Tris), phosphate buffer, o-phenanthroline solution, etc., all of the above reagents are of analytical grade.

[0077] 2. Extraction of bacterial culture from samples

[0078] Different fractions of the bacterial strain were prepared according to the method described by Gao Li'e (Gao Li'e. Study on the diversity and antioxidant characteristics of lactic acid bacteria in traditionally fermented yak milk in the Qinghai-Tibet Plateau [D]. Lanzhou University, 2020. DOI:10.27204 / d.cnki.glzhu.2020.002996.). The strain was inoculated at 2% (v / v) on LB liquid medium and cultured at 37℃ for 24 h. After centrifugation at 8000g for 10 min at 4℃, the supernatant and bacterial cells were collected separately. The supernatant was designated as fermentation supernatant (FS). The obtained bacterial cells were washed 2–3 times with sterile water, resuspended in PBS, and the density was adjusted to 1×10⁻⁶. 9 The CFU / mL was divided into two equal parts. One part was used as a bacterial suspension (IC), and the other part was sonicated under ice bath conditions (250W, sonication for 5s, 10s interval, for a total of 10min). The mixture was centrifuged at 8000g for 10min, and the supernatant was collected to obtain the cell-free extract (CFE).

[0079] 3. In vitro antioxidant activity assay

[0080] 3.1 Determination of DPPH free radical scavenging ability

[0081] Take 1 mL of sample and place it in a test tube. Add 2 mL of DPPH anhydrous ethanol solution (concentration of 0.2 mmol / L), mix well, and react at room temperature in the dark for 30 min. After centrifugation at 8000g for 10 min at 4℃, take the supernatant and measure its absorbance at 517 nm. Zero the instrument with deionized water. Perform 3 replicates for each group and calculate the average value.

[0082] DPPH clearance rate (%) = [1 - (Sample A - Blank A) / Control A] × 100%

[0083] (Blank group: DPPH was replaced with an equal volume of anhydrous ethanol; Control group: Sample solution was replaced with an equal volume of distilled water, and the blank was zeroed using an equal volume of distilled water and anhydrous ethanol mixture.)

[0084] Preparation of liquid: 0.2 mmol / L DPPH anhydrous ethanol solution: Weigh 50 mg DPPH, add 634 mL anhydrous ethanol, and shake well.

[0085] 3.2 Determination of hydroxyl radical (·OH) scavenging activity (Fenton method)

[0086] The Fenton method utilizes H₂O₂ and Fe₂O₃. 2+ The mixture undergoes a Fenton reaction, generating highly reactive ·OH groups. Salicylic acid can capture these ·OH groups and produce a colored substance (dihydroxybenzoic acid), which has maximum absorption at 510 nm. However, if a certain amount of a scavenging agent is added, it will compete with the reaction of salicylic acid, thus reducing the amount of the colored substance (dihydroxybenzoic acid) produced and causing a change in the absorbance of the solution. The activity of the antioxidant can then be measured based on the change in absorbance.

[0087] H2O2 + Fe 2+ →·OH+OH - +Fe 3+

[0088] Preparation of liquids: ①FeSO4 solution: Accurately weigh 0.05g of ferrous sulfate heptahydrate into a 100mL volumetric flask, add distilled water to the mark, and shake well to obtain a 1.8mmol / L ferrous sulfate solution;

[0089] ② Salicylic acid-ethanol solution: Accurately weigh 0.0249 g of salicylic acid and place it in a 100 mL volumetric flask. Add anhydrous ethanol to the mark and shake well until stable to obtain a 1.8 mmol / L salicylic acid-ethanol solution.

[0090] ③ 0.3% H2O2 solution: Accurately measure 1.0 mL of 30% hydrogen peroxide into a 100 mL volumetric flask, add distilled water to the mark, and mix well to obtain a 0.3% H2O2 solution.

[0091] The specific steps are as follows: Add 2 mL of 1.8 mmol / L FeSO4 solution, 1 mL of sample, and 0.1 mL of 0.3% H2O2 solution to a test tube sequentially, shake well, then add 1.5 mL of 1.8 mmol / L salicylic acid-ethanol solution, shake well, and let stand at 30℃ for 30 min. Measure the absorbance Ai of the sample group at 510 nm. Replace the H2O2 solution in the sample group with an equal volume of distilled water, and react again as a sample reference group, measuring its absorbance Aj. Replace the sample in the sample group with an equal volume of distilled water, and react again as a blank control group, measuring its absorbance Ao. Calculate the hydroxyl radical scavenging rate using the following formula:

[0092] Hydroxyl radical scavenging rate (%) = [Ao - (Ai - Aj) / Ao] × 100%

[0093] 3.3 Superoxide anion radical (O2) - • Determination of scavenging activity (pyrogallol method)

[0094] Phloroglucinol undergoes auto-oxidation readily under alkaline conditions, producing colored intermediate products and superoxide anions (O2). - ·, O2 - It also catalyzes auto-oxidation reactions; the amount of O2 produced is used to determine its effectiveness. - The amount of · generated. When antioxidant substances are added, the auto-oxidation of pyrogallol can be weakened, thus allowing the reduction of colored substances to be used as a measure of antioxidant activity.

[0095] Preparation of the liquid: ① Pyrogallol: Accurately weigh 0.315 g of pyrogallol and place it in a 100 mL volumetric flask. Then add 10 mL of 0.1 mol / L hydrochloric acid, and finally add distilled water to the mark. Shake well to obtain 25 mmol / L pyrogallol. Pyrogallol should be prepared and used immediately and reacted at 20 °C.

[0096] ②Tris-HCl: Accurately weigh 3.0285g of Tris reagent into a 500mL volumetric flask, add 114.5mL of 0.1mol / L hydrochloric acid, and finally add distilled water to the mark. Shake well to obtain 0.05mol / L Tris-HCl buffer solution.

[0097] The specific steps are as follows: Take out the test tube and add 7.5 mL of 0.05 mol / L Tris-HCl buffer solution. Preheat in a 25°C water bath for 20 min. Then add 1 mL of sample solution and 0.5 mL of 25 mmol / L pyrogallol solution, mix, and react in a 25°C water bath for 5 min. Add 1 mL of concentrated HCl to terminate the reaction. This is the sample group. Measure the absorbance Ai of the solution at 320 nm using a UV-Vis spectrophotometer. For the sample control group, use the same volume of distilled water instead of pyrogallol solution and measure the absorbance Aj. For the blank control group, use the same volume of distilled water instead of the sample solution and measure the absorbance Ao. Calculate O2 using the following formula. - • Clearance rate:

[0098] Superoxide anion radical scavenging rate (%) = [Ao - (Ai - Aj)] / Ao × 100%

[0099] 3.4 Determination of reducing power

[0100] Preparation of the liquids: ① 0.2 mol / L, pH 6.6 phosphate buffer solution: First, prepare solution A (0.2 M Na2HPO4 (17.19 g Na2HPO4·12H2O + 240 mL H2O) and solution B (0.2 M NaH2PO4 (11.23 g NaH2PO4·2H2O + 360 mL H2O)); take 40 mL of solution A and 60 mL of solution B, mix them to obtain a 0.2 mol / L, pH 6.6 phosphate buffer solution.

[0101] ②1% Potassium ferricyanide: 1g potassium ferricyanide + 100mL H2O;

[0102] ③10% trichloroacetic acid: 1 mL trichloroacetic acid + 9 mL H2O;

[0103] ④ 0.1% ferric chloride: 0.1g ferric chloride + 100mL H2O.

[0104] Take 0.5 mL of sample and place it in a test tube. Add 0.5 mL of 0.2 mol / L phosphate buffer solution (pH 6.6), then add 0.5 mL of 1% potassium ferricyanide. Incubate at 50°C for 20 min, then rapidly cool in an ice bath. Add 0.5 mL of 10% trichloroacetic acid. Centrifuge at 4000 rpm for 10 min. Take 1 mL of the supernatant, add 1 mL of distilled water and 1 mL of 0.1% ferric chloride, mix well, and incubate at room temperature for 10 min. Measure the absorbance at 700 nm. Perform three replicates for each sample and calculate the average value. A higher absorbance indicates a stronger reducing power of the sample.

[0105] The results are shown in Table 7. The fermentation supernatant (FS), bacterial suspension (IC), and cell-free extract (CFE) of the porcine Bacillus subtilis YB-X-721 of the present invention all have certain DPPH free radical, superoxide anion free radical, and hydroxyl free radical scavenging abilities and certain reducing abilities, indicating that the strain has certain antioxidant activity in vitro.

[0106] Table 7

[0107]

[0108] Example 3

[0109] An application of a porcine-derived Bacillus in antioxidant activity includes:

[0110] Step 1: Mix corn gluten meal, quercetin, and PBS buffer, place in a sweep-frequency pulsed sonication device, set the sonication parameters, and perform sweep-frequency pulsed sonication for 10 minutes. Then add konjac glucomannan and continue sweep-frequency pulsed sonication for another 10 minutes to obtain a nutrient solution. Add the nutrient solution to LB liquid medium at 15% (v / v), stir well, and autoclave at 121℃ for 20 minutes to obtain the fermentation medium. The mass-volume ratio of corn gluten meal, quercetin, konjac glucomannan, and PBS buffer is 8g:2g:1g:100mL. The sweep-frequency pulsed sonication parameters are: temperature 25℃, ultrasonic power 600W, frequency 28kHz, sweep amplitude ±2kHz, sweep period 300ms, pulsed sonication time 200s, and interval time 10s.

[0111] Step 2: Inoculate Bacillus subtilis YB-X-721 strain at 2% (v / v) into fermentation medium, incubate at 37℃ for 24h, centrifuge at 8000g for 10min, collect the supernatant at 4℃, and obtain YB-X-721 fermentation supernatant.

[0112] In this embodiment, during the cultivation of the strain, the prepared nutrient solution is added to the LB liquid medium. The resulting fermentation medium is more conducive to the growth and metabolism of porcine Bacillus, promotes the secretion of metabolites with good antioxidant capacity by the strain, and enhances the antioxidant capacity of the fermentation supernatant. In particular, when preparing the nutrient solution, it is subjected to frequency-sweeping pulse ultrasound treatment. The synergistic effect of pulse ultrasound and frequency-sweeping ultrasound is used to improve the mixing efficiency and the uniformity and dispersion of each substance in the nutrient solution, and the strain has more sufficient contact with each substance.

[0113] Example 4

[0114] An application of a porcine-derived Bacillus in antioxidant activity includes:

[0115] Step 1: Mix corn gluten meal, quercetin, and PBS buffer, and sonicate with pulsed sonication for 10 min. Then add konjac glucomannan and continue sonicating with pulsed sonication for another 10 min to obtain a nutrient solution. Add the nutrient solution to LB liquid medium at 15% (v / v), stir well, and autoclave at 121℃ for 20 min to obtain the fermentation medium. The mass-to-volume ratio of corn gluten meal, quercetin, konjac glucomannan, and PBS buffer is 8 g:2 g:1 g:100 mL. The pulsed sonication parameters are: temperature 25℃, ultrasonic power 600 W, frequency 28 kHz, pulsed sonication time 200 s, and interval time 10 s.

[0116] Step 2: Inoculate Bacillus subtilis YB-X-721 strain at 2% (v / v) into fermentation medium, incubate at 37℃ for 24h, centrifuge at 8000g for 10min, collect the supernatant at 4℃, and obtain YB-X-721 fermentation supernatant.

[0117] Example 5

[0118] An application of a porcine-derived Bacillus in antioxidant activity includes:

[0119] Step 1: Mix corn gluten powder and PBS buffer, place in a sweep pulse sonication device, set the sonication parameters, and perform sweep pulse sonication for 10 min. Then add konjac glucomannan and continue sweep pulse sonication for another 10 min to obtain a nutrient solution. Add the nutrient solution to LB liquid medium at 15% (v / v), stir well, and autoclave at 121℃ for 20 min to obtain the fermentation medium. The mass-to-volume ratio of corn gluten powder, konjac glucomannan, and PBS buffer is 8 g:1 g:100 mL. The sweep pulse sonication parameters are: temperature 25℃, ultrasonic power 600 W, frequency 28 kHz, sweep amplitude ±2 kHz, sweep period 300 ms, pulse sonication time 200 s, and interval time 10 s.

[0120] Step 2: Inoculate Bacillus subtilis YB-X-721 strain at 2% (v / v) into fermentation medium, incubate at 37℃ for 24h, centrifuge at 8000g for 10min, collect the supernatant at 4℃, and obtain YB-X-721 fermentation supernatant.

[0121] Example 6

[0122] An application of a porcine-derived Bacillus in antioxidant activity includes:

[0123] Step 1: Mix corn gluten meal, quercetin, and PBS buffer, place in a sweep-frequency pulsed sonication device, set the sonication parameters, and perform sweep-frequency pulsed sonication for 10 minutes. Then add konjac glucomannan and continue sweep-frequency pulsed sonication for another 10 minutes to obtain a nutrient solution. Add the nutrient solution to LB liquid medium at 15% (v / v), stir well, and autoclave at 121℃ for 20 minutes to obtain the fermentation medium. The mass-volume ratio of corn gluten meal, quercetin, konjac glucomannan, and PBS buffer is 8g:2g:1g:100mL. The sweep-frequency pulsed sonication parameters are: temperature 25℃, ultrasonic power 600W, frequency 28kHz, sweep amplitude ±2kHz, sweep period 300ms, pulsed sonication time 200s, and interval time 10s.

[0124] Step 2: Inoculate Bacillus subtilis YB-X-721 strain at 2% (v / v) into fermentation medium, incubate at 37℃ for 24h, centrifuge at 8000g for 10min, collect the supernatant at 4℃, and obtain YB-X-721 fermentation supernatant.

[0125] Step 3: Inoculate Bacillus tequilensis strain YB-2 (preservation number CCTCC No: M 20221531) at 2% (v / v) into fermentation medium, incubate at 37℃ for 24h, centrifuge at 8000g for 10min, collect the supernatant at 4℃, and obtain YB-2 fermentation supernatant.

[0126] Step 4: Mix the fermentation supernatant of YB-X-721 and the fermentation supernatant of YB-2 at a volume ratio of 3:1 to obtain a compound fermentation supernatant.

[0127] In this embodiment, YB-X-721 fermentation supernatant and YB-2 fermentation supernatant are combined to form a composite fermentation supernatant, which further enhances the antioxidant capacity.

[0128] The in vitro antioxidant activity of the fermentation supernatants prepared in Examples 3-5 and the composite fermentation supernatant prepared in Example 6 was determined, and the results are shown in Table 8. It can be seen that the antioxidant capacity of Examples 3-6 is higher than that of Example 2 compared with the fermentation supernatant in Example 2, and the composite fermentation supernatant of Example 6 has the best antioxidant capacity.

[0129] Table 8

[0130]

[0131] Example 7

[0132] Application of a porcine-derived Bacillus in antioxidant activity

[0133] The in vivo antioxidant activity of the porcine-derived Bacillus subtilis YB-X-721 of this invention in animals:

[0134] 1. Preparation of probiotic solution

[0135] The strain was inoculated into LB solid medium and activated for three generations. A probiotic solution was prepared using a 20L automated fermenter. The viable count of the produced solution was calculated using the plate count method. The viable count of *Bacillus subtilis* YB-X-721 in the sample was 1 × 10⁻⁶. 10 CFU / g.

[0136] 2. Animal experimental treatment

[0137] Eighteen healthy, 28-day-old weaned Rongchang piglets with an average weight of 6.9-7.1 kg were selected. Based on the principle of similar weight and a consistent male-to-female ratio, the piglets were randomly divided into three groups, with six piglets per pen (half male, half female). The control group was fed a basal diet, while the YB-X-721 group had YB-X-721 probiotic liquid added to their basal diet to adjust the live bacteria count to 1×10⁻⁶. 7 CFU / g, the commercial probiotic group was adjusted to a live bacteria count of 1×10⁻⁶ by adding Yisheng Kangfuhe microbial preparation (Guangdong Boda Technology) to the basal diet. 7 CFU / g, pre-feeding period of 3 days, formal experimental period of 28 days. All test pigs were fed and managed according to the standard pig farm practices. During the experiment, immunization, deworming and disinfection procedures were carried out according to the normal pig farm procedures. At the end of the experimental feeding, 5 mL of blood was collected from the anterior vena cava of each group, placed in a serum tube for coagulation, centrifuged at 3000 r / min for 10 min, and the serum was separated and stored at -20℃ for later testing.

[0138] 3. Growth performance

[0139] On the mornings of days 1 and 28 of the experiment, piglets were weighed on an empty stomach. Feed intake was recorded daily and diarrhea was observed throughout the experiment. The average daily feed intake, average daily weight gain, and feed conversion ratio were calculated based on feed intake and weight gain over 28 days.

[0140] Diarrhea rate (%) = Total number of diarrhea episodes / (Total number of experimental pigs × Total number of experimental days) × 100%

[0141] 4. Antioxidant capacity test

[0142] The collected piglet serum samples were tested using an antioxidant capacity assay kit, and five indicators were measured: total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), and malondialdehyde (MDA).

[0143] The results of the growth performance test of weaned piglets are shown in Table 8. Compared with the control group, there were no significant differences in birth weight and body weight on day 28 of the YB-X-721 group (P>0.05), but the average daily weight gain was significantly increased, while the average daily feed intake, feed conversion ratio, and diarrhea rate were significantly decreased. This indicates that adding YB-X-721 to the feed improved the growth performance of weaned piglets. Furthermore, the effect of the porcine Bacillus subtilis YB-X-721 of this invention on improving the growth performance of piglets is close to that of commercial probiotics.

[0144] Table 8

[0145]

[0146] In the table, different letters in the superscript of data in the same row indicate significant differences (P < 0.05); the same letter or no letter in the superscript indicates no significant differences (P > 0.05).

[0147] The results of the antioxidant capacity assay are shown in Table 9. The activities of T-SOD, GSH-PX, and T-AOC in the serum of piglets in the YB-X-721 group were significantly higher than those in the control group (P<0.05), while the MDA content was lower. This indicates that YB-X-721 improved the antioxidant capacity of weaned piglets. Furthermore, the antioxidant capacity-enhancing effect of the porcine Bacillus subtilis YB-X-721 of this invention on piglets is close to that of commercial probiotics.

[0148] Table 9

[0149]

[0150] In the table, different letters in the superscript of data in the same row indicate significant differences (P < 0.05); the same letter or no letter in the superscript indicates no significant differences (P > 0.05).

[0151] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. The application of a porcine-derived Bacillus in antioxidant activity, characterized in that, The porcine-derived Bacillus is used to prepare antioxidant products; the antioxidant products are microbial agents or animal feed additives; the amount of animal feed additive added is such that the viable count of porcine-derived Bacillus in the feed is 0.5 × 10⁻⁶. 7 ~2×10 7 CFU / g; The porcine-derived Bacillus subtilis YB-X-721, classified as Bacillus subtilis, was deposited at the China General Microbiological Culture Collection Center on November 10, 2023, with accession number CGMCC No. 28942; the 16S rDNA sequence of the porcine-derived Bacillus is shown in SEQ ID NO.

1. The antioxidant product contains Bacillus subtilis YB-X-721, fermentation supernatant of Bacillus subtilis YB-X-721, or a compound fermentation supernatant of Bacillus subtilis YB-X-721 and Bacillus tequilensis YB-2. The method for preparing the supernatant of the combined fermentation of Bacillus subtilis YB-X-721 and Bacillus tequilensis YB-2 includes the following steps: Step 1: Mix corn gluten meal, quercetin, and PBS buffer, place in a sweep-frequency pulse sonication device, set the sonication parameters, and perform sweep-frequency pulse sonication. Then add konjac glucomannan and continue sweep-frequency pulse sonication to obtain a nutrient solution. Add the nutrient solution to LB liquid culture medium, stir well, and autoclave at 121°C for 20 minutes to obtain the fermentation medium. The mass-to-volume ratio of corn gluten meal, quercetin, konjac glucomannan, and PBS buffer is 5-15. g:1~3g:0.5~2g:50~300mL; the nutrient solution is added to LB liquid culture medium at 10~30% (v / v); the parameters of the swept-frequency pulsed ultrasound treatment are: temperature 15~30℃, ultrasound power 400~800W, frequency 22~40kHz, swept-frequency amplitude ±0.5~±2kHz, swept-frequency period 100~500ms, pulsed ultrasound time 100~300s, interval time 5~15s, treatment time 5~30min; Step 2: Inoculate Bacillus subtilis YB-X-721 strain at 1~3% (v / v) into fermentation medium, incubate at 37℃ for 24h, centrifuge at 8000g for 10min, collect the supernatant at 4℃, and obtain YB-X-721 fermentation supernatant. Step 3: Inoculate Bacillus tequilensis YB-2 strain at 1~3% (v / v) into fermentation medium, incubate at 37℃ for 24h, centrifuge at 8000g for 10min, collect the supernatant at 4℃, and obtain YB-2 fermentation supernatant; Step 4: Mix the fermentation supernatant of YB-X-721 and the fermentation supernatant of YB-2 at a volume ratio of 2~5:1 to obtain a compound fermentation supernatant.

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