A Rongchang pig-derived Lactobacillus reuteri and its application in piglet intestinal anti-oxidation

Abstract

The present invention discloses a Rongchang pig-source Lactobacillus reuteri and its application in piglet intestinal antioxidant treatment. The Lactobacillus reuteri is classified and named Lactobacillus reuteri RS‑X‑811, which was registered and preserved in the General Microbiology Center of China National Microbiological Culture Collection on November 23, 2023, with the preservation number CGMCC No.29110. The present invention uses fresh feces of Chongqing local Rongchang pigs as samples, screens out a Lactobacillus reuteri RS-X-811, which has a certain inhibitory effect on Escherichia coli, Staphylococcus aureus and Salmonella, and has excellent acid resistance and choline resistance. In addition, the fermentation supernatant, bacterial suspension and cell-free extract of RS-X-811 have certain DPPH free radical, superoxide anion free radical, hydroxyl free radical scavenging ability and certain reducing ability. Adding RS-X-811 to piglet feed can significantly promote the intestinal development of weaned piglets, improve the absorption capacity of the intestine, and also improve the antioxidant capacity of the intestine of weaned piglets.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to a Rongchang pig-derived Lactobacillus reuteri and application thereof in piglet intestinal antioxidant activity. Background Art

[0002] When piglets are weaned, the conversion of food types and the oxidative stress response it triggers will cause a decline in the intestinal health function of piglets and a significant change in the composition of intestinal flora within a certain period of time. During this period, the diarrhea rate of piglets increased significantly, the growth performance decreased, and the number and proportion of probiotics such as lactic acid bacteria decreased significantly, all of which would affect the health and growth of pigs. Improving and maintaining intestinal health and promoting the stability and balance of intestinal microecological flora are one of the key factors to ensure the rapid growth of piglets in the early stage. In order to reduce the occurrence of diseases during breeding, antibiotics are used in large quantities in production, but long-term use of antibiotics destroys the balance of intestinal microbial flora in piglets, reduces the body's immune ability, and produces drug resistance to pathogens. Probiotics have become one of the best choices for "antibiotic replacement" due to their advantages such as non-toxicity, no residue, not easy to develop drug resistance, anti-intestinal infection and immune promotion. Therefore, the development of efficient and green antibiotic substitutes that can improve the antioxidant capacity of piglets is an urgent problem to be solved in the breeding industry. Summary of the invention

[0003] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0004] In order to achieve these objectives and other advantages of the present invention, a Rongchang pig-derived Lactobacillus reuteri is provided, which is classified and named Lactobacillus reuteri RS-X-811, and was registered and preserved in the General Microbiological Center of China National Microbiological Culture Collection on November 23, 2023, with a preservation number of CGMCC No.29110.

[0005] Preferably, the Rongchang pig-derived Lactobacillus reuteri is derived from fresh feces of Rongchang pigs.

[0006] Preferably, the 16S rDNA sequence of the Rongchang pig-derived Lactobacillus reuteri is as shown in SEQ ID NO.1.

[0007] A use of the Rongchang pig-derived Lactobacillus reuteri as described above in inhibiting Escherichia coli, Staphylococcus aureus or Salmonella.

[0008] A use of the Rongchang pig-derived Lactobacillus reuteri as described above in the preparation of acid-resistant and / or bile-resistant products.

[0009] Preferably, the survival rate of the Rongchang pig-derived Lactobacillus reuteri is 115.62% after being in an environment of pH = 2 for 4 hours; the survival rate of the Rongchang pig-derived Lactobacillus reuteri is 124.71% after being in an environment of pH = 3 for 4 hours; the survival rate of the Rongchang pig-derived Lactobacillus reuteri is 148.33% after being in an environment of 0.15% bile salt for 4 hours; the survival rate of the Rongchang pig-derived Lactobacillus reuteri is 66.63% after being in an environment of 0.3% bile salt for 4 hours.

[0010] An application of the Rongchang pig-derived Lactobacillus reuteri as described above in promoting intestinal development in piglets.

[0011] Preferably, the Rongchang pig-derived Lactobacillus reuteri is used to prepare a medicine or feed additive for promoting intestinal development in piglets.

[0012] Preferably, the feed additive is added in an amount such that the number of live Lactobacillus reuteri from Rongchang pigs in the feed is 0.5 to 2×10 7 CFU / g.

[0013] An application of the Rongchang pig-derived Lactobacillus reuteri as described above in the intestinal antioxidant effect of piglets.

[0014] Preferably, the Rongchang pig-derived Lactobacillus reuteri is used to prepare a medicine or feed additive for improving the antioxidant capacity of the piglet intestine.

[0015] Preferably, the feed additive is added in an amount such that the number of live Lactobacillus reuteri from Rongchang pigs in the feed is 0.5 to 2×10 7 CFU / g.

[0016] Preferably, the strain is fermented and cultured using a modified MRS liquid medium to prepare a fermentation supernatant with high antioxidant activity, and the specific steps include:

[0017] Step 1, adding Bletilla striata polysaccharide to MRS liquid culture medium at a mass volume ratio of 1-3 g:1 L, performing frequency sweep pulse ultrasonic treatment for 8-15 min, and sterilizing at 121° C. for 20 min to obtain a modified MRS liquid culture medium; wherein the frequency sweep pulse ultrasonic treatment parameters are: temperature 20-30° C., ultrasonic power 500-800 W, frequency 68 kHz, frequency sweep width ±1-2 kHz, frequency sweep period 100-500 ms, pulse ultrasonic time 100-200 s, and interval time 3-10 s;

[0018] Step 2: The strain was inoculated into a modified MRS liquid medium at 2% (v / v), cultured at 37°C for 24 h, centrifuged at 8000 g for 10 min at 4°C, and the supernatant and bacterial cells were collected separately. The supernatant was the fermentation supernatant.

[0019] The present invention at least includes the following beneficial effects: the present invention uses fresh feces of Chongqing local Rongchang pigs as samples to screen out a strain of Lactobacillus reuteri RS-X-811, which has a certain inhibitory effect on Escherichia coli, Staphylococcus aureus and Salmonella, and has excellent acid resistance and choline resistance; in addition, the fermentation supernatant, bacterial suspension and cell-free extract of RS-X-811 have a certain DPPH free radical, superoxide anion free radical, hydroxyl free radical scavenging ability and a certain reducing ability; adding RS-X-811 to the feed of piglets can increase the height of the villi of the small intestine of weaned piglets and reduce the depth of the crypts, which can significantly promote the intestinal development of weaned piglets and improve the absorption capacity of the intestine. At the same time, it can also increase the activity of superoxide dismutase and glutathione peroxidase in the small intestinal mucosa of piglets, reduce the content of malondialdehyde, and improve the antioxidant capacity of the intestine of weaned piglets.

[0020] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a comparison chart of the inhibition zones of Rongchang pig-derived Lactobacillus reuteri RS-X-811, RS-M-123, RS-X-615 and RS-X-123 of the present invention, wherein: A is Escherichia coli; B is Salmonella; C is Staphylococcus aureus; 1: RS-X-811; 2: RS-M-123; 3: RS-X-615; 4: RS-X-123;

[0022] Figure 2 This is a Gram staining image of Rongchang pig-derived Lactobacillus reuteri RS-X-811 of the present invention;

[0023] Figure 3 The genetic evolution tree of Rongchang pig-derived Lactobacillus reuteri RS-X-811 of the present invention;

[0024] Figure 4 This is the growth curve of Rongchang pig-derived Lactobacillus reuteri RS-X-811 of the present invention. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0026] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0027] In the following embodiments:

[0028] MRS solid medium: peptone 10g, beef extract 10g, yeast powder 5g, K2HPO4 2g, diammonium citrate 2g, sodium acetate 5g, glucose 20g, Tween-80 1mL, MgSO4·7H2O 0.58g, MnSO4·4H2O 0.25g, agar 18g, distilled water 1000mL;

[0029] MRS liquid culture medium: peptone 10 g, beef extract 10 g, yeast powder 5 g, K2HPO4 2 g, diammonium citrate 2 g, sodium acetate 5 g, glucose 20 g, Tween-80 1 mL, MgSO4·7H2O 0.58 g, MnSO4·4H2O 0.25 g, distilled water 1000 mL.

[0030] Example 1

[0031] A Rongchang pig-source Lactobacillus reuteri, characterized in that its classification name is Lactobacillus reuteriRS-X-811, which has been registered and preserved in the General Microbiological Center of China National Microbiological Culture Collection on November 23, 2023, with a preservation number of CGMCC No.29110.

[0032] 1. Screening and separation of porcine lactic acid bacteria

[0033] Fresh pig manure from Chongqing Rongchang pigs was used as a sample. 0.1 g of pig manure was aseptically weighed and gradiently diluted with PBS solution. The dilution was evenly spread on MRS solid culture medium containing calcium carbonate and cultured at 37°C for 24 h. A single colony with a calcium-dissolving circle was picked and streaked into MRS solid culture medium and cultured at 37°C for 24 h. This process was repeated for three times until a pure strain was obtained. Four Gram-positive lactic acid bacteria were initially isolated and numbered RS-X-811, RS-M-123, RS-X-615 and RS-X-123.

[0034] 2. Screening of antagonistic pathogenic bacteria characteristics of porcine lactic acid bacteria

[0035] After the sterilized MRS broth solid medium is cooled to about 50°C, pour it into a sterile culture dish. After the plate is naturally dried, transfer 0.1 mL of indicator bacteria solution (10 8 CFU / mL) was evenly spread on the plate, and after natural drying, a hole was punched on the plate, and 0.1 mL of the test bacterial solution (10 8CFU / mL), let it stand, and after the bacterial solution penetrated into the plate, culture it at 37℃ for 24h, measure the size of the inhibition zone, and the indicator bacteria are Escherichia coli, Staphylococcus aureus and Salmonella respectively; through the inhibition of Escherichia coli, Staphylococcus aureus and Salmonella, two lactic acid bacteria with good antibacterial effect were initially screened, namely RS-X-811 and RS-M-123, among which RS-X-811 had the best antibacterial effect. The antibacterial results are shown in Table 1, Figure 1 The dominant strains that passed the antibacterial test were preserved for the next step of rescreening for tolerance to the digestive tract environment.

[0036] Table 1

[0037]

[0038] Through the above-mentioned screening of the antagonistic pathogenic bacteria characteristics of porcine lactic acid bacteria, it can be known that the Rongchang porcine Lactobacillus reuteri RS-X-811 can be used in the preparation of products that inhibit Escherichia coli, Staphylococcus aureus or Salmonella.

[0039] 3. Screening of acid and bile resistance of porcine lactic acid bacteria

[0040] Bile salt tolerance test:

[0041] RS-X-811 and RS-M-123 were inoculated into MRS liquid culture medium respectively. After culturing for 12-24 h, the bacterial solution was inoculated into MRS liquid culture medium with different bile salt levels (0.15% and 0.3%) at a ratio of 5% (v / v) and placed at 37°C for 4 h. 100 μL of bacterial solution was diluted 10-fold at 0, 2, and 4 h, and 10 -5 The gradient dilutions were spread on MRS plates, cultured at 37°C for 24 h, and the colonies were counted; each sample was replicated 3 times.

[0042] Survival rate (%) = "2h (or 4h)" colony count / "0h" colony count × 100%

[0043] Acid resistance test:

[0044] RS-X-811 and RS-M-123 were inoculated into MRS liquid culture medium respectively. After culturing for 12-24 hours, the bacterial solution was inoculated into MRS liquid culture medium with pH of 2 and 3 at a ratio of 5% (v / v) and placed at 37°C for 4 hours. 100 μL of bacterial solution was diluted 10-fold at 0, 2 and 4 hours respectively. 10 -5 The gradient dilutions were spread on MRS plates, cultured at 37°C for 24 h, and the colonies were counted; each sample was replicated 3 times.

[0045] Survival rate (%) = "2h (or 4h)" colony count / "0h" colony count × 100%

[0046] In order to further screen the candidate strains for tolerance to the digestive tract environment, an acid and bile salt resistance experiment was conducted, and the experimental results are shown in Table 2. In the environment of pH 2 and 3, the survival rate of RS-X-811 was the highest. In summary, RS-X-811 has the best acid resistance, and the survival rate after 4 hours is greater than 100%, indicating that RS-X-811 has a certain growth in this environment; in the environment of 0.15% bile salt and 0.3% bile salt, RS-X-811 has the best tolerance, among which RS-X-811 has a 2h and 4h survival rate in the environment of 0.15% bile salt greater than 100%, indicating that RS-X-811 has a certain growth in this environment. Based on the results of acid and bile salt resistance, RS-X-811 has good acid and bile salt resistance characteristics, and is determined as a target strain, which can be used in the preparation of acid-resistant and / or bile salt-resistant products.

[0047] Table 2

[0048]

[0049]

[0050] Example 2

[0051] Identification of Lactobacillus RS-X-811 from pigs:

[0052] 1. Morphological identification

[0053] In a sterile operating table, the purified RS-X-811 was streaked on an MRS plate and cultured at 37°C for 24 hours. The size, shape, glossiness, color, transparency and other characteristics of the single colony were observed and recorded. All isolates were subjected to Gram staining. The staining steps were referred to the staining kit, and the bacterial morphology was observed and recorded under a microscope.

[0054] The colony morphology of RS-X-811 strain is shown in Table 3; Gram staining observation shows that the screened lactic acid bacteria are rod-shaped, such as Figure 2 shown.

[0055] Table 3

[0056] surface color Raised / flat shape edge Viscosity Diameter(mm) transparency RS-X-811 smooth cream Raised Round rule Not sticky 1.0-1.5 opaque

[0057] 2. Identification of physiological and biochemical characteristics

[0058] The physiological and biochemical characteristics of RS-X-811 strain were tested according to the steps in the biochemical detection kit, and the physiological and biochemical characteristics of the strain are shown in Table 4. With reference to the "Common Bacterial System Identification Manual" and the "Berger's Bacterial Identification Manual" for comparative analysis, RS-X-811 was preliminarily determined to be a lactic acid-producing Lactobacillus bacterium.

[0059] Table 4

[0060] project result project result Catalase - Sorbitol - VP reaction - Mannitol - Methyl red reaction + glucose + Starch hydrolysis - Raffinose + Cellobiose - sucrose - maltose + gelatin -

[0061] 3. Molecular Biology Identification

[0062] PCR amplification was performed using the bacterial identification 16S rDNA universal primers 27F and 1492R. The primer sequences are shown in Table 5. The primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.;

[0063] Table 5

[0064]

[0065]

[0066] The PCR amplification template was fresh bacterial solution after activation, the system was 50 μL, and the reaction system was shown in Table 6;

[0067] Table 6

[0068]

[0069] The PCR amplification program was as follows: 95°C for 5 min; 95°C for 45 s; 55°C for 45 s; 72°C for 1 min; repeated for 35 cycles; 72°C for 8 min. The PCR amplification products were identified by 1% agarose gel electrophoresis and sequenced at Shanghai Bio-Technology Co., Ltd., and BLAST comparison analysis was performed in GenBank to draw a genetic evolution tree, such as Figure 3 The results showed that RS-X-811 and the reference strain Lactobacillus reuteri (MW 866759) were on the same branch and had the closest affinity, so RS-X-811 was determined to be Lactobacillus reuteri. The 16SrDNA sequence of Rongchang pig-derived Lactobacillus reuteri RS-X-811 obtained by sequencing was shown in SEQ ID NO.1.

[0070] Example 3

[0071] Analysis on the biological characteristics of Lactobacillus reuteri RS-X-811 from Rongchang pigs

[0072] After the first generation of bacterial activation, the inoculum was inoculated into 100 mL of MRS liquid medium at a rate of 2% (v / v), and cultured at 37°C with shaking. Every 4 hours, 5 mL of bacterial solution was taken for testing, and the concentration (OD value) of the bacterial solution was measured at a wavelength of 600 nm using an ultraviolet spectrophotometer, and the data were recorded for a total of 24 hours. Figure 4 As shown, by measuring the growth curve of Rongchang pig-derived Lactobacillus reuteri RS-X-811, it can be found that RS-X-811 enters the logarithmic growth phase after 2 hours and enters the stable phase after 10 hours.

[0073] Example 4

[0074] Application of Rongchang pig-derived Lactobacillus reuteri RS-X-811 in anti-oxidation

[0075] In vitro antioxidant activity of Rongchang pig-derived Lactobacillus reuteri RS-X-811 of the present invention:

[0076] 1. Preparation of bacterial solution and in vitro antioxidant assay

[0077] 1. Reagents

[0078] Anhydrous ethanol, potassium ferricyanide, PBS buffer, acetic acid, ferric chloride, ascorbic acid, hydrogen peroxide, ferrous sulfate, 3,5-dinitrosalicylic acid (DNS), pyrogallol, tris(hydroxymethylaminomethane)-hydrochloric acid buffer solution (Tris), phosphate buffer, o-phenanthroline solution, all of the above reagents were of analytical grade.

[0079] 2. Extraction of sample bacterial solution

[0080] The different components of the strain were prepared according to the method of Gao Li'e (Gao Li'e. Study on the Diversity and Antioxidant Properties of Lactic Acid Bacteria in Traditional Fermented Yak Milk on the Qinghai-Tibet Plateau [D]. Lanzhou University, 2020. DOI: 10.27204 / d.cnki.glzhu.2020.002996.). The strain was inoculated in MRS liquid medium at 2% (v / v), cultured at 37°C for 24h, centrifuged at 8000g for 10min, and the supernatant and bacteria were collected separately at 4°C. The supernatant was the fermentation supernatant (FS). The obtained bacteria were washed 2 to 3 times with sterile water, resuspended in PBS, and the density was adjusted to 1×10 9 CFU / mL, and it was equally divided into two parts, one part was used as bacterial suspension (IC), and the other part was ultrasonically disrupted in an ice bath (250 W, ultrasonic for 5 s, interval of 10 s, total 10 min), centrifuged at 8000 g for 10 min, and the supernatant was collected to obtain the cell-free extract (CFE).

[0081] 3. In vitro antioxidant activity assay

[0082] 3.1 Determination of DPPH free radical scavenging ability

[0083] Take 1 mL of the sample and place it in a test tube. Add 2 mL of DPPH anhydrous ethanol solution (concentration is 0.2 mmol / L). After mixing, react at room temperature in the dark for 30 min. Centrifuge at 8000 g for 10 min at 4°C, take the supernatant, measure its absorbance at 517 nm, adjust to zero with deionized water, repeat 3 times for each group, and calculate the average value.

[0084] DPPH removal rate (%) = [1-(A sample-A blank) / A control] × 100%

[0085] (Blank group: DPPH was replaced by an equal volume of anhydrous ethanol; control group: the sample solution was replaced by an equal volume of distilled water, and the blank was adjusted to zero with an equal volume of a mixture of distilled water and anhydrous ethanol)

[0086] Liquid preparation: 0.2mmol / L DPPH anhydrous ethanol solution: weigh 50mg DPPH, add 634mL anhydrous ethanol, and shake well.

[0087] 3.2 Determination of hydroxyl radical (·OH) scavenging activity (Fenton method)

[0088] The Fenton method uses H2O2 and Fe 2+ The mixture undergoes a Fenton reaction, generating ·OH with high reactivity, and salicylic acid can capture ·OH and produce a colored substance (dihydroxybenzoic acid), which has a maximum absorption at 510nm. However, if a certain amount of a substance with scavenging ability is added, it will compete with the reaction of salicylic acid, resulting in a decrease in the amount of colored substance (dihydroxybenzoic acid) produced, causing a certain change in the absorbance of the solution. The activity of the antioxidant can then be measured based on the change in absorbance.

[0089] H2O2+Fe 2+ → OH+OH - +Fe 3+

[0090] Liquid preparation: ①FeSO4 solution: accurately weigh 0.05g of ferrous sulfate heptahydrate and place it in a 100mL volumetric flask, add distilled water to the mark, and shake well to obtain a 1.8mmol / L ferrous sulfate solution;

[0091] 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, shake well and stabilize to obtain a 1.8 mmol / L salicylic acid-ethanol solution;

[0092] ③0.3% H2O2 solution: Accurately measure 1.0mL of 30% hydrogen peroxide and place it in a 100mL volumetric flask, add distilled water to the scale, and mix well to obtain 0.3% H2O2 solution.

[0093] The specific steps are as follows: add 2mL of 1.8mmoL / L FeSO4 solution, 1mL of sample, and 0.1mL of 0.3% H2O2 solution to the test tube in turn, shake well, then add 1.5mL of 1.8mmol / L salicylic acid-ethanol solution, shake well, let stand at 30℃ for 30min, and measure the sample group absorbance Ai at 510nm; replace the H2O2 solution in the sample group with an equal amount of distilled water, react, and use it as the sample reference group and measure its absorbance Aj; replace the sample in the sample group with an equal amount of distilled water, react, and use it as the blank control group to measure the absorbance Ao. Calculate the hydroxyl radical scavenging rate according to the following formula:

[0094] Hydroxyl free radical scavenging rate (%) = [Ao-(Ai-Aj) / Ao] × 100%

[0095] 3.3 Superoxide anion free radical (O2 - ·) Determination of scavenging activity (pyrogallol method)

[0096] Pyrogallol is very easy to self-oxidize in an alkaline environment, producing colored intermediates and superoxide anions O2 - ·, O2 - It also catalyzes the self-oxidation reaction, and the amount of O2 can be determined based on the amount of colored substances produced. - When adding substances with antioxidant activity, the self-oxidation of pyrogallol can be weakened, so that the antioxidant activity can be judged by the trend of reducing colored substances.

[0097] Liquid preparation: ① Pyrogallol: Accurately weigh 0.315g of pyrogallol and place it in a 100mL volumetric flask, then add 10mL of 0.1mol / L hydrochloric acid, and finally add distilled water to the scale, shake well, and you will get 25mmol / L of pyrogallol; pyrogallol should be prepared and used immediately, and react at 20℃;

[0098] ②Tris-HCl: Accurately weigh 3.0285g of Tris reagent and place it in a 500mL volumetric flask. Then add 114.5mL of 0.1mol / L hydrochloric acid. Finally, add distilled water to the scale and shake well to obtain 0.05mol / L Tris-HCl buffer.

[0099] The specific steps are as follows: take out the test tube and add 7.5mL 0.05mol / L Tris-HCl buffer solution, place it in a 25℃ water bath to preheat for 20min, then add 1mL sample solution and 0.5mL 25mmol / L pyrogallol solution respectively, mix them and place them in a 25℃ water bath to react for 5min, add 1mL concentrated HCl to terminate the reaction, and use the sample group to measure the absorbance Ai of the solution at 320nm wavelength under a UV-visible spectrophotometer; the sample reference group uses the same volume of distilled water to replace the pyrogallol solution and measure the absorbance Aj of the solution; the blank control group uses the same volume of distilled water to replace the sample solution and measure the absorbance Ao of the solution. Calculate O2 according to the following formula - Clearance rate:

[0100] Superoxide anion free radical scavenging rate (%) = [Ao-(Ai-Aj)] / Ao×100%

[0101] 3.4 Determination of reducing capacity

[0102] Liquid preparation: ① 0.2 mol / L, pH 6.6 phosphate buffer solution: first prepare 0.2M Na2HPO4 (17.19 g Na2HPO4·12H2O+240 mL H2O) and 0.2M NaH2PO4 (11.23 g NaH2PO4·2H2O+360 mL H2O) of solution A and solution B, respectively; take 40 mL of solution A and 60 mL of solution B, and mix them to obtain a 0.2 mol / L, pH 6.6 phosphate buffer solution;

[0103] ②1% potassium ferrocyanide: 1g potassium ferrocyanide + 100mL H2O;

[0104] ③10% trichloroacetic acid: 1 mL trichloroacetic acid + 9 mL H2O;

[0105] ④0.1% ferric chloride: 0.1g ferric chloride + 100mL H2O.

[0106] Take 0.5mL of sample and place it in a test tube, add 0.5mL of phosphate buffer solution with a concentration of 0.2mol / L and pH 6.6, then add 0.5mL of 1% potassium ferricyanide, place in a 50℃ water bath for 20min, then quickly cool in an ice bath, then add 0.5mL of 10% trichloroacetic acid, centrifuge at 4000r / min for 10min, take 1mL of supernatant, add 1mL of distilled water and 1mL of 0.1% ferric chloride, mix well, place at room temperature for 10min, measure its absorbance at a wavelength of 700nm, repeat 3 times for each sample, and calculate the average value. The larger the absorbance, the stronger the reducing ability of the sample to be tested.

[0107] The results are shown in Table 7. The fermentation supernatant (FS), bacterial suspension (IC) and cell-free extract (CFE) of Rongchang porcine Lactobacillus reuteri RS-X-811 all have certain DPPH free radical, superoxide anion free radical, hydroxyl free radical scavenging ability and certain reducing ability, indicating that the strain has certain antioxidant activity in vitro.

[0108] Table 7

[0109]

[0110]

[0111] Example 5

[0112] Step 1, adding Bletilla striata polysaccharide to MRS liquid culture medium at a mass volume ratio of 2g:1L, performing frequency sweep pulse ultrasonic treatment for 10min, and sterilizing at 121°C for 20min to obtain improved MRS liquid culture medium; wherein the frequency sweep pulse ultrasonic treatment parameters are: temperature 25°C, ultrasonic power 600W, frequency 68kHz, frequency sweep width ±2kHz, frequency sweep period 200ms, pulse ultrasonic time 100s, and interval time 5s;

[0113] Step 2: The strain was inoculated into a modified MRS liquid medium at 2% (v / v), cultured at 37°C for 24h, centrifuged at 8000g for 10min, and then at 4°C, the supernatant and the cells were collected respectively, and the supernatant was the fermentation supernatant; the rest of the method was the same as in Example 4;

[0114] The preparation method of Bletilla striata polysaccharide comprises the following steps: placing Bletilla striata tubers in a freezer and freezing at -15°C for 4 hours; crushing the Bletilla striata tubers at a temperature of -15°C and a pressure of 0.1 MPa; spray drying, passing through a 100-mesh sieve, and obtaining Bletilla striata powder; taking 20 parts of Bletilla striata powder by weight and adding them to 800 parts of distilled water, stirring at 100°C and 500 rpm for 60 minutes for the first extraction, centrifuging at 3000 rpm for 10 minutes, collecting the supernatant, adding the solid residue to 600 parts of distilled water, stirring at 100°C and 500 rpm for 60 minutes for the second extraction, and centrifuging at 3000 rpm for 10 minutes. 000rpm for 10 min, collect the supernatant, add the solid residue into 600 parts of distilled water, stir at 100℃ and 500rpm for 60min for the third extraction, centrifuge at 3000rpm for 10min, collect the supernatant, combine the supernatants obtained three times, and perform rotary evaporation to 100 parts of the supernatant, then add 3 times the volume of anhydrous ethanol to the supernatant after rotary evaporation for alcohol precipitation for 2h, centrifuge at 3000rpm for 10min, discard the supernatant, pre-freeze, freeze-vacuum dry, and crush at 10000rpm for 3min to obtain Bletilla striata polysaccharide.

[0115] In this embodiment, the MRS liquid culture medium is improved by adding Bletilla striata polysaccharide. The improved MRS liquid culture medium is more suitable for the growth and metabolism of Rongchang pig-derived Lactobacillus reuteri RS-X-811, metabolizes products with high antioxidant activity, and improves the antioxidant activity of the fermentation supernatant. At the same time, swept frequency pulse ultrasound is used to improve the uniformity and dispersion of various substances in the culture medium, which is conducive to full contact of microorganisms.

[0116] Example 6

[0117] Step 1, adding Bletilla striata polysaccharide into MRS liquid culture medium at a mass volume ratio of 2g:1L, stirring evenly, and sterilizing by high pressure at 121°C for 20min to obtain a modified MRS liquid culture medium;

[0118] Step 2: The strain was inoculated into a modified MRS liquid medium at 2% (v / v), cultured at 37°C for 24h, centrifuged at 8000g for 10min, and at 4°C, the supernatant and the cells were collected separately, and the supernatant was the fermentation supernatant; the rest of the method was the same as in Example 4.

[0119] The in vitro antioxidant activity test results of the fermentation supernatants of Examples 5-6 are shown in Table 8. It can be seen that the DPPH free radical, superoxide anion free radical, hydroxyl free radical scavenging ability and reducing ability of the fermentation supernatants of Examples 5-6 are higher than those of Example 4, among which the fermentation supernatant of Example 5 has the highest antioxidant activity.

[0120] Table 8

[0121] Antioxidant index Example 4 Example 5 Example 6 DPPH free radical scavenging rate (%) 88.89±1.64 93.26±1.22 91.65±2.05 Superoxide anion free radical scavenging rate (%) 70.57±9.95 78.35±3.58 75.43±5.30 Hydroxyl free radical scavenging rate (%) 75.98±2.88 80.76±2.17 78.37±3.42 Reducing ability (OD700) 0.066±0.002 0.078±0.003 0.070±0.002

[0122] Example 7

[0123] Application of Rongchang pig-derived Lactobacillus reuteri RS-X-811 in promoting intestinal development of piglets and in anti-oxidation of piglet intestines

[0124] 1. Preparation of probiotic solution

[0125] The strain was inoculated into MRS solid medium for activation for three generations, and RS-X-811 probiotic solution was prepared using a 20L automatic fermentation tank (37°C, pH=6.0-7.0, 24h). The number of viable bacteria in the produced bacterial solution was calculated by the plate count method. The number of viable bacteria of Rongchang pig-derived Lactobacillus RS-X-811 in the sample was 1×10 10 CFU / g.

[0126] 2. Animal Experimentation

[0127] Eighteen healthy Rongchang local piglets with an average weight of 6.5-7.0 kg and 28 days of weaning were selected. Based on the principle of similar weight and consistent ratio of male and female piglets, the experimental piglets were randomly divided into three groups, with one pen in each group and 6 piglets in each pen, half male and half female. The control group was fed a basic diet, and the RS-X-811 group was fed a basic diet with RS-X-811 probiotic solution added to the basic diet to adjust the number of live bacteria in the feed to 1×10 7 CFU / g, lactic acid bacteria group of a certain manufacturer added lactic acid bacteria of a certain manufacturer (Yihong Bio, lactic acid bacteria) to the basic diet to adjust the number of viable bacteria in the feed to 1×10 7 CFU / g, pre-feeding period of 3 days, formal test period of 28 days. The test pigs were fed and managed according to the routine pig farm, and the immunization, deworming and disinfection procedures during the test were carried out according to the normal procedures of the pig farm. At the end of the test feeding, 5mL of blood was collected from the anterior vena cava of each group, placed in a serum tube for coagulation, centrifuged at 3000r / min for 10min, and the serum was separated and stored at -20℃ for testing.

[0128] 3. Detection of small intestinal tissue development

[0129] The collected samples of each segment of the piglet small intestine were fixed in paraformaldehyde, then embedded and sectioned, and stained with HE. The height of intestinal villi and the depth of crypts were observed and measured, and the ratio of villus height to crypt depth was calculated.

[0130] 4. Determination of antioxidant capacity

[0131] The collected piglet small intestinal mucosal samples were tested using an antioxidant capacity index detection kit, and a total of three indicators were measured: superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and malondialdehyde (MDA).

[0132] 5. Results Analysis

[0133] 5.1 Effect of dietary supplementation of Rongchang pig-derived Lactobacillus reuteri RS-X-811 on intestinal development in weaned Rongchang piglets

[0134] As shown in Table 9, the addition of RS-X-811 to the diet increased the height of the villi in the small intestine of weaned piglets (P<0.05) and reduced the depth of the crypts, but the difference was not significant (P>0.05). In addition, the addition of RS-X-811 to the diet significantly increased the villus height / crypt depth values ​​of the jejunum and ileum of piglets (P<0.05). This shows that Rongchang pig-derived Lactobacillus reuteri RS-X-811 can significantly promote the intestinal development of weaned piglets and improve the absorption capacity of the intestine.

[0135] Table 9

[0136]

[0137] Note: Data in the same row do not contain the same lowercase letters, which indicates a significant difference (P<0.05); data with the same letters or no letters indicate no significant difference (P>0.05); the same as in the following table.

[0138] 5.2 Effect of dietary supplementation of Rongchang pig-derived Lactobacillus reuteri RS-X-811 on intestinal antioxidant capacity of weaned Rongchang piglets

[0139] Biochemical indices related to oxidative stress in the small intestinal mucosa were detected, among which SOD and GSH-Px were indicators of antioxidant capacity, and MDA was an indicator of oxidative damage.

[0140] As shown in Table 10, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in the small intestinal mucosa of piglets in the RS-X-811 group and the lactic acid bacteria group of a certain manufacturer were higher than those in the control group, while the malondialdehyde (MDA) content in the small intestinal mucosa of the two experimental groups was significantly lower than that in the control group (P<0.05), and there was no significant difference in these indicators between the RS-X-811 group and the lactic acid bacteria group of a certain manufacturer. This shows that Rongchang pig-derived Lactobacillus reuteri RS-X-811 improves the antioxidant capacity of the intestine of weaned piglets and achieves the effect of existing commercial probiotic lactobacilli.

[0141] Table 10

[0142]

[0143] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A Rongchang pig-derived Lactobacillus reuteri, characterized in that: Its classification name is Lactobacillus reuteriRS-X-811, and it was registered and deposited at the General Microbiology Center of China Culture Collection Administration on November 23, 2023, with the deposit number CGMCC No.29110.

2. The Rongchang pig-derived Lactobacillus reuteri according to claim 1, characterized in that: The Rongchang pig-derived Lactobacillus reuteri is derived from fresh feces of Rongchang pigs.

3. The Rongchang pig-derived Lactobacillus reuteri according to claim 1, characterized in that: The 16S rDNA sequence of the Rongchang pig-derived Lactobacillus reuteri is shown in SEQ ID NO.

1.

4. A use of the Rongchang pig-derived Lactobacillus reuteri as claimed in claim 1 in the preparation of a medicine or feed additive for promoting intestinal development in piglets.

5. A use of the Rongchang pig-derived Lactobacillus reuteri as claimed in claim 1 in the preparation of a medicine or feed additive for improving the intestinal antioxidant capacity of piglets, characterized in that: The Rongchang pig-derived Lactobacillus reuteri is fermented and cultured using a modified MRS liquid culture medium to prepare a fermentation supernatant with high antioxidant activity.

6. The use according to claim 4, characterized in that The amount of feed additive added is 0.5~2×10 7 CFU / g.

7. The use according to claim 5, characterized in that The specific steps of preparing the fermentation supernatant with high antioxidant activity include: Step 1, adding Bletilla striata polysaccharide to MRS liquid culture medium at a mass volume ratio of 1-3 g:1 L, performing frequency sweep pulse ultrasonic treatment for 8-15 min, and sterilizing at 121° C. and high pressure for 20 min to obtain a modified MRS liquid culture medium; wherein the frequency sweep pulse ultrasonic treatment parameters are: temperature 20-30° C., ultrasonic power 500-800 W, frequency 68 kHz, frequency sweep width ±1-2 kHz, frequency sweep period 100-500 ms, pulse ultrasonic time 100-200 s, and interval time 3-10 s; Step 2: Rongchang pig-derived Lactobacillus reuteri strain was inoculated into modified MRS liquid culture medium at 2% (v / v), cultured at 37°C for 24h, centrifuged at 8000g for 10min, and collected the supernatant and bacteria at 4°C, respectively. The supernatant was the fermentation supernatant, which was used to prepare drugs or feed additives for improving the intestinal antioxidant capacity of piglets.

8. The use according to claim 5, characterized in that The amount of feed additive added is 0.5~2×10 7 CFU / g.

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