1. PlantLactobacillus plantarum (II)Lactobacillus plantarum) Kmust-Li01, its preservation number in China general microbiological culture Collection center is CGMCC No. 21266.

2. A process for the high-density fermentation of Lactobacillus plantarum Kmust-Li01, according to claim 1, characterized in that: inoculating lactobacillus plantarum Kmust-Li01 seed liquid into an MRS liquid culture medium according to an inoculation amount of 2-5% in volume ratio, fermenting under the conditions of 35-38 ℃ and pH 6.2-6.4, stirring and dissolved oxygen amount being less than 1%, in the fermentation process, when the fermentation culture is carried out for 7-8 hours and the residual glucose amount is 0.04-0.06%, supplementing glucose solution at the rate of 5-8 mL/L.h, after the fermentation culture is carried out for 11-12 hours and the residual glucose amount is 0.10-0.15%, supplementing glucose solution at the rate of 10-15 mL/L.h, continuing to ferment for 17-18 hours and when the residual glucose amount is 0.12-0.15%, supplementing glucose solution at the rate of 9-12 mL/min, continuing to ferment for 1-2 hours, and completing the fermentation, wherein the concentration of the glucose solution is 900-1100 g/L.

3. A process for the high-density fermentation of Lactobacillus plantarum Kmust-Li01, according to claim 1, characterized in that: fermenting under the conditions of 35-38 ℃, pH 6.2-6.4, stirring and dissolved oxygen amount less than 1%, in the fermentation process, when fermentation culture is carried out for 7-8 h and the residual glucose amount is 0.04-0.06%, supplementing a glucose solution at the rate of 5-8 mL/L.h, after fermentation culture is carried out for 11-12 h and the residual glucose amount is 0.10-0.15%, supplementing the glucose solution at the rate of 10-15 mL/L.h, after fermentation is carried out for 14-15 h and the residual glucose amount is 0.05-0.10%, supplementing the glucose solution at the rate of 15-18 mL/L.h, after fermentation is carried out for 17-18 h and the residual glucose amount is 0.12-0.15%, supplementing the glucose solution at the rate of 9-12 mL/min, continuing fermentation for 1-2 h, and completing fermentation, wherein the concentration of the glucose solution is 900-1100 g/L.

4. The high-density fermentation method according to claim 2, wherein: centrifuging the fermentation liquor by 5000-12000 g to remove supernatant, adding a freeze-drying protective agent with the mass being 3-5 times that of the fermentation liquor into the thallus, uniformly mixing, and freeze-drying to obtain freeze-dried fungus powder, wherein the freeze-dried fungus powder is prepared

The freeze-drying protective agent is prepared by 8-12% of skim milk powder, 3-7% of trehalose, 3-7% of glucose, 0.5-1.5% of vitamin C and 1000mL of distilled water through sterilization at 115 ℃ for 20 min.

5. Use of lactobacillus plantarum Kmust-Li01 according to claim 1 for the preparation of a feed additive capable of inhibiting pathogenic microorganisms.

6. Use according to claim 5, characterized in that: the pathogenic microorganism is Escherichia coli (Escherichia coli) Aeromonas hydrophila (b) ((b))Aeromonas hydrophila) Pseudomonas aeruginosaPseudomonas aeruginosa) Streptococcus agalactiae (1)Streptococcus agalactiae) Staphylococcus aureus (1)Staphylococcus aureus) Shigella bacteria (A), (B), (C)Shigella Castellani）。

Background

Since the 20 th century, with the discovery of antibiotics, the great progress of human medicine and livestock and poultry breeding is promoted, so that antibiotic medicines become the first choice for resisting and treating bacteria. The use of a large amount of antibiotics, although beneficial to the breeding industry, leads to the appearance of a large amount of drug-resistant strains along with the non-standard use of antibiotics, so that the search for antibiotic substitutes is a problem which needs to be solved urgently.

The probiotics can be directly fed to animals, and has the characteristics of safety, reliability, excellent performance and the like. The probiotics plays an important role in disease prevention, treatment and the like, is mainly positioned in the oral cavity, intestinal tract and reproductive tract of animals, has the effects of improving the intestinal micro-ecological environment, balancing the flora dynamics, correcting the intestinal flora disorder, enhancing the immune function, promoting the proliferation of beneficial bacteria in the intestinal tracts of livestock, preventing or inhibiting the propagation of harmful bacteria and the like. It is symbiotic and co-evolutionary with the host, and once the balance is broken, the health of animals is harmed. The lactobacillus plantarum is a main probiotic flora in human and animal intestinal tracts, can be used as a food auxiliary material for preparing functional food and regulating the intestinal flora of human and animals. In addition, the lactobacillus plantarum is used for animal breeding, has no residue and no toxic or side effect, can improve the breeding ecological environment to achieve the aim of ecological prevention and control, can enhance the animal immunocompetence and meat quality as a feed additive, realizes the benign development of animal breeding production, and obtains better ecological benefit and economic benefit.

At present, the production of domestic microecological preparations mainly adopts a common fermentation technology, the production efficiency is relatively low, the nutrient components of a culture medium can not be completely utilized, the high-density fermentation of lactobacillus plantarum is still difficult to realize at present, and the main problem difficult to realize is that the fermented culture medium and the fermentation method have insufficient nutrition and metabolite accumulation in the later fermentation stage to inhibit the growth of thalli, and the lactobacillus plantarum is usually subjected to anaerobic fermentation, and the high-density fermentation is difficult to realize due to the inconvenience of material filling and supplement.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a lactobacillus plantarum (A)Lactobacillus plantarum) Kmust-Li01, which is preserved in China general microbiological culture Collection center on 30.11.2020, with the preservation number of CGMCC No.21266, address: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

The lactobacillus plantarum is a gram-positive bacterium separated from intestinal mucosa of a healthy pig, is cultured in an MRS liquid culture medium, the cells are short rod-shaped, and are cultured on an MRS agar culture medium plate, the bacterial colony is milky white, the surface is smooth, the protrusion is obvious, the edge is neat, and the bacterial colony is opaque and glossy; extracting total DNA of the strain, amplifying a target fragment by using a bacterial 16SrDNA universal primer, and recovering, cloning and sequencing the fragment; the similarity alignment of 16SrDNA was performed using BLAST of NCBI on the sequence of the bacterial 16SrDNA gene fragment, and the result showed that the known sequence homology with Lactobacillus plantarum was 99.82%, which was identified as Lactobacillus plantarum.

The invention also aims to provide the high-density fermentation method of the lactobacillus plantarum Kmust-Li01, which comprises the steps of inoculating lactobacillus plantarum Kmust-Li01 seed liquid into an MRS liquid culture medium according to the inoculation amount of 2-5% of the volume ratio, fermenting under the conditions of 35-38 ℃, pH 6.2-6.4, stirring and dissolved oxygen amount less than 1%, wherein in the fermentation process, when the fermentation culture is carried out for 7-8 h and the residual glucose amount is 0.04-0.06%, the glucose is supplemented at the rate of 5-8 mL/L.h, after the fermentation culture is carried out for 11-12 h and the residual glucose amount is 0.10-0.15%, the glucose is supplemented at the rate of 10-15 mL/L.h, after the fermentation is carried out for 17-18 h and the residual glucose amount is 0.12-0.15%, the glucose is supplemented at the rate of 9-12 mL/min, the fermentation is carried out for 1-2 h, and the fermentation is completed, the number of viable bacteria in the fermentation liquor can reach 10¹¹The magnitude is obviously improved by 30 times compared with the common fermentation viable count.

The lactobacillus plantarum Kmust-Li01 fermentation liquor is centrifuged by 5000-12000 g to remove supernatant, a freeze-drying protective agent with the mass 3-5 times of that of the lactobacillus plantarum Kmust-Li01 fermentation liquor is added into the bacteria, the bacteria are uniformly mixed and then freeze-dried to obtain freeze-dried bacteria powder, wherein the freeze-drying protective agent comprises 8-12% of skim milk powder, 3-7% of trehalose, 3-7% of glucose, 0.5-1.5% of vitamin C and 1000mL of distilled water, and the freeze-dried bacteria powder is prepared by sterilizing the bacteria at 115 ℃ for 20 min.

The seed solution is prepared by inoculating lactobacillus plantarum Kmust-Li01 to an MRS liquid culture medium and culturing for 12-18 h at 37 ℃.

The invention also aims to apply the lactobacillus plantarum Kmust-Li01 in inhibiting pathogenic microorganisms, and the lactobacillus plantarum Kmust-Li01 can be used as a food auxiliary material, an environmental disinfectant or a feed additive, and the strain can be used for treating escherichia coli ((Escherichia coli))Escherichia coli) Aeromonas hydrophila (b) ((b))Aeromonas hydrophila) Pseudomonas aeruginosaPseudomonas aeruginosa) Streptococcus agalactiae (1)Streptococcus agalactiae) Staphylococcus aureus (1)Staphylococcus aureus) Shigella bacteria (A), (B), (C)Shigella Castellani) The 6 strains have good bacteriostatic activity, and the sensitivity is high or extremely sensitive;

the invention has the advantages and technical effects that: the invention obtains a new lactobacillus plantarum from healthy pig intestinal tract, which can effectively inhibit pathogenic microorganisms; the invention provides a new way for ecological breeding of livestock and poultry breeding; compared with the common fermentation technology, the fermentation method provided by the invention is more efficient, and the viable count of lactobacillus plantarum fermented by the common fermentation technology can only reach 10⁹The number of viable bacteria in the fermentation liquid prepared by the fermentation method can reach 10¹¹Magnitude above; the method is simple, easy to operate and suitable for industrial production and market popularization and application.

Drawings

FIG. 1 is a schematic representation of the colony of Lactobacillus plantarum Kmust-Li 01;

FIG. 2 is a microscopic view of Lactobacillus plantarum Kmust-Li 01;

FIG. 3 is a graph showing the growth of Lactobacillus plantarum Kmust-Li 01;

FIG. 4 shows the results of acid and alkali resistance experiment of Lactobacillus plantarum Kmust-Li 01;

FIG. 5 shows the results of the salt tolerance test of Lactobacillus plantarum Kmust-Li 01;

FIG. 6A tree was developed phylogenetically by Lactobacillus plantarum Kmust-Li 01.

Detailed Description

The present invention is further illustrated by the following figures and examples, but the scope of the present invention is not limited thereto, and the methods in the examples are performed according to the conventional methods unless otherwise specified, the reagents used are commercially available reagents or prepared according to the conventional methods, and the media are commercially available products and used according to the instructions.

Example 1: isolation and characterization of Lactobacillus plantarum

1. Homogenizing intestinal mucosa of healthy pig, diluting with 10 times of sterile normal saline, and collecting 10 times of the diluted solution^-6～10^-8Coating the diluent in the dilution range on an MRS solid plate culture medium, and culturing at the constant temperature of 37 ℃ for 24 hours; selecting typical single colony, and repeatedly streaking and separating to obtain pure bacterial strain; the MRS liquid culture medium comprises 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 801 mL/L of Tween and 1000mL of distilled water, the pH value is adjusted to be 6.2-6.4, and the sterilization is carried out for 20min at the temperature of 115 ℃.

The strain is cultured in an MRS liquid culture medium, cells are in a short rod shape, as shown in figure 2, and cultured on an MRS agar culture medium plate, bacterial colonies are milky white, smooth in surface, obvious in protrusion, neat in edge, opaque and glossy, as shown in figure 1.

2. Growth Curve determination

Inoculating the purified strain into an MRS liquid culture medium, carrying out shake culture for 12h in a constant-temperature shaking table at 37 ℃ and 150rpm, taking the culture solution every 2h from 0h in the culture process, measuring the concentration (OD value) of bacterial liquid at the wavelength of 600nm, correcting the zero point of a spectrophotometer by taking the culture solution without the bacterial liquid as a reference to eliminate errors introduced by an instrument, drawing a growth curve chart of the bacillus licheniformis according to the concentration of the bacterial liquid, wherein the logarithmic growth phase of the bacillus licheniformis is 12h, and the growth curve is shown in figure 3.

3. Physiological and biochemical identification

According to the requirements of physiological and biochemical identification tests of lactobacillus, gram staining, mobility, salt resistance (0, 5%, 7%, 10%, 12% and 15% NaCl), acid and alkali resistance (3, 5, 6, 7, 8, 9 and 10), heat resistance (35 ℃, 45 ℃ and 55 ℃), nitrate reduction tests, sugar fermentation acid production, sugar fermentation gas production, catalase, starch hydrolysis tests, gelatin liquefaction tests, VP tests, MR tests, catalase tests, hydrogen sulfide production tests and arginine double hydrolysis tests are respectively carried out, and compared with physiological and biochemical indexes of lactobacillus plantarum mode strains in Bergey bacteria identification handbook (eighth edition), lactobacillus plantarum Kmust-Li01 has stronger tolerance in the range of pH7-10, as shown in figure 4; Kmust-Li01 has better salt tolerance at a salt concentration of 5%, see FIG. 5; the rest of the experimental results are shown in table 1;

TABLE 1 physiological and biochemical experimental results of Lactobacillus plantarum Kmust-Li01

Note: "-" represents a negative reaction, "+" represents a positive reaction, and "W" represents a weaker reaction;

4. 16S rRNA Gene sequence analysis

Carrying out 16S rDNA molecular biology identification on the finally screened strain; extracting total DNA of the strain by using a bacterial genome DNA extraction kit (Beijing of Tiangen Biochemical technology Co., Ltd.), and amplifying a 16S rDNA fragment by using universal primers 27F (5-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5'-GGTTACCTTGTTAGGACTT-3'); the reaction sequence for PCR was as follows: pre-denaturation at 94 ℃ for 4 min; annealing at 94 deg.C for 30s and 50 deg.C for 1min, extending at 72 deg.C for 2min, and repeating for 30 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃; after the size of the PCR product is detected and verified by agarose gel electrophoresis, a target band is purified and handed to Shanghai biological engineering Co., Ltd for Sanger sequencing; obtaining a sequencing sequence, performing Blast comparison in a GenBank database (http:// Blast. ncbi. nlm. nih. gov/Blast. cgi) of a National Center for Biotechnology information (National Center for Biotechnology Inrmmion) website, and determining the seed name according to the known species with the highest score; sequencing the 16S rDNA fragment obtained by PCR amplification to 1095bp,see sequence table 1; using BLAST alignment, MEGA constructed a Neighbor-Joining tree (FIG. 6), which was clustered with known L.plantarum in one branch, andLactobacillus plantarum 7029 andLactobacillus plantarum 6694 the homology is high, and the strain separated from intestinal tract of healthy pig is Lactobacillus plantarum (by combining morphological observation, physiological and biochemical experiment and molecular biological detection sequencing results)Lactobacillus plantarum）。

5. Experiment of bacteriostatic Activity

Performing antibacterial experiment by double-layer plate method, preparing LB solid plate and LB semisolid culture medium, culturing Kmust-Li01 to logarithmic phase, centrifuging at 8000r/min for 10min, and filtering the supernatant with 0.22 μm filter membrane; preparing bacteria liquid of pathogenic bacteria escherichia coli, aeromonas hydrophila, pseudomonas aeruginosa, streptococcus agalactiae, staphylococcus aureus and shigella. Uniformly mixing 100 mu L of pathogenic bacteria liquid and 4mL of semisolid culture medium, pouring the mixture into an LB solid plate, placing a sterilized Oxford cup on a double-layer plate by using forceps, adding 200 mu L of Kmust-Li01 supernatant into the Oxford cup, respectively taking kanamycin added with 50 mu g/mL and a sterilized PBS buffer solution as positive control and negative control, setting 3 pathogenic bacteria in parallel in each group, placing the plate in a refrigerator at 4 ℃ for diffusion for 4h, then transferring the plate into a constant-temperature incubator at 37 ℃ for culture for 24h, measuring the diameter of an inhibition zone, and taking the average diameter of the inhibition zone as the final diameter of the inhibition zone, wherein the experimental results are shown in Table 2, Kmust-Li01 has sensitivity to six pathogenic bacteria, the positive control is extremely sensitive to the pathogenic bacteria, and the negative control is not sensitive to the pathogenic bacteria.

TABLE 2 Lactobacillus plantarum Kmust-Li01 bacteriostatic activity

Note: >20 is extremely sensitive; 15-20 is highly sensitive; 10-14 is medium sensitive; <10 is low sensitivity; 0-8 is insensitive.

Example 2: three-stage differentiated feeding high-density fermentation culture lactobacillus plantarum Kmust-Li01

(1) Lactobacillus plantarum high-density fermentation MRS liquid culture medium component and content

10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate and 801 mL/L of tween; preparing a fermentation culture medium, and reducing the temperature of the culture medium to the temperature required by culture;

(2) inoculating lactobacillus plantarum Kmust-Li01 with the inoculation amount of 5.0% to an MRS liquid culture medium, and culturing at 37 ℃ for 15h to obtain a seed solution;

(3) inoculating the seed solution obtained in the step (2) into the fermentation culture medium obtained in the step (1) with an inoculation amount of 5.0%, stirring at a rotating speed of 150rpm at 37 ℃, and fermenting under the conditions that the dissolved oxygen is less than 1%, wherein the pH value is maintained at 6.2-6.4 in the fermentation process, and when the pH value is less than 6.2, automatically feeding ammonia water or sodium hydroxide serving as an alkaline neutralizing agent; adding 50% glucose solution when pH is higher than 6.4; in the fermentation process, glucose solution is added for feeding, the concentration of the glucose solution is 1000g/L, when the fermentation culture is carried out for 7 hours and the residual glucose amount is 0.05 percent, the glucose solution is fed at the rate of 5 mL/L.h, after the fermentation culture is carried out for 12 hours and the residual glucose amount is 0.10 percent, the glucose solution is fed at the rate of 12 mL/L.h, after the fermentation is continued for 17 hours and the residual glucose amount is 0.12 percent, the glucose solution is fed at the rate of 10 mL/L.h, the fermentation is continued for 2 hours, the fermentation is finished, the viable bacteria number of the Lactobacillus plantarum Kmust-Li01 in the fermentation broth is 1.3 multiplied by 10, and the viable bacteria number is 1.3 multiplied by 10¹¹cfu/mL；

(4) Centrifuging the fermentation liquor by 5000-12000 g to remove supernatant, adding a freeze-drying protective agent with the mass 4 times that of the fermentation liquor into the thalli, uniformly mixing, freezing and drying to obtain freeze-dried fungus powder, wherein the freeze-drying protective agent comprises 10% of skim milk powder, 5% of trehalose, 5% of glucose, 1% of vitamin C and 1000mL of distilled water, and sterilizing at 115 ℃ for 20 min.

Example 3: three-stage differentiated feeding high-density fermentation culture lactobacillus plantarum Kmust-Li01

(1) Lactobacillus plantarum high-density fermentation MRS liquid culture medium component and content

10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate and 801 mL/L of tween; preparing a fermentation culture medium, and reducing the temperature of the culture medium to the temperature required by culture;

(2) activating the lactobacillus plantarum Kmust-Li01 which is frozen and preserved for three times on an MRS agar plate culture medium, selecting a single colony to inoculate in a deep MRS liquid test tube, inoculating to the MRS liquid culture medium with the inoculum size of 5.0 percent after culturing for 18 hours at 37 ℃, and taking the inoculated strain as a seed solution after culturing for 12 hours at 37 ℃;

(3) inoculating the seed solution in the step (2) into the fermentation culture medium in the step (1) with an inoculation amount of 5.0%, stirring at a rotating speed of 150rpm at 37 ℃, and fermenting under the conditions that the dissolved oxygen is less than 1%, wherein the pH value is maintained at 6.2-6.4 in the fermentation process, and when the pH value exceeds 6.2, adding glucose liquid; when the pH value is lower than 6.4, adding ammonia water or sodium hydroxide as alkaline neutralizing agent; the fermentation process is not aerated, the glucose solution is added for supplementing materials in the fermentation process, the concentration of the glucose solution is 1000g/L, and the glucose supplementing mode is as follows: when the fermentation culture is carried out for 8 hours and the residual quantity of the glucose is 0.04 percent, the glucose solution is supplemented at the speed of 8 mL/L.h, after the fermentation culture is carried out for 12 hours and the residual quantity of the glucose is 0.12 percent, the glucose solution is supplemented at the speed of 15 mL/L.h, after the fermentation is continued for 18 hours and the residual quantity of the glucose is 0.13 percent, the glucose solution is supplemented at the speed of 10 mL/L.h, the fermentation is continued for 1 hour, the fermentation is finished, the viable count of the lactobacillus plantarum Kmust-Li01 in the fermentation broth is 1.2 multiplied by 10, and the viable count of the lactobacillus plantarum in the fermentation broth is 1.2 multiplied by 10¹¹cfu/mL。

Example 4: four-stage differential feed supplement high-density fermentation culture lactobacillus plantarum

(1) Lactobacillus plantarum high-density fermentation MRS liquid culture medium component and content

10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate and 801 mL/L of tween; preparing a fermentation culture medium, and reducing the temperature of the culture medium to the temperature required by culture;

(2) activating the lactobacillus plantarum Kmust-Li01 which is frozen and preserved for three times on an MRS agar plate culture medium, selecting a single colony to inoculate in a deep MRS liquid test tube, inoculating to the MRS liquid culture medium with the inoculum size of 5.0 percent after culturing for 18 hours at 37 ℃, and taking the inoculated strain as a seed solution after culturing for 12 hours at 37 ℃;

(3) inoculating the seed solution in the step (2) into the fermentation culture medium in the step (1) with an inoculation amount of 5.0%, stirring at a rotating speed of 150rpm at 37 ℃, and fermenting under the conditions that the dissolved oxygen is less than 1%, wherein the pH value is maintained at 6.2-6.4 in the fermentation process, and when the pH value exceeds 6.2, adding glucose liquid; when the pH value is lower than 6.4, adding ammonia water or sodium hydroxide as alkaline neutralizing agent; the fermentation process is not aerated, the glucose solution is added for supplementing materials in the fermentation process, the concentration of the glucose solution is 1000g/L, and the glucose supplementing mode is as follows: fermenting and culturing for 7h, when the residual amount of glucose is 0.05%, starting feeding at the rate of 6 mL/L.h, fermenting and culturing for 12h, when the residual amount of glucose is 0.13%, feeding at the rate of 12 mL/L.h, after fermenting for 15h, when the residual amount of glucose is 0.05%, feeding at the rate of 18 mL/L.h, after continuing fermenting for 17h, when the residual amount of glucose is 0.12%, feeding at the rate of 10mL/min, and after continuing fermenting for 1.5h, finishing the fermentation, wherein the viable count of Lactobacillus plantarum Kmust-Li01 in the fermentation broth is 1.9 multiplied by 10¹¹cfu/mL。

Sequence listing

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