<i>Lactiplantibacillus plantarum </i>GOLDGUT-LP618 having function of resisting salmonella infection and application thereof

Abstract

The present invention relates to the technical field of probiotics, in particular to a Lactiplantibacillus plantarum GOLDGUT-LP618 having a function of resisting Salmonella infection and application thereof. The Lactiplantibacillus plantarum GOLDGUT-LP618 provided by the present invention is preserved in China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 28399. The strain has high resistance to acid and bile salt and can adapt to the digestive tract environment. The strain can inhibit Salmonella infection, relieve diarrhea, inflammation and intestinal damage caused by Salmonella infection, and promote intestinal health. The strain can be used for preparing functional foods, dietary supplements or drugs that can resist Salmonella infection and improve diarrhea and inflammation caused by Salmonella infection, and has good application prospects.

Claims

1. A method of preparing a microbial preparation for resisting Salmonella infection, wherein the microbial preparation comprising a Lactiplantibacillus plantarum GOLDGUT-LP618 strain, which was deposited at the China General Microbiological Culture Collection Center (CGMCC), with a deposition number of CGMCC No. 28399; wherein the method comprising steps: (a) inoculating the Lactiplantibacillus plantarum GOLDGUT-LP618 strain into an MRS liquid medium to form a mixture; (b) incubating the mixture at 37 C. to obtain an enriched solution; (c) performing a tenfold gradient on the enrichment solution to generate dilutions with gradient concentrations of 10.sup.5, 10.sup.6, and 10.sup.7; (d) applying 100 L of each of the dilutions to sterile a first MRS solid medium and incubating under aerobic conditions at 37 C. for 24 to 48 hours, then single colonies are formed; (e) selecting the single colonies; and (f) repeatedly streaking the selected colonies on a second MRS solid medium for purification to obtain a purified strain.

Description

DESCRIPTION OF DRAWINGS

(1) To more clearly describe the technical solutions in the present invention or in the prior art, the drawings required to be used in the description of the embodiments or the prior art will be simply presented below. Obviously, the drawings in the following description are some embodiments of the present invention, and for those ordinary skilled in the art, other drawings can also be obtained according to these drawings without contributing creative labor.

(2) FIG. 1 shows the colony morphology of Lactiplantibacillus plantarum GOLDGUT-LP618 in embodiment 1 of the present invention.

(3) FIG. 2 shows the gram staining diagram of Lactiplantibacillus plantarum GOLDGUT-LP618 in embodiment 1 of the present invention with a magnification of 1000 times.

(4) FIG. 3 shows the CFU counting results before and after acid-resistant culture of Lactiplantibacillus plantarum GOLDGUT-LP618 in embodiment 2 of the present invention; the data are from three repeated experiments; the error line represents the standard deviation; a statistical analysis method is t test; and ns represents that p value is greater than 0.05.

(5) FIG. 4 shows the CFU counting results before and after incubation and culture of simulated intestinal fluid of Lactiplantibacillus plantarum GOLDGUT-LP618 in embodiment 3 of the present invention; the data are from three repeated experiments; the error line represents the standard deviation; a statistical analysis method is t test; and ns represents that p value is greater than 0.05.

(6) FIG. 5 shows the detection results of the inhibition ability of the supernatant of Lactiplantibacillus plantarum GOLDGUT-LP618 to the growth of Salmonella in embodiment 4 of the present invention, wherein MRS represents negative control and LP618 represents Lactiplantibacillus plantarum GOLDGUT-LP618: the data are from four repeated experiments; the error line represents the standard deviation; a statistical analysis method is ANOVA; and **** represents that p value is less than 0.0001.

(7) FIG. 6 shows the concentration detection results of acetic acid (A) and propionic acid (B) in the supernatant of Lactiplantibacillus plantarum GOLDGUT-LP618 in embodiment 5 of the present invention, wherein Control represents a control strain; the data are from three repeated experiments; the error line represents the standard deviation; a statistical analysis method is t test; and **** represents that p value is less than 0.0001.

(8) FIG. 7 shows a weight change curve (A), a weight gain rate (B) and a fecal score (C) of mice in embodiment 6 of the present invention; the error line represents the standard deviation; a statistical analysis method is a one-way ANOVA; * represents that p value is less than 0.05; ** represents that p value is less than 0.01; *** represents that p value is less than 0.001; and **** represents that p value is less than 0.0001.

(9) FIG. 8 shows the detection results of the number of blood immune cells in mice in embodiment 7 of the present invention; the error line represents the standard deviation; a statistical analysis method is a one-way ANOVA; ns represents that p value is greater than 0.05; * represents that p value is less than 0.05; ** represents that p value is less than 0.01; and **** represents that p value is less than 0.0001.

(10) FIG. 9 shows the detection results of ELISA of serum cytokines in mice in embodiment 7 of the present invention; the error line represents the standard deviation; a statistical analysis method is a one-way ANOVA; ns represents that p value is greater than 0.05; * represents that p value is less than 0.05; and ** represents that p value is less than 0.01.

(11) FIG. 10 shows the H&E staining diagram of the jejunum and ileum tissue of mice in embodiment 8 of the present invention, with a magnification of 40 times.

(12) FIG. 11 shows the quantitative analysis results of villus length (A), crypt depth (B) and pile to crypt ratio (C) of jejuna and ilea of mice in embodiment 8 of the present invention; the error line represents the standard deviation; a statistical analysis method is a one-way ANOVA; ns represents that p value is greater than 0.05; ** represents that p value is less than 0.01; and *** represents that p value is less than 0.001.

(13) FIG. 12 shows the detection results of content of short-chain fatty acid in colon contents of mice in embodiment 9 of the present invention; the error line represents the standard deviation; a statistical analysis method is a one-way ANOVA; ns represents that p value is greater than 0.05; * represents that p value is less than 0.05; ** represents that p value is less than 0.01; and **** represents that p value is less than 0.0001.

(14) In FIGS. 7-12, NC represents a blank control group, STMC represents a model group, POS represents a positive drug group, and LP618 represents a probiotic treatment group.

DETAILED DESCRIPTION

(15) To make purposes, technical solutions and advantages of the present invention more clear, the technical solutions in the present invention will be clearly and fully described below in combination with the drawings in the present invention. Apparently, the described embodiments are merely part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those ordinary skilled in the art without contributing creative labor will belong to the protection scope of the present invention.

(16) Salmonella enterica subsp. Enterica BNCC292614 is used in the following embodiment, and purchased from BeNa Culture Collection.

Embodiment 1 Isolation and Identification of Lactiplantibacillus plantarum GOLDGUT-LP618

(17) 1. Isolation of Lactiplantibacillus plantarum GOLDGUT-LP618

(18) 1.1 Sample Source

(19) Lactiplantibacillus plantarum GOLDGUT-LP618 is isolated from a healthy human gut.

(20) 1.2 Preparation of Medium

(21) The medium used for sample isolation and strain screening is an MRS solid medium. The formula of an MRS liquid medium is as follows: 10.0 g/L of casease digest, 10.0 g/L of beef powder, 4.0 g/L of yeast powder, 2.0 g/L of triammonium citrate, 5.0 g/L of sodium acetate, 0.2 g/L of magnesium sulfate, 0.05 g/L of manganese sulfate, 20.0 g/L of glucose, 2.0 g/L of dipotassium hydrogen phosphate, and 1.0 g/L of Twain-80, and 1.5% agar is added to prepare the MRS solid medium, with pH=5.70.2.

(22) 1.3 Isolation of Strains

(23) 1 g of sample was put into 10 mL of MRS liquid medium, mixed evenly and then cultured at 37 C.; the enrichment solution after culture was absorbed for 10-fold gradient dilution; 100 L of bacteria solution with 10.sup.5, 10.sup.6 and 10.sup.7 dilution gradients was selected and coated on a petri dish containing the sterile MRS solid medium, and subjected to stationary culture at 37 C. under aerobic conditions for 24-48 h; and after obvious single colonies were formed, typical colonies were selected, and scribed and purified on the MRS solid plate medium for several times until the morphologies of colonies on the whole plate were consistent; and the cultures were identified by strains.

(24) 2. Identification of Lactiplantibacillus plantarum GOLDGUT-LP618

(25) 2.1 Colony Features

(26) After the Lactiplantibacillus plantarum GOLDGUT-LP618 was cultured in the MRS solid medium for 24 h, the colonies were small, milky white, round, raised and smooth, as shown in FIG. 1.

(27) 2.2 Microscopic Morphology

(28) Colony smear of Lactiplantibacillus plantarum GOLDGUT-LP618: the gram stain is positive; bacteria are rod-shaped and arranged in single, pair or chain, as shown in FIG. 2.

(29) 2.3 16S rDNA Identification

(30) Identification company: Tsingke Biotechnology Co., Ltd.

(31) Identification sequence: as shown in SEQ ID NO. 1.

(32) Identification results: According to the sequence comparison results and physiological and biochemical results of the strain, the strain was determined to be Lactiplantibacillus plantarum.

Embodiment 2 Test of Gastric Acid Resistance of Lactiplantibacillus plantarum GOLDGUT-LP618

(33) The overall pH condition of the human stomach environment is strongly acidic, so the acid resistance of the strain is an important index to evaluate whether the strain can survive and colonize in the stomach acid environment. Artificial gastric juice was used to simulate the gastric environment. 100 mL of prepared artificial gastric juice reserve was taken, 1.9 mL of IM hydrochloric acid was added to the artificial gastric juice, and a bottle of digestive enzyme was added to obtain the simulated gastric juice. After 4 mL of Lactiplantibacillus plantarum GOLDGUT-LP618 bacteria solution was washed and resuspended with PBS, 4 mL of simulated gastric juice was added, blown, mixed evenly and co-incubated anaerobically for 2 h at 37 C. The bacteria solution before (0 h) and after (2 h) incubation of the simulated gastric juice was gradiently diluted to 10.sup.5. Three concentrations of 10.sup.3, 10.sup.4 and 10.sup.5 were taken, and the diluent was coated on the MRS agar plate. After anaerobic culture at 37 C. for 48 h, colony count was performed. It can be seen from FIG. 3 that after the incubation and culture of the simulated gastric juice, the viable count of Lactiplantibacillus plantarum GOLDGUT-LP618 is not decreased, which indicates that the strain has good gastric acid resistance.

Embodiment 3 Test of Bile Salt Resistance of Lactiplantibacillus plantarum GOLDGUT-LP618

(34) The bile salt resistance of the strain was tested by simulated intestinal fluid. 100 mL of prepared artificial intestinal fluid reserve was taken, and 1 bottle of digestive enzyme was added and mixed evenly to obtain simulated intestinal fluid. After 4 mL of Lactiplantibacillus plantarum GOLDGUT-LP618 bacteria solution was washed and resuspended with PBS, 4 mL of simulated intestinal fluid was added, blown, mixed evenly and co-incubated anaerobically for 2 h at 37 C. The bacteria solution before (0 h) and after (2 h) incubation was gradiently diluted to 10.sup.5. Three concentrations of 10.sup.3, 10.sup.4 and 10.sup.5 were taken, and the diluent was coated on the MRS agar plate. After anaerobic culture at 37 C. for 48 h, colony count was performed. It can be seen from FIG. 4 that after the incubation and culture of the simulated intestinal fluid, the viable count of Lactiplantibacillus plantarum GOLDGUT-LP618 is not decreased, which indicates that the strain has good bile salt resistance.

Embodiment 4 Test of In Vitro Antibacterial Ability of Lactiplantibacillus plantarum GOLDGUT-LP618

(35) The Lactiplantibacillus plantarum GOLDGUT-LP618 was transferred into a fresh MRS medium at an inoculation rate of 2% (v/v), subjected to anaerobic culture at 37 C. for 48 h, and centrifuged at 4000 rpm for 10 min. The supernatant was filtered by a 0.22 m filter membrane and sterilized for later use.

(36) Salmonella enterica subsp. Enterica was transferred into a fresh LB medium at an inoculation rate of 2%. After the OD value of the cultured bacteria solution was adjusted to 1.6 by an LB medium, 100 L of Salmonella solution was taken, added to 100 mL of LB solid medium which was sterilized and cooled to 50 C., then mixed evenly and poured onto a plate. After the medium in the plate is solidified, the plate was punched by a hole puncher, and the distance of hole centers was not less than 25 mm. 200 L of filtered and sterilized supernatant of Lactiplantibacillus plantarum GOLDGUT-LP618 was added. The samples were naturally diffused at 4 C. for 3 h, and then cultured at 37 C. overnight for 24-48 h. The diameter of an inhibition zone was measured. The MRS medium was used as negative control and ampicillin (25 g/mL) was used as positive control.

(37) It can be seen from FIG. 5 that the supernatant of the Lactiplantibacillus plantarum GOLDGUT-LP618 can effectively inhibit the growth of Salmonella.

Embodiment 5 Determination of Acid Production Ability of Lactiplantibacillus plantarum GOLDGUT-LP618

(38) The Lactiplantibacillus plantarum GOLDGUT-LP618 was transferred into a fresh MRS medium at an inoculation rate of 4% (v/v), subjected to anaerobic culture at 37 C. for 24 h, and centrifuged at 4000 rpm for 10 min. The supernatant was taken. The content of short-chain fatty acid in the supernatant was determined by an HPLC method. A control strain was a Lactiplantibacillus plantarum with low acid production obtained in this screening experiment.

(39) It can be seen from FIG. 6 that the Lactiplantibacillus plantarum GOLDGUT-LP618 has high production ability of acetic acid and propionic acid.

Embodiment 6 Improvement of Diarrhea Symptoms in Mice Infected with Salmonella by Lactiplantibacillus plantarum GOLDGUT-LP618

(40) To assess the ability of Lactiplantibacillus plantarum GOLDGUT-LP618 to resist Salmonella infection in vivo, a diarrhea model of mice infected with Salmonella typhimurium was used. After one week of adaptive feeding. 6-week-old male BALB/c mice at SPF level were fed with 5 mg/mL streptomycin water for two days, and then fed with normal water and food for one day. After fasting and water deprivation for 4 h, each mouse was gavaged with 100 L of 5% sodium bicarbonate solution, and then each mouse was gavaged with 210.sup.8 CFU Salmonella typhimurium and continuously gavaged with Salmonella typhimurium for three days to induce diarrhea. The mice with diarrhea induced by gavaging with Salmonella typhimurium were divided into three groups. In a model group (STMC group), each mouse was gavaged with 200 L of normal saline daily. In a positive drug group (POS group), each mouse was gavaged with 200 L of levofloxacin solution daily, and the dose was 8 mg/kg. In a probiotic treatment group (LP618 group), each mouse was gavaged with 200 L of bacteria solution of Lactiplantibacillus plantarum GOLDGUT-LP618 daily, and the viable count was 510.sup.8 CFU per mouse. Continuous gavage intervention was performed for 5 days. The mice in a blank control group (NC group) were not gavaged with Salmonella typhimurium and modeled, but gavaged with the same amount of normal saline daily as control.

(41) A in FIG. 7 shows weight changes in the four groups of mice during modeling and intervention. The weight of the mice in the model group is decreased significantly, and the weight of the mice in the positive drug group and the probiotic treatment group are decreased slowly. After the intervention of positive drugs and probiotics, the weight gain rate of the mice is decreased significantly more slowly than that of the model group (B in FIG. 7).

(42) During the intervention, the diarrhea status of the mice was assessed in the form of fecal scores by observing the defecation status of the mice. A score of 0 indicates normal stool; a score of 1 indicates mild diarrhea; and a score of 2 indicates moderate diarrhea. It can be seen from C in FIG. 7 that the mice in the blank control group have no diarrhea, and the mice in the model group have diarrhea of different degrees. After the intervention of the positive drugs, the diarrhea status of the mice is significantly improved, and the diarrhea symptom of the mice after the intervention of Lactiplantibacillus plantarum GOLDGUT-LP618 is also significantly improved.

Embodiment 7 Reduction of Inflammatory Reaction of Mice Infected with Salmonella by Lactiplantibacillus plantarum GOLDGUT-LP618

(43) To assess the inflammatory reaction of Salmonella typhimurium to the intestinal tract of the mice, the blood of the mice was taken for blood routine analysis. In case of bacterial infection, the number of white blood cells and lymphocytes is decreased and the number of neutrophils and intermediate cells is increased. It can be seen from FIG. 8 that compared with the NC group, after the mice of the STMC group are infected with Salmonella typhimurium, the total number of white blood cells is decreased, the ratio of lymphocytes is significantly decreased, and the number of neutrophils and the ratio of intermediate cells are significantly increased, which indicates that the mice have the inflammatory reaction due to the infection by Salmonella typhimurium. After the intervention of the positive drugs, the total number of white blood cells and the ratio of lymphocytes are increased significantly, and the ratio of intermediate cells and neutrophils are decreased significantly, which indicates that the inflammatory reaction caused by Salmonella is alleviated. Similarly, after the intervention of Lactiplantibacillus plantarum GOLDGUT-LP618, the ratio of lymphocytes is increased significantly, the ratio of intermediate cells and the ratio of neutrophils are decreased significantly; and the total number of white blood cells is increased but not significantly. This indicates that the inflammation caused by Salmonella infection is controlled to a certain extent after gavaging with Lactiplantibacillus plantarum GOLDGUT-LP618.

(44) Mouse serum was taken for ELISA analysis, and the results were shown in FIG. 9. The content of TGF-, as an immunosuppressive cytokine, was slightly decreased in the STMC group after the mice were infected with Salmonella, and significantly increased after the intervention of Lactiplantibacillus plantarum GOLDGUT-LP618 (A in FIG. 9). In addition, compared with the STMC model group, after the intervention of Lactiplantibacillus plantarum GOLDGUT-LP618, the contents of inflammatory factors IL-6 and IL-17 were significantly decreased (B and C in FIG. 9). The expression of serum ferritin (ST) was closely related to the infiltration of inflammatory cells. Compared with the STMC group, the ST content was significantly decreased after gavaging with Lactiplantibacillus plantarum GOLDGUT-LP618 (D in FIG. 9). Immunoglobulin A (IgA) can affect the immune response in the intestinal microenvironment. Compared with the STMC group, the IgA content in the intervention group of Lactiplantibacillus plantarum GOLDGUT-LP618 was significantly increased (E in FIG. 9).

(45) To sum up, Lactiplantibacillus plantarum GOLDGUT-LP618 can alleviate intestinal inflammation induced by Salmonella infection by reducing the level of proinflammatory factors in serum, increasing the level of anti-inflammatory factors, reducing ST content and promoting the secretion of IgA.

Embodiment 8 Protective Effect of Lactiplantibacillus plantarum GOLDGUT-LP618 on Intestinal Mucosa of Mice

(46) To assess whether Lactiplantibacillus plantarum GOLDGUT-LP618 improves the intestinal damage of the mice caused by infection with Salmonella typhimurium, the jejunum and ileum tissue was taken for tissue section and H&E staining analysis, and the results were shown in FIG. 10. In the NC group, the intestinal wall had no void and the Lamina propria structure was intact and normal; in the STMC group, the jejunal wall was thinner and had void and the Lamina propria structure was loose; intestinal villi became short and lost the normal shape; intestinal channels became wide and incomplete; and a few inflammatory cells were infiltrated. Compared with the model group, after the intervention of the positive drugs and the Lactiplantibacillus plantarum GOLDGUT-LP618, the intestinal morphology was changed significantly; the intestinal tissue morphology was recovered to a certain extent; the length of the intestinal villi tended to be normal, and the structure was relatively complete. The void in the intestinal wall was restored; the Lamina propria structure was normal; the mucosa was intact; and the cells were not obviously infiltrated.

(47) The length of the villi, the depth of crypt and the ratio of the villi to the crypt were quantitatively analyzed, and the results were shown in FIG. 11. Compared with the NC group, after infection with Salmonella in the STMC group, the villus length of the jejunum was significantly reduced, and the depth of the crypt was not significantly increased, but the ratio of the villi to the crypt was significantly decreased, which indicated that Salmonella infection seriously damaged the intestinal structure and affected the structural integrity of the intestinal mucosa. Compared with the STMC group, the length of the intestinal villi and the ratio of the villi to the crypt were significantly increased in the positive drug group and the intervention of Lactiplantibacillus plantarum GOLDGUT-LP618. This indicates that the intervention of the Lactiplantibacillus plantarum GOLDGUT-LP618 can significantly promote villus development, improve intestinal health, and alleviate intestinal damage caused by Salmonella infection.

Embodiment 9) Increase of Content of Intestinal Short-Chain Fatty Acid in Mice by Lactiplantibacillus plantarum GOLDGUT-LP618

(48) To verify whether the colonized Lactiplantibacillus plantarum GOLDGUT-LP618 increases the content of the intestinal short-chain fatty acid in mice, the short-chain fatty acid was detected from the colon contents of the mice. The results were shown in FIG. 12. Compared with the NC group, after infection with Salmonella typhimurium, the content of acetic acid in the STMC group was significantly decreased, the content of propionic acid was significantly increased, and the content of butyric acid was not significantly different. Compared with the STMC group, the intervention of the positive drug group and the Lactiplantibacillus plantarum GOLDGUT-LP618 significantly increased the content of acetic acid. Compared with the STMC group, the intervention of the Lactiplantibacillus plantarum GOLDGUT-LP618 did not change the content of propionic acid and butyric acid.

(49) In conclusion, the present invention obtains a strain of Lactiplantibacillus plantarum through isolation and screening, and the strain is named Lactiplantibacillus plantarum GOLDGUT-LP618. The strain has good resistance to stomach acid and bile salt. The Lactiplantibacillus plantarum GOLDGUT-LP618 can produce short-chain fatty acid in vitro and in vivo. The Lactiplantibacillus plantarum GOLDGUT-LP618 has the ability to inhibit Salmonella in vitro and can relieve diarrhea, intestinal inflammation and intestinal damage caused by Salmonella typhimurium in vivo. After the mice are infected with Salmonella typhimurium, colonized Lactiplantibacillus plantarum GOLDGUT-LP618 can improve the intestinal barrier, promote the intestinal health, and reduce diarrhea and inflammatory reaction. Therefore, the Lactiplantibacillus plantarum GOLDGUT-LP618 is a strain that can relieve the bacterial diarrhea caused by Salmonella infection and has anti-inflammatory function.

(50) Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit the technical solutions. Although the present invention is described in detail by referring to the above embodiments, those ordinary skilled in the art shall understand that the technical solutions recorded in the above embodiments can be still amended, or some technical features therein can be replaced equivalently. These amendments or replacements do not enable the essence of the corresponding technical solutions to depart from the spirit and the scope of the technical solutions in the embodiments of the present invention.