Porcine-derived gamma-aminobutyric acid-producing lactobacillus reuteri for relieving vomitoxin toxicity and application of lactobacillus reuteri

Abstract

The invention discloses an application of a product of Limosilbacillus reuteri LR1 (Limosilbacillus reuteri LR1) for producing gamma-aminobutyric acid (gamma-aminobutyric acid) from pigs in prevention and treatment of vomitoxin toxicity and a preparation method of the Limosilbacillus reuteri LR1. The strain is preserved in Guangdong Microbiological Culture Collection Center, the preservation number is GDMCC.No: 64522, and the preservation date is April 17, 2024. The strain is separated and purified from a pig cecum content sample, the performance of gamma-aminobutyric acid produced by fermentation of the strain is stable, and the yield of gamma-aminobutyric acid is 0.6 g / L. The strain has a good protection effect on prevention and treatment of vomitoxin toxicity, can be applied to preparation of products for prevention and treatment of animal vomitoxin toxicity and strains for fermentation production of GABA, can be applied to different fields of veterinary microecologics, feed production and the like, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a strain and its application, in particular to the application of a gamma-aminobutyric acid (GABA)-producing Lactobacillus reuteri LR1 from pig intestine in preventing and treating vomitoxin toxic products and feed additives, and belongs to the technical field of microorganisms. BACKGROUND

[0002] Deoxynivalenol (DON) is a mycotoxin produced by Fusarium and other fungi, belonging to the B-type trichothecene family of toxins, and is one of the most serious mycotoxins in feed pollution. When livestock and poultry ingest feed contaminated with DON, they may experience acute and chronic poisoning symptoms such as vomiting, anorexia, diarrhea, and weight loss. Pigs are the most sensitive animals to DON, and the intestinal tract is the primary target organ for DON. Currently, there is a lack of effective antidotes for DON. The widespread contamination of DON in feed has seriously threatened the healthy development of the pig farming industry. Therefore, effectively preventing and treating DON toxin contamination is of great significance for reducing the toxicity of DON to the intestinal tract of livestock and poultry and promoting the healthy breeding of livestock and poultry. Although current research can use physical, chemical, and biological methods to detoxify DON in feed, both physical and chemical methods have limitations. DON has stable physicochemical properties, and physical adsorption is not effective. Adsorption methods can also cause the loss of nutritional substances in feed. In order to preserve nutritional substances, there may be mycotoxin residues or even toxin concentration during the detoxification process of feed. Residual chemicals in the chemical method can cause side effects in animals and change the color and odor of feed. Using microbial strains to degrade or alleviate the toxicity of DON is considered a promising green control strategy. Lactic acid bacteria are important probiotics, and some lactic acid bacteria have the ability to produce gamma-aminobutyric acid (GABA). GABA has many functions such as increasing animal feed intake, improving brain function, and enhancing immune function. GABA-producing lactic acid bacteria are expected to become effective biological agents for preventing and treating the toxic damage of DON to livestock and poultry. Currently, there is a lack of GABA-producing lactic acid bacteria strains from pig intestinal tracts that can prevent and treat DON toxicity. SUMMARY

[0003] To solve the problem of the lack of GABA-producing lactic acid bacteria strains from pig intestinal tracts that can prevent and treat DON toxicity in the prior art, the present application provides a method for screening a GABA-producing lactic acid bacteria strain from pig intestinal tracts and its application in alleviating DON toxicity. The lactic acid bacteria are isolated from pig cecal contents, can metabolize glutamic acid to produce GABA, and significantly reduce the toxic effects of DON. The strain is classified and named as Lactobacillus reuteri LR1 (Lactobacillus reuteri LR1). Limosilactobacillus reuteriLactobacillus reuteri LR1 has been deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 64522, and the deposit date is April 17, 2024. The core of this invention lies in providing the application of Lactobacillus reuteri LR1 in the prevention and control of DON toxicity. This strain can maintain good growth under DON-containing conditions and continuously synthesize GABA, enabling its fermentation production of GABA. This strain exhibits good acid resistance, resistance to artificial gastrointestinal fluids, and safety characteristics, effectively preventing and controlling the toxic effects of vomitoxin in animals. It can be applied to various fields such as veterinary microecological preparations and feed production, showing broad application prospects. Attached Figure Description

[0004] Figure 1 This is a Gram staining image of Lactobacillus reuteri LR1; Figure 2 It is a 16S gene phylogenetic tree of Lactobacillus reuteri based on the 16S rRNA gene sequence; Figure 3 Figure a shows the growth curve and tolerance test results of Lactobacillus reuteri LR1; Figure b shows the survival rate of Lactobacillus reuteri LR1 after 4 hours of treatment with artificial gastric fluid and artificial small intestinal fluid; Figure c shows the survival rate of Lactobacillus reuteri LR1 after 4 hours of treatment with different pH culture media. Figure 4 Figure 1 shows the high-performance liquid chromatogram of Lactobacillus reuteri LR1 fermentation broth. Figure 2a is the chromatogram of GABA standard, and Figure 3b is the chromatogram of Lactobacillus reuteri LR1 fermentation broth. Figure 5 Lactobacillus reuteri LR1 can effectively alleviate the toxicity of vomiting toxin. Figure a shows the body weight of mice challenged with the toxin, Figure b shows the expression of the mouse intestinal tight junction protein Occludin, and Figure c shows the hematoxylin-eosin staining of the mouse jejunum and colon. Detailed Implementation

[0005] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0006] I. Screening and Identification of Lactobacillus reuteri LR1 1. Screening for Lactobacillus reuteri LR1 The *Lactobacillus reuteri* LR1 strain obtained by screening in this invention was isolated and purified from porcine cecal contents samples. The specific isolation and purification process is as follows: (1) Take 10 g of pig cecum content sample, then add the sample to 10 mL of 0.85% sterile NaCl solution to prepare a turbid liquid.

[0007] (2) The prepared turbid liquid is gradient diluted, then 100 μL of the diluted 10 5 times bacterial liquid is uniformly coated on MRS solid culture medium with a coating rod, and the MRS plate is inverted and cultured in a 37℃ anaerobic incubator for 24 h.

[0008] (3) Single colonies are randomly picked from the MRS plate, and the single colonies are inoculated on new MRS solid culture medium by plate streaking, inverted and cultured in a 37℃ anaerobic incubator for 24 h, and the single colonies at the last streaking site are selected for preservation and bacterial identification, which are round, medium-sized, convex in the middle, with neat edges, white and yellowish, and moist and opaque surface.

[0009] 2. Identification of Lactobacillus reuteri LR1 (1) Observation of colony morphology and cell shape Gram staining method: ① Initial staining: Add crystal violet dye to the fixed bacterial film, cover for 1 minute, and then rinse gently with water. Lactic acid bacteria start to be stained purple. ② Decolorization: This is a key step. Add 95% alcohol for decolorization, and control the time to be 20-30 seconds. After the flowing alcohol is basically colorless, wash with water immediately. Gram-positive bacteria (such as lactic acid bacteria) have thick cell walls, and the purple color is not easily washed off at this step. ③ Counterstaining: Add saffron (or carmine) dye, cover for about 1 minute, then wash with water and dry. ④ Microscopic examination: After the glass slide is dried, observe under the oil lens of the microscope.

[0010] (2) Whole genome sequencing identification The strain obtained by the final screening of the application is inoculated into MRS liquid culture medium, and cultured at 37℃ for 12 h to obtain a bacterial liquid. The bacterial liquid is sent to Pishonino Biotechnology (Shanghai) Co., Ltd. for second-generation + third-generation whole genome sequencing identification analysis.

[0011] II. Culture of Lactobacillus reuteri LR1 1. Solid culture of Lactobacillus reuteri LR1 MRS solid culture medium is prepared for Lactobacillus reuteri LR1, the pH is adjusted to 5.7±0.2, 121℃ high pressure sterilization for 15 min, and cooled to 45℃ in a clean bench. The purified Lactobacillus reuteri LR1 is inoculated on the MRS plate, and cultured at 37℃ for 48 h.

[0012] 2. Liquid culture of Lactobacillus reuteri LR1 Prepare 100 mL MRS liquid medium, adjust pH to 5.7±0.2, 121°C high pressure sterilization for 15 min, after sterilization, cool to room temperature for standby. From MRS plate, pick up 1 ring of independent bacteria and inoculate into MRS liquid medium, seal with plastic wrap, incubate at 37°C for 24 h.

[0013] 3. Fermentation of Lactobacillus reuteri LR1 Prepare 200 mL MRSG basic fermentation medium, adjust pH to 5.7±0.2, 121°C high pressure sterilization for 15 min, after sterilization, cool to room temperature for standby. Take the 16 h cultured bacteria solution and inoculate into MRSG basic fermentation medium with 5% inoculation amount, seal with plastic wrap, incubate at 37°C for 24 h.

[0014] Three, biological characteristics detection of Lactobacillus reuteri LR1 1. Growth curve Add the culture solution of isolated strain into MRS liquid medium (2% w / v), measure the absorbance at 600 nm every 2 hours.

[0015] 2. Artificial gastric juice and artificial small intestinal juice resistance Resuspend the activated strain with PBS, prepare bacteria solution with concentration of 1x10 8 CFU / mL, take 100 μL bacteria solution into 900 μL artificial gastric juice (SGF) and PBS solution respectively, incubate at 37°C for 3 h, then take 100 μL culture solution from each into 900 μL artificial intestinal juice (SIF) or PBS solution respectively, continue to incubate at 37°C for 24 h. Then take 50 μL of PBS solution, artificial gastric juice and artificial intestinal juice incubated bacteria solution, gradient dilute and spread on MRS agar medium, incubate at 37°C for 36 h, then count the number of colonies. Take PBS solution treatment as control group, calculate the relative survival rate of strain in artificial gastric juice and artificial intestinal juice respectively. Survival rate is calculated as follows: Survival rate (%) = control group OD600 / experimental group OD600x100%.

[0016] 3. Acid resistance Adjust MRS culture solution to pH=2.5, pH=3.5, pH=4, pH=4.5. Add bacteria suspension into MRS culture solution (10% w / v), incubate at 37°C for 4 h, take normal MRS culture solution as control. Then measure the absorbance of each solution at 600 nm with spectrophotometer.

[0017] Four, detection of GABA in fermentation product Quantitative detection of GABA by HPLC method. Chromatographic conditions: C18 column, mobile phase A is 4.1 g.L-1 Sodium acetate buffer-saline-acetonitrile (95:5) as mobile phase B; linear gradient elution program: 0-15 min, 5%-10% B, 15-20 min, 10%-100% B, 100% B to equilibrate the column. UV detection wavelength 254 nm, flow rate 1.0 mL.min -1 Column temperature 30°C, injection volume 10 μL.

[0018] Five, the evaluation of the protective effect of Lactobacillus reuteri LR1 on DON toxicity (1) Animal model experiment DON challenge mice experiment: mice with similar body weight and good growth status were randomly divided into 2 groups, set as control group, DON group, 5 mice in each group. The initial body weight and feed weight of mice were recorded before the experiment. The mice in the DON group were given 4 mg / (kg.bw) DON by gavage every day for one week. LR1 compensation mice experiment: mice were randomly divided into 4 groups, set as control group, LR1 group, DON group and LR1+DON (LD) group. The control group mice were fed with basic diet every day; the LR1 group was given 1 x 10 9 CFU / d LR1 lactobacillus by gavage every day for 14 days; the DON group mice were given 4 mg / (kg.bw) DON by gavage every day from the 8th day for 7 days; the LD group mice were given 1 x 10 9 CFU / d LR1 by gavage every day for one week, and 1 x 10 9 CFU / d LR1 and 4 mg / (kg.bw) DON by gavage every day from the 8th day. The mice were given drugs at regular times every day during the experiment, and the body weight of the mice was recorded.

[0019] (2) Hematoxylin-eosin staining of intestinal tissue The intestinal contents of mice were washed clean with sterile PBS, and 1 cm of jejunum, ileum and colon tissues from the four groups (control group, DON group, LR1 group and LR1+DON group) were taken and fixed in 4% paraformaldehyde for 24 h; the tissues were sent to Wuhan Saiver Biological Technology Co., Ltd. for hematoxylin-eosin staining, and observed under a microscope after the slices were prepared.

[0020] (3) Detection of mouse intestinal tissue samples The protein level of Occludin in the jejunum and colon tissues of mice was detected by Western Blotting. Occludin is a transmembrane protein that plays a role in maintaining the stability and barrier function of tight junctions, and verifies the protective effect of LR1 on the intestine.

[0021] Five, results The results of Gram staining of LR1 strain are shown in Figure 1. Figure 1As shown, the bacterial cells are blue-violet, which is a typical gram-positive bacterium. The bacterial morphology is regular and slender rod-shaped, and most of them are distributed in single or pair form.

[0022] The ANI value of the whole genome sequence of the strain was analyzed using fastANI software, and the results showed that the strain LR1 was most closely related to Limosilactobacillus reuteri W. reuteri subsp. reuteri, and the ANI value was 96.0424. The 16S phylogenetic tree of the strain LR1 based on the 16S rRNA gene sequence is shown in Figure 2 As shown, the strain LR1 is most closely related to Limosilactobacillus reuteri , and the strain screened in the application can be identified as Limosilactobacillus reuteri , and is named Limosilactobacillus reuteri LR1.

[0023] The growth curve results of the strain are shown in Figure 3 a, and the strain LR1 starts to enter the exponential growth phase after 4 h of inoculation, and enters the stationary phase at 14 h. The survival rate is shown in Figure 3 b, and the survival rate of the strain LR1 inoculated in artificial gastric juice and artificial small intestinal juice is close to 100%, indicating that the strain has good resistance to gastric juice and intestinal juice. The survival rate results are shown in Figure 3 c, and more than 90% of the lactic acid bacteria can survive after being inoculated in a medium with pH 4.5 for 4 h; more than 80% of the lactic acid bacteria can survive after being inoculated in a medium with pH 4 for 4 h; nearly 60% of the lactic acid bacteria can survive after being inoculated in a medium with pH 3.5 for 4 h; and about half of the bacteria can survive after being inoculated in a medium with pH 2.5 for 4 h, indicating that the strain has good acid resistance.

[0024] The HPLC detection results are shown in Figure 4 . As can be seen from Figure 4 , by comparing with the GABA standard, it is found that the strain LR1 contains GABA in the fermentation product (fermentation broth) fermented in the MRSG fermentation medium, and the content of GABA is calculated to be 0.6 g / L.

[0025] After the strain LR1 was administered to mice, the body weight change is shown in Figure 5 a, during the first week of pretreatment, the mice in the LR1 group and the LD group had normal mental and growth states, and there was no obvious change in body weight and food intake compared with the control group. After one week of pretreatment, the DON group and the LD group started to be administered with DON. The body weight and food intake of the mice were monitored daily, and the results showed that compared with the CON group, the body weight of the mice in the DON group decreased, and the food intake decreased; there was no obvious difference in the LR1 group. Compared with the DON group, the LD group can significantly improve the decrease of the body weight of the mice. As shown in Figure 5b, compared with the control group, the protein level of Occludin in the jejunum and colon tissues of the DON group mice decreased significantly. Compared with the DON group, the protein level of Occludin in the intestinal tract of the LD group mice increased, which indicated that LR1 can alleviate the intestinal barrier damage caused by DON. As shown in Figure 5 c, the intestinal tract HE staining pathological section results of the mice showed that DON infection caused the villi of the jejunum of the mice to become short, and the intestinal epithelial cells to fall off, while in the jejunum LR1 group and the LD group, the morphological structure of the small intestine remained complete, the villi did not show atrophy and shortening, and were arranged in order; the number of the colon goblet cells of the DON infection group reduced, and there was inflammatory cell infiltration, while the LR1 supplement can reduce the inflammatory cells and restore the number of the goblet cells. The above results showed that LR1 has safety, and no intestinal pathological damage was found in the mice by gavage, and the supplement of LR1 can effectively antagonize the intestinal toxicity of DON to the mice.

[0026] Six, conclusion In summary, the above-mentioned pig-derived gamma-aminobutyric acid-producing lactic acid bacteria LR1 and its application in preventing and treating emetic toxin toxicity can effectively antagonize the intestinal toxicity of DON, and therefore can be applied to different fields such as veterinary microbial preparations, feed, etc.

Claims

1. A gamma-aminobutyric acid-producing Lactobacillus reuteri LR1, characterized in that, The Lactobacillus reuteri LR1 is isolated and purified from a pig cecal content sample, can metabolically produce gamma-aminobutyric acid, and is classified and named as Lactobacillus reuteri LR1 Limosilactobacillus reuteri LR1, with a preservation number of GDMCC.No: 64522, a preservation date of April 17, 2024, a preservation unit of Guangdong Microbial Culture Collection Center, and a preservation place of Guangdong, China.

2. The Lactobacillus reuteri of claim 1 Limosilactobacillus reuteri LR1, characterized in that, The bacteria can effectively alleviate the toxicity of vomitoxin.

3. An inoculant characterized in that: The bacterial agent contains the Lactobacillus reuteri LR1 of claim 1.

4. Use of the Lactobacillus reuteri LR1 of claim 1 or the bacterial agent of claim 2 in the preparation of a product for preventing and treating the toxicity of vomitoxin.

5. A preparation for the control of mycotoxicosis characterized by: The active ingredient of the product comprises the Lactobacillus reuteri LR1 of claim 1.

6. Use of the Lactobacillus reuteri LR1 of claim 1 in the preparation of an animal feed additive.

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