Lactobacillus plantarum CQPC09 and its application in preparing products for preventing and / or treating livestock diarrhea

Abstract

The present invention provides a Lactobacillus plantarum CQPC09 and its application in the preparation of products for preventing and / or treating livestock diarrhea, belonging to the field of microbial technology. The Lactobacillus plantarum CQPC09 in the present invention has a preservation number of CGMCC No.14958. The bacteria can maintain a high survival rate in artificial gastric juice with pH=3.0 and 0.3% bile salt, and to a certain extent can alleviate the limited weight gain caused by diarrhea, showing a lower diarrhea index, and significantly improving the level of intestinal mucosal immunoglobulins. This shows that the bacteria can improve the damaged intestinal barrier, repair the intestinal mucosal barrier, and reduce the permeability of the intestinal mucosa, effectively preventing or treating livestock diarrhea.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Lactobacillus plantarum CQPC09 and application thereof in preparing products for preventing and / or treating livestock diarrhea. Background Art

[0002] Diarrhea is a common disease in livestock. In severe cases, it can cause the death of a large number of livestock. It is one of the important constraints affecting the development of the global animal husbandry. Generally speaking, livestock diarrhea is caused by pathogenic Escherichia coli, which not only affects the physiological functions of animals and leads to decreased production performance, but in severe cases it can even cause the death of livestock, causing significant economic losses to farmers. At present, the main treatment for livestock diarrhea is the use of antibiotics, such as gentamicin and oxytetracycline. However, the use of antibiotics can lead to the problem of bacterial resistance. Escherichia coli diarrhea is a zoonosis. Due to the widespread use of antibiotics in animal husbandry, the cross-transmission of resistance of pathogens between animals and plants has caused widespread public concern. Large-scale use of antibiotics may also affect the intestinal microbiota, leading to an imbalance in the flora, thereby reducing the body's immune capacity. Therefore, the need to develop green, efficient, and low-residue drugs or biological agents is becoming increasingly urgent.

[0003] As a natural biological agent with no toxic side effects, probiotics have been proven to solve livestock diarrhea problems to a certain extent, regulate the intestinal environment of animals, promote animal health, and increase the economic benefits of farmers. Therefore, their application in animal husbandry is becoming more and more extensive, especially for the treatment of diarrhea and other related diseases. However, the probiotics that can be actually used in animal husbandry are still relatively limited, and there is an urgent need to develop new probiotics that can effectively prevent or treat diarrhea symptoms. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide Lactobacillus plantarum CQPC09 and its use in the preparation of products for preventing and / or treating livestock diarrhea. The Lactobacillus plantarum CQPC09 provided by the present invention can effectively prevent or treat diarrhea symptoms in livestock.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A strain of Lactobacillus plantarum CQPC09, wherein the deposit number of the Lactobacillus plantarum CQPC09 is CGMCC No.14958, and the classification name is Lactobacillus plantarum It was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on November 24, 2017. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] In certain technical solutions, the nucleotide sequence of 16S rDNA of the Lactobacillus plantarum CQPC09 is as shown in SEQ ID No.1.

[0008] In certain technical solutions, the colonies of the Lactobacillus plantarum CQPC09 are white or milky white in color, round in shape, with neat edges and a moist and smooth surface.

[0009] In certain technical solutions, the survival rate of the Lactobacillus plantarum CQPC09 in artificial gastric juice at pH=3.0 can reach 95.08%.

[0010] In certain technical solutions, the survival rate of the Lactobacillus plantarum CQPC09 in 0.3% bile salt can reach 55.97%.

[0011] The present invention also provides an application of Lactobacillus plantarum CQPC09 described in the above technical solution in the preparation of products for regulating gastrointestinal health.

[0012] The present invention also provides an application of Lactobacillus plantarum CQPC09 described in the above technical scheme in preparing a product for preventing and / or treating diarrhea.

[0013] The present invention also provides an application of Lactobacillus plantarum CQPC09 described in the above technical solution in preparing a product for preventing and / or treating livestock diarrhea.

[0014] In certain technical solutions, the livestock preferably includes but is not limited to cattle, sheep, and pigs.

[0015] In certain technical solutions, the content of Lactobacillus plantarum CQPC09 in the product is preferably 1×10 8 CFU / mL~1×10 9 CFU / mL.

[0016] In certain technical embodiments, the diarrhea preferably includes but is not limited to diarrhea caused by Escherichia coli.

[0017] Beneficial technical effect: The present invention provides a Lactobacillus plantarum CQPC09 and its application in preparing a product for preventing and / or treating livestock diarrhea. The Lactobacillus plantarum CQPC09 in the present invention is deposited with CGMCC No. 14958 and is classified as Lactobacillus plantarum, was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 24, 2017, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Lactobacillus plantarum CQPC09 can maintain a high survival rate in artificial gastric juice with pH = 3.0 and 0.3% bile salt, and to a certain extent can alleviate the limited weight gain caused by diarrhea, showing a lower diarrhea index and significantly improving the level of intestinal mucosal immunoglobulin. This shows that the bacteria can improve the damaged intestinal barrier, repair the intestinal mucosal barrier, and reduce the permeability of the intestinal mucosa, effectively preventing or treating livestock diarrhea. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the colony morphology of strain CQPC09;

[0019] Figure 2 is the Gram staining result of strain CQPC09;

[0020] Figure 3 The figure is an agarose gel electrophoresis diagram of the amplified product of strain CQPC09; wherein, M is 2000 bp DNA Ladder; 0 is the negative control group; 09 represents the strain numbered CQPC09, and other numbers represent other isolated strains. DETAILED DESCRIPTION

[0021] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial channels; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0022] Example 1

[0023] 1. Strain Isolation and Identification

[0024] 1.1 Experimental Materials

[0025] 40 mL of kimchi water was collected from naturally fermented farmers in Nan'an District, Chongqing. The water was placed in a sterile centrifuge tube, placed in a food sampling box, and stored in a 4°C refrigerator in the laboratory for later use.

[0026] 1.2 Strain isolation and identification

[0027] 1.2.1 Strain isolation and purification

[0028] Take 1 mL of kimchi water sample and dilute it 10 times to 10 with sterile saline. -6 , then take 10 -4 , 10 -5 , 10-6 100 μL of 3 gradient bacterial solutions were spread on MRS plate medium and cultured at 37℃ for 24h~48h, and the colony morphology was observed and recorded. Colonies with different morphologies on the plate were picked up and separated by streaking on MRS plate medium. After culturing at 37℃ for 48h, single colonies with different morphologies on the plate were picked up again and separated by streaking on MRS plate medium. This was repeated 2 to 3 times until pure single colonies with consistent morphology were obtained.

[0029] 1.2.2 Preliminary identification of strains

[0030] Pure colonies on the plate were picked and inoculated into 5 mL of MRS liquid medium and cultured at 37°C for 24 h.

[0031] Colony morphology Figure 1 As shown, the colonies of CQPC09 bacteria are mostly white or milky white in color, round in shape, with neat edges and a moist and smooth surface.

[0032] Take 1 mL of the above-mentioned bacterial culture medium into a sterile centrifuge tube, centrifuge at 4000 r / min for 10 min, discard the upper layer of culture medium, resuspend the bacterial pellet in sterile physiological saline and perform Gram staining microscopy. Gram staining microscopy is positive. Under a 100x oil microscope, the cell morphology of CQPC09 strain is as follows: Figure 2 As shown, the cell morphology includes long rods and short rods, and budding reproduction does not exist.

[0033] After preliminary identification, CQPC09 was initially identified as lactic acid bacteria.

[0034] 1.2.3 Molecular identification

[0035] The purified suspected target strain was inoculated into MRS broth and cultured at 37°C for 18-24 hours. DNA was extracted using a bacterial genomic DNA extraction kit (item number: K0512, Thermo Fisher Scientific, USA). The extracted DNA was numbered and stored in a -20°C freezer for later use.

[0036] The extracted DNA was subjected to PCR amplification, including 1 μL of upstream primer 27F (5'-AGA GTT TGA TCC TGGCTC AG-3', SEQ ID No.2), 1 μL of downstream primer 1495R (5'-CTA CGG CTA CCTTGT TAC GA-3', SEQ ID No.3), 12.5 μL of 2×Taq plus Buffer, 1 μL of template DNA, and the system was supplemented to 25 μL with sterile ddH2O. Sterile ultrapure water was used to replace template DNA as a negative control. The amplification conditions were: 94℃ 5min; 94℃ 30s, 55℃ 30s, 72℃ 1min, a total of 29 cycles, and finally 72℃ extension for 5min.

[0037] 5 μL of amplified product was taken for agarose gel electrophoresis detection, the agarose concentration was 1.5%, and the electrophoresis conditions were 110V, 45min. The successfully detected PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the successfully sequenced sequences were compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI.

[0038] The results of 16S rDNA amplification products were detected by 1.5% agarose gel. Figure 3 The lane with sterile ultrapure water as negative control had no bands, indicating that there was no contamination during the PCR amplification process. The length of the specific amplified fragment in lane 09 was about 1500bp, which was consistent with the expected amplified fragment length.

[0039] The sequencing result of the 16S rDNA amplification product is shown in SEQ ID No. 1:

[0040]

[0041] As shown in Table 1, CQPC09 was sequenced successfully. After comparison, the homology between CQPC09 and the known Lactobacillus plantarum in the Gene Bank database reached 99%, indicating that it was Lactobacillus plantarum. The bacterium was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 24, 2017, with the deposit number CGMCC No.14958.

[0042] Table 1 16S rDNA sequence analysis results of bacteria

[0043]

[0044] 2 In vitro resistance screening

[0045] 2.1 Ability to tolerate 0.3% bile salts

[0046] Porcine bile salt was added to MRS-THIO medium (MRS broth containing 0.2% sodium thioglycolate) to make the concentration 0.3%, sterilized at 121℃ for 15min, and 5mL of activated bacteria were inoculated at 2% (v / v) into MRS-THIO medium without bile salt (0.0%) and MRS-THIO medium containing 0.3% bile salt, respectively. Blank medium (MRS-THIO medium without bacteria) was used as control. After culturing at 37℃ for 24h, the OD values ​​of the above different concentrations of culture medium were measured. 600nm The tolerance of the strain to bile salts was calculated according to the following formula:

[0047]

[0048] 2.2 Artificial gastric juice tolerance test

[0049] Preparation of artificial gastric juice: Artificial gastric juice is composed of 0.2% NaCl and 0.35% pepsin. The NaCl and pepsin required for the test are weighed according to the corresponding mass-to-volume ratio for preparation. The pH of the prepared artificial gastric juice is adjusted to 3.0 with 1 mol / L HCl, and then filtered with a 0.22 μm filter membrane for sterilization and set aside.

[0050] In the clean bench, 5 mL of the cultured bacterial culture was taken into a 10 mL sterile centrifuge tube, centrifuged at 3000 r / min for 10 min, the upper culture medium was discarded and the bacteria were collected, and an equal volume (5 mL) of sterile saline was added to mix to make a bacterial suspension, and then 1 mL of the bacterial suspension was mixed with 9 mL of artificial gastric juice with pH = 3.0. At this time, 1 mL of the above mixture was taken as the sample of artificial gastric juice treatment for 0 h, and the remaining 9 mL of the mixture was placed in a constant temperature water bath shaker (37 ° C, 150 r / min) and cultured for 3 h. The samples of 0 h and 3 h were diluted 10 times in a gradient, and the number of viable bacteria was determined by plate coating method with a suitable gradient. The samples were cultured at 37 ° C for 48 h on MRS solid culture medium, and the survival rate (%) was calculated according to the following formula:

[0051]

[0052] 2.3 In vitro resistance results

[0053] The in vitro resistance results are shown in Table 2. As shown in Table 2, the survival rate of CQPC09 Lactobacillus plantarum in artificial gastric juice with pH = 3.0 reached 95.08%; the survival rate of CQPC09 Lactobacillus plantarum in 0.3% bile salt was relatively high, reaching 55.97%, indicating that it has a strong ability to tolerate bile salt.

[0054] Table 2 Survival rate of CQPC09 in pH=3.0 artificial gastric juice and 0.3% bile salt

[0055]

[0056] 3 Animal experiments

[0057] 3.1 Experimental animals and strains

[0058] Fifty C57BL / 6 mice weighing 20±2g and aged 6 weeks were selected as experimental animals, half of which were male and half were female. The mice were allowed to eat and drink freely and were kept in the SPF barrier environment of the animal laboratory of Chongqing Engineering Laboratory for Functional Food Research and Development of Chongqing Second Normal University. They were fed SPF mouse feed purchased from Chongqing Tengxin Biotechnology Co., Ltd. and drinking laboratory pure water. The litter used was sterilized by high pressure and changed every three days. After changing the litter, the breeding room was disinfected with 84 disinfectant and irradiated with ultraviolet light once a week. Enteropathogenic Escherichia coli Escherichia coli EPEC (strain number: CICC 10663), purchased from China Industrial Microbiology Culture Collection Center.

[0059] 3.2 Main reagents and instruments

[0060] Mouse secretory immunoglobulin (sIgA) and polymeric immunoglobulin receptor (pIgR) ELISA kits, RNA extraction kits, reverse transcription kits, real-time fluorescence quantitative PCR kits, and real-time fluorescence quantitative PCR instrument were purchased from Thermo Fisher Scientific.

[0061] 3.3 Establishment and treatment of Escherichia coli diarrhea mouse model

[0062] Fifty mice were randomly divided into five groups, namely, normal group, model group, antibiotic (enrofloxacin) group, CQPC09 low-concentration gavage group and CQPC09 high-concentration gavage group, with 10 mice in each group. The mice in the normal group were intraperitoneally injected with 0.5 mL of normal saline, and the mice in the other groups were intraperitoneally injected with 0.5 mL of 1×10 8 After the mice showed diarrhea symptoms, the mice in the enrofloxacin group were intragastrically administered with 0.2 mL of enrofloxacin solution at a dose of 10 mg / kg, and the mice in the CQPC09 low-concentration gavage group (CQPC09L) and the CQPC09 high-concentration gavage group (CQPC09H) were intragastrically administered with 1×10 8 CFU / mL and 1×10 9 CFU / mL of CQPC09 bacterial suspension was used for continuous treatment for 7 days.

[0063] 3.4 Statistics of feed intake, body weight and diarrhea index

[0064] During the experiment, the clinical symptoms, body weight, and food intake of mice were recorded every day. On the third day of treatment, the mice were housed in single cages with filter paper as pads, and the diarrhea index was calculated.

[0065] Diarrhea index = loose stool rate × average loose stool grade

[0066] The loose stool rate is the ratio of the loose stool number of a single mouse to the total number of stools. The loose stool grade is assessed by the diameter of the loose stool stain on the filter paper: the stain diameter is less than 1.0 cm, which is graded as 1; the stain diameter is 1.0 cm to 1.9 cm, which is graded as 2; the stain diameter is 2 cm to 3 cm, which is graded as 3; the stain diameter is greater than 3 cm, which is graded as 4.

[0067] Sample collection: After 7 days of the experiment, the mice were dissected and the tissues 2 cm from the middle of the duodenum and ileum were taken. The dissolved contents were washed with saline and the mucosa of the duodenum and ileum were scraped and stored at -80°C for later use.

[0068] 3.5 Intestinal mucosal immunoglobulin levels

[0069] The ileal mucosa of mice was obtained, and the levels of sIgA and pIgR in the intestinal mucosa were determined according to the instructions of the kit.

[0070] 3.6 Expression levels of intestinal mucosal tight junction proteins

[0071] The mouse duodenal mucosa was obtained, and total RNA was extracted according to the kit instructions. After the concentration was determined, it was reverse transcribed into cDNA. The relative expression levels of intestinal tight junction proteins claudin-1, occludin and ZO-1 mRNA were determined using a real-time fluorescence quantitative PCR kit. The primer information is shown in Table 3. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. PCR reaction conditions: 95℃, 30s; 95℃, 5s, 60℃, 30s, a total of 40 cycles; 95℃, 15s; 60℃, 60s; 95℃, 15s. GAPDH was used as the internal reference gene, and 2 -△△ct The relative expression of genes was calculated.

[0072] Table 3 Primer information

[0073]

[0074] 3.7 Data Statistical Analysis

[0075] The experimental data were analyzed by One-way ANOVA using SPSS 20.0 software, and multiple comparisons between groups were performed using Tukeytest and LSD method. The experimental results are expressed as mean ± standard deviation. P <0.05 indicated significant difference.

[0076] 4 Animal Experiment Results

[0077] 4.1 Food intake of mice

[0078] The average food intake of mice after treatment is shown in Table 4. As shown in Table 4, the food intake of mice in the normal group was significantly higher than that of the other groups, while that of the model group was significantly lower than that of the other groups ( P <0.05). The enrofloxacin group was slightly lower than the CQPC09H group, but significantly higher than the CQPC09L group ( P <0.05). The experimental results show that the antibiotic enrofloxacin and the probiotic CQPC09 can improve the feeding of diarrheal mice and facilitate the recovery of the mice, and the effect of CQPC09 is better than that of enrofloxacin. This shows that after CQPC09 treatment, the recovery of diarrheal mice has been improved.

[0079] Table 4 Statistics of average food intake of mice after treatment

[0080]

[0081] Note: Data in the same column with different letters on their shoulders indicate significant differences ( P <0.05); the same letters on shoulders indicate no significant difference ( P >0.05), the same below.

[0082] 4.2 Mouse weight

[0083] After intraperitoneal injection of E. coli, the weight of mice in the normal group continued to increase normally, while the weight of mice in the model group decreased (Table 5). Enrofloxacin and CQPC09 can inhibit the weight loss caused by intraperitoneal injection of E. coli, and CQPC09H has the best effect, while enrofloxacin is better than CQPC09L.

[0084] In this study, mice were injected intraperitoneally with bacterial liquid, and their weight dropped significantly. After two days of drug treatment, the weight of the mice gradually recovered. The weight of mice in the normal group continued to increase, while the weight of mice in the model group increased slowly or even decreased. This shows that CQPC09 can alleviate the limited weight gain caused by diarrhea to a certain extent, showing the effect of treating diarrhea.

[0085] Table 5 Initial body weight, final body weight and average weight gain of mice

[0086]

[0087] 4.3 Diarrhea Index

[0088] The model group showed the highest loose stool rate and diarrhea index (Table 6). Enrofloxacin and CQPC09 could downregulate the high loose stool rate and diarrhea index caused by E. coli, and CQPC09H had the most significant downregulation effect. CQPC09 showed a lower diarrhea index, which suggested that CQPC09 played a role in inhibiting diarrhea caused by E. coli.

[0089] Table 6 Loose stool rate and diarrhea index of mice after 3 days of treatment

[0090]

[0091] 4.4 Levels of immunoglobulins in the intestinal mucosa of mice

[0092] The normal group showed the highest sIgA and pIgR levels, and the sIgA and pIgR levels of the CQPC09H group were higher than those of the enrofloxacin group and the CQPC09L group (Table 7).

[0093] The level of intestinal mucosal immunoglobulin in the model group mice was significantly lower than that in the CQPC09 treatment group and the control group, indicating that under the influence of E. coli, the intestinal mucosa of mice was damaged and the immune function of the intestinal mucosa decreased. The level of intestinal mucosal immunoglobulin in mice treated with CQPC09 was significantly higher than that in the model group, indicating that CQPC09 can effectively promote the secretion of sIgA and pIgR in the intestinal mucosa. sIgA is an important defense factor in the mucosal immune system, and pIgR, as a transport receptor for sIgA, can maintain the balance of the mucosal surface, resist the invasion of external pathogens, and promote the repair of the intestinal mucosa. CQPC09 can significantly enhance this process.

[0094] Table 7 Immunoglobulin levels in mouse intestinal mucosa

[0095]

[0096] 4.5 Expression levels of tight junction proteins in mouse intestinal tissue

[0097] Table 8 shows that the relative expression levels of claudin-1, occludin and ZO-1 mRNA in the normal group of mice were significantly higher than those in the other groups ( P <0.05), the above expression intensity of CQPC09H group was significantly higher than that of CQPC09L group ( P <0.05), slightly higher than the enrofloxacin group, but no significant difference ( P >0.05).

[0098] The results showed that the relative expression levels of tight junction proteins claudin-1, occludin and ZO-1 in the intestinal tissue of the model group mice were significantly lower than those of the other groups receiving drug treatment, indicating that when the intestinal mucosal barrier of mice was damaged, the relative expression of tight junction proteins in the intestinal tissue decreased, resulting in increased intestinal mucosal permeability, which in turn caused diarrhea. This study showed that after treatment with CQPC09, the relative expression of tight junction proteins claudin-1, occludin and ZO-1 mRNA increased, and it was able to repair intestinal villus damage, indicating that CQPC09 can improve the damaged intestinal barrier, repair the intestinal mucosal barrier, and reduce the permeability of the intestinal mucosa, thereby alleviating the diarrhea symptoms of mice. In particular, enrofloxacin, as an antibiotic veterinary drug, is often used to treat diarrhea. CQPC09 shows better effects than enrofloxacin, and is non-toxic and has no side effects. It can avoid the abuse of antibiotics, while improving feeding quality and reducing costs. CQPC09 can be used as a substitute for livestock antibiotics.

[0099] Table 8 Relative expression of claudin-1, occludin and ZO-1 mRNA

[0100]

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus plantarum ( Lactobacillus plantarum ) CQPC09, characterized in that The Lactobacillus plantarum CQPC09 has a deposit number of CGMCC No. 14958, and was deposited in the General Microbiology Center of the China Culture Collection Administration on November 24, 2017. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The use of Lactobacillus plantarum CQPC09 according to claim 1 in preparing a product for treating diarrhea in livestock, characterized in that: The diarrhea is caused by Escherichia coli.

3. The use according to claim 2, characterized in that: The livestock include but are not limited to cattle, sheep, and pigs.

4. The use according to claim 2, characterized in that: The content of Lactobacillus plantarum CQPC09 in the product is 1×10 8 CFU / mL~1×10 9 CFU / mL.

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