CN107502576B - Lactobacillus pentosus and application thereof in inhibiting salmonella - Google Patents
Lactobacillus pentosus and application thereof in inhibiting salmonella Download PDFInfo
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Abstract
The invention discloses lactobacillus pentosus and application thereof in inhibiting salmonella, belonging to the technical field of microorganisms. The invention provides a pharmaceutical composition, which contains Lactobacillus pentosus (Lactobacillus pentosus) AT6 and active ingredients thereof, and can be used for inhibiting the growth of salmonella; the lactobacillus pentosus AT6 can reduce adhesion and invasion of salmonella to HT-29 cells, reduce the load of salmonella in intestinal contents and organs of mice, and slow down death of mice caused by salmonella.
Description
Technical Field
The invention relates to lactobacillus pentosus and application thereof in inhibiting salmonella, belonging to the technical field of microorganisms.
Background
Salmonella (Salmonella) is a generic term for a variety of animal and human diseases caused by bacteria of the genus Salmonella. The host of salmonella includes mammals, birds, reptiles, fishes, humans, etc., and the host is wide and can cause various diseases. The severity of the disease ranges from chronic to acute, from local infection to systemic sepsis, which can severely cause death. The salmonellosis is a global disease, the geographical position distribution is very wide, and most of all countries in the world have salmonellosis, which brings serious economic loss and potential safety hazard.
Salmonella infections in humans are often seen in acute cases of infection, manifested by colic, diarrhea, nausea, vomiting, fever, and the like. The degree of morbidity is related to the salmonella serotype and the dose of infection.
At present, various medicines for treating pathogenic bacteria infection, such as antibiotics and the like, can effectively relieve the disease symptoms after the pathogenic bacteria infection, such as salmonella and the like, are infected. Some traditional Chinese medicine formulas can effectively relieve pathogenic bacteria infection symptoms. However, antibiotics can kill a large amount of intestinal symbiotic bacteria and cause intestinal flora disorder while treating pathogenic bacteria infection, and the use of antibiotics not only has side effects on human bodies, but also can flow into the environment to cause the pressure of the antibiotics in the environment to be increased and destroy the ecological balance. The action and effect of traditional Chinese medicine treatment are not ideal, and the problem of hysteresis is the same as that of antibiotic treatment.
Disclosure of Invention
The first purpose of the invention is to provide lactobacillus pentosus (Lactobacillus pentosus), which has been preserved in China general microbiological culture Collection center (CGMCC) in 29 months in 2017, wherein the preservation number is CGMCC No.13956, and the preservation address is Beijing City Shangyang district Beichen Xilu No.1 institute No. 3, China academy of sciences microbial research institute.
It is a second object of the present invention to provide a pharmaceutical composition comprising the lactobacillus pentosus (lactobacillus pentosus) and/or a metabolite thereof.
In one embodiment of the invention, the content of Lactobacillus pentosus is not less than 1 × 108CFU/mL。
In one embodiment of the invention, the content of Lactobacillus pentosus is not less than 1 × 108CFU/g。
In one embodiment of the present invention, the metabolite is a supernatant collected after inoculating lactobacillus pentosus into MRS medium and centrifuging.
In one embodiment of the invention, the pharmaceutical composition consists of lactobacillus pentosus and a pharmaceutically acceptable carrier.
In one embodiment of the present invention, the pharmaceutically acceptable carrier is one or more carriers selected from the group consisting of fillers, wetting agents, disintegrants, binders, lubricants, and flavoring agents, which are generally used pharmaceutically.
The third purpose of the invention is to provide the application of the lactobacillus pentosus in inhibiting salmonella biomass.
In one embodiment of the invention, the lactobacillus pentosus is inoculated into an MRS culture medium, cultured at 35-37 ℃ for 14-36 h, and then added into a liquid, solid or semisolid containing salmonella.
In one embodiment of the invention, the concentration ratio of lactobacillus pentosus to salmonella is 1: 1-100.
The invention also provides a food or health-care product prepared from the lactobacillus pentosus.
Has the advantages that: the lactobacillus pentosus AT6 culture supernatant is co-cultured with the salmonella, so that the growth of the salmonella can be inhibited, and the biomass is reduced by 82.9%; the lactobacillus pentosus AT6 can reduce the adhesion and invasion of the salmonella to HT-29 cells, and can reduce the adhesion rate by 75.0 percent and the invasion rate by 89.3 percent. The intake of lactobacillus pentosus AT6 reduces the detection rate of salmonella in liver and spleen from 60% and 50% to 0% and 0% respectively, and reduces the amount of salmonella in ileum, caecum and colon contents by 71.0%, 67.7% and 84.3% respectively. The lactobacillus pentosus AT6 can slow down the death of mice caused by salmonella, and the survival rate of the mice is improved from 40% to 65% AT 15 days.
Biological material preservation
The lactobacillus pentosus provided by the invention is preserved in China general microbiological culture Collection center in 29 months in 2017, wherein the preservation number is CGMCC No.13956, the preservation address is Beijing City Zhongyang district Beicheng Xilu No.1, the institute of microbiology of China academy of sciences.
Drawings
FIG. 1 is a graph showing the inhibitory effect of a supernatant of Lactobacillus pentosus AT6 culture on Salmonella in LB medium; wherein the AT6 group is co-cultured by Lactobacillus pentosus AT6 and Salmonella; the LGG group is co-cultured by Lactobacillus rhamnosus LGG and salmonella; MRS group is blank control group, and MRS culture medium and salmonella are co-cultured; the HCl-3.81 group is characterized in that MRS and salmonella are co-cultured, the pH value of the MRS is adjusted to 3.81 by hydrochloric acid; the LA-3.81 group is co-cultured with MRS and Salmonella, the pH of which is adjusted to 3.81 by DL-lactic acid;
FIG. 2 shows the effect of Lactobacillus pentosus AT6 on inhibiting adhesion of Salmonella to HT-29 cells, wherein CON group is control group, blank 1640 medium is added to six-well plate, and 1.0 × 10 is added8CFU Salmonella, LGG group indicates addition of 1.0 × 10 to six well plates8Adding 1.0 × 10 into CFU Lactobacillus rhamnosus LGG8CFU Salmonella, AT6 group indicates addition of 1.0 × 10 to six well plates8CFU Lactobacillus pentosus AT6, and adding 1.0 × 108CFU salmonella;
FIG. 3 shows the effect of Lactobacillus pentosus AT6 in inhibiting Salmonella invasion of HT-29 cells, wherein CON group is control group, blank 1640 medium is added to six-well plate, and 1.0 × 10 is added8CFU Salmonella, LGG group indicates addition of 1.0 × 10 to six well plates8Adding 1.0 × 10 into CFU Lactobacillus rhamnosus LGG8CFU Salmonella, AT6 group indicates addition of 1.0 × 10 to six well plates8CFU Lactobacillus pentosus AT6, and adding 1.0 × 108CFU salmonella;
FIG. 4 is the effect of Lactobacillus pentosus AT6 on Salmonella burden in the liver and spleen of mice infected with Salmonella; wherein, lever-con represents the salmonella load of the liver of the model group; the lever-LGG represents the hepatic salmonella load of the LGG treated group of lactobacillus rhamnosus; liver-AT6 represents hepatic salmonella load in lactobacillus pentosus AT 6-treated group; spleen-con represents the splenic salmonella load of the model group; spleen-LGG represents the splenic salmonella load of the LGG treated group of Lactobacillus rhamnosus; spleen-AT6 represents splenic salmonella load of lactobacillus pentosus AT 6-treated group;
FIG. 5 is the effect of Lactobacillus pentosus AT6 on the Salmonella load in intestinal contents of Salmonella-infected mice; wherein Ileum-con represents the Salmonella load in Ileum contents of the model group; Ileum-LGG represents the salmonella load in Ileum contents of a Lactobacillus rhamnosus LGG-treated group; Ileum-AT6 represents the salmonella load in Ileum contents of lactobacillus pentosus AT 6-treated group; cecum-con represents the salmonella loading in the cecal contents of the model group; Cecum-LGG represents the salmonella loading in the cecal content of the LGG treated group of lactobacillus rhamnosus; Cecum-AT6 represents the Salmonella load in the cecal content of the Lactobacillus pentosus AT 6-treated group; colon-con represents the Salmonella load in the contents of the model Colon; Colon-LGG represents the salmonella load in Colon contents of the LGG-treated group of Lactobacillus rhamnosus; Colon-AT6 indicates Salmonella load in Colon contents treated with Lactobacillus pentosus AT 6;
FIG. 6 is a graph showing that ingestion of Lactobacillus pentosus AT6 enhances survival of mice after Salmonella typhimurium infection; wherein, the control group is perfused with PBS; model group Salmonella gavage; in the LGG group, firstly, the lactobacillus rhamnosus LGG is gavaged, and then, the salmonella is gavaged; in the AT6 group, Lactobacillus pentosus was first gavaged, followed by Salmonella gavage.
Detailed Description
MRS liquid medium (per 1L): 10g of tryptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 2g of diammonium hydrogen citrate, 5g of anhydrous sodium acetate, 2g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate and 801mL of Tween, adding water to 1000mL, adjusting the pH to 6.2-6.4, carrying out moist heat sterilization at 115 ℃ for 20 min.
MRS solid medium (per L): agar powder 15g/L is added into the liquid culture medium.
LB medium (per L): 5.0g of yeast powder, 10.0g of tryptone and 5.0g of sodium chloride. Adding water 1L, and performing moist heat sterilization at 121 deg.C for 15 min.
Activating strains: and selecting a small amount of bacteria liquid from the environment-friendly bacteria tube by using an inoculating loop, and marking in an MRS solid flat plate. Culturing in a 37 ℃ incubator until a single colony grows.
And (3) strain culture: and picking a single colony on the MRS solid culture medium by using an inoculating loop to an MRS liquid culture medium, and carrying out static culture at 37 ℃ for 14-18 h. Then, 1% of the inoculum size is transferred to a liquid MRS culture medium, and the static culture is carried out for 14-18h at 37 ℃. For each activation, the seeds are inoculated with 1 percent of inoculum size and cultured for 14-18h at 37 ℃.
And (3) measuring the bacterial load: after the mice are anesthetized and sacrificed, taking the contents of ileum, caecum and colon, weighing, diluting in a gradient way and counting; taking the liver and the spleen, homogenizing, diluting in a gradient manner, and counting.
The counting method comprises the following steps: the samples were spread on a MacConkey agar plate containing 50mg/L streptomycin and cultured at 37 ℃ for about 12 hours until colonies grew.
The lactobacillus pentosus (lactobacillus pentosus) provided by the present invention is abbreviated as lactobacillus pentosus AT6 in the following examples. Since no literature report on the inhibition of the salmonella by the lactobacillus pentosus is available at present, the lactobacillus rhamnosus capable of inhibiting the salmonella is taken as a control.
Example 1: co-culture of supernatant of Lactobacillus pentosus AT6 culture with Salmonella
(1) Preparation of culture supernatant of Lactobacillus pentosus AT6
After the passage of Lactobacillus pentosus AT6 and Lactobacillus rhamnosus LGG (i.e., Lactobacillus rhamnosus ATCC53103), the culture was cultured AT 37 ℃ for 18 hours and centrifuged, and the pH of the culture supernatant was measured while adjusting the MRS broth to the same pH (pH3.81) as that of the AT6 culture supernatant with hydrochloric acid or DL-lactic acid as a control group. The culture supernatant and the control group were filtered through a sterile filter with a pore size of 0.22 μm.
(2) Preparation of Salmonella cultures
The culture medium of the salmonella typhimurium SL1344 is an LB culture medium, and after the salmonella is activated and cultured in the LB culture medium by the inoculum size of 2 percent (2mL/100mL), the salmonella is shake-cultured for 12h at 37 ℃ and 225 rpm.
(3) Co-culture of supernatant of Lactobacillus pentosus AT6 culture with Salmonella
50. mu.L of culture supernatant of Lactobacillus pentosus AT6 and Lactobacillus rhamnosus LGG or control group MRS medium was added to 5mLLB broth, followed by 50. mu.L of 2.0 × 109CFU/mL of Salmonella were co-cultured. Standing at 37 deg.C for 24h, and measuring OD of co-culture at different times600。
The results are shown in FIG. 1: AT the time of 4h culture, AT6 culture supernatant, LGG culture supernatant, MRS medium, MRS adjusted to pH3.81 with HCL, and OD of MRS group adjusted to pH3.81 with DL-lactic acid were added6000.026, 0.037, 0.574, 0.278, 0.042, respectively; OD of AT6, LGG, MRS, HCl-3.81 and LA-3.81 groups AT 12h6000.038, 0.178, 0.705, 0.525, 0.233, respectively; OD of AT6, LGG, MRS, HCl-3.81 and LA-3.81 groups in 24h culture600Respectively 0.133, 0.315, 0.781, 0.603 and 0.246, which shows that the lactobacillus pentosus AT6 can obviously inhibit the growth of the salmonella, and the inhibition effect is even better than that of the organic acid.
Example 2: inhibition of Salmonella adhesion HT-29 cells by Lactobacillus pentosus AT6
(1) After passaging Lactobacillus pentosus AT6 and Lactobacillus rhamnosus LGG, it was cultured AT 37 ℃ for 18h in MRS medium, centrifuged, washed three times with PBS, and the cells were resuspended to 1.0 × 10 in 1640 medium (purchased from Gibco)8CFU/mL。
(2) Activating and culturing salmonella in LB culture medium, standing and culturing for 12h, washing three times with PBS, and suspending the thallus to 1.0 × 10 with 1640 culture medium8CFU/mL。
(3) Preparation of HT-29 cells: the medium required for HT-29 growth was 1640 medium supplemented with 5% (v/v) Fetal Bovine Serum (FBS) and no penicillin/streptomycin addition. After activation of HT-29 cells, they were grown to a monolayer in 6-well plates and ready for use.
(4) Measurement of adhesion inhibition: washing HT-29 cells with PBS for three times, adding 1mL of Lactobacillus pentosus AT6, 1mL of Lactobacillus rhamnosus LGG or 1640 culture medium (control group) into each well, and culturing for 1h in a carbon dioxide incubator; adding 1mL of the salmonella suspension cultured in the step 2 into each hole, and culturing for 1h in a carbon dioxide incubator; washing with PBS for three times, adding 0.5% TritonX-100 into each well, and blowing and mixing well with a pipette after 20 min; the diluted solution is diluted in gradient, poured by LB solid medium added with streptomycin 50mg/L and counted after colonies grow out.
The CON group is a control group, and 1mL1640 culture medium is added into each hole of a 6-hole plate before adding the salmonella; LGG group was prepared by adding 1mL1 per well in 6 well plates before Salmonella.0×108CFU/mL of LGG bacterial suspension of Lactobacillus rhamnosus, AT6 group is prepared by adding 1mL of 1.0 × 10 per well in 6-well plate before adding Salmonella8CFU/mL of Lactobacillus pentosus AT6 bacterial suspension.
As shown in FIG. 2, the LOG values of the numbers of Salmonella adhesion to cells per well of the CON, LGG and AT6 groups were 6.20, 5.98 and 5.81, respectively.
Example 3: inhibition of Salmonella invasion of HT-29 cells by Lactobacillus pentosus AT6
(1) After passaging Lactobacillus pentosus AT6 and Lactobacillus rhamnosus LGG, it was cultured AT 37 ℃ for 18h in MRS medium, centrifuged, washed three times with PBS, and the cells were resuspended to 1.0 × 10 in 1640 medium (purchased from Gibco)8CFU/mL。
(2) Activating and culturing salmonella in LB culture medium, standing and culturing for 12h, washing three times with PBS, and suspending the thallus to 1.0 × 10 with 1640 culture medium8CFU/mL。
(3) Preparation of HT-29 cells: the medium required for HT-29 growth was 1640 medium supplemented with 5% (v/v) Fetal Bovine Serum (FBS) and no penicillin/streptomycin addition. After activation of HT-29 cells, they were grown to a monolayer in 6-well plates and ready for use.
(4) Invasion inhibition experiment: washing HT-29 cells with PBS for three times, adding 1mL of Lactobacillus pentosus AT6, 1mL of Lactobacillus rhamnosus LGG or 1640 culture medium (control group) into each well, and culturing for 1h in a carbon dioxide incubator; adding 1mL of the salmonella suspension cultured in the step 2 into each hole, and culturing for 1h in a carbon dioxide incubator; washing with PBS for three times, adding 1mL gentamicin into each well, and treating at 37 deg.C for 45 min; washing with PBS for three times; adding 0.5% TritonX-100 into each hole, and blowing and stirring uniformly by a pipette after 20 min; the diluted solution is diluted in gradient, poured by LB solid medium added with streptomycin 50mg/L and counted after colonies grow out.
CON group is control group, 1mL1640 culture medium is added into each well of 6-well plate before adding salmonella, and LGG group is 1mL1.0 × 10 added into each well of 6-well plate before adding salmonella8CFU/mL of LGG bacterial suspension of Lactobacillus rhamnosus, AT6 group is prepared by adding 1mL of 1.0 × 10 per well in 6-well plate before adding Salmonella8CFU/mL of Lactobacillus pentosus AT6 bacterial suspension.
As shown in FIG. 3, the LOG values of the numbers of Salmonella adhesion to cells per well of the CON, LGG and AT6 groups were 6.04, 5.68 and 5.49, respectively.
Example 4: influence of lactobacillus pentosus AT6 on bacterial load of intestinal contents and detection rate of salmonella in liver and spleen of mice infected by salmonella
The mice are of C57BL/6 strain, female, 6-8w, SPF grade, 10 mice per group, divided into 3 groups.
Model group-PBS gavage 10 days, 0.1 mL/day, 11 days Salmonella gavage 1.0 × 106CFU/mouse；
Prevention group-Lactobacillus pentosus AT6 gavage for 10 days, 0.1mL (5 × 10)8CFU)/day, day 11 Salmonella gavage 1.0 × 106CFU/mouse；
Control group-Lactobacillus rhamnosus LGG gavage 10 days, 0.1mL (5 × 10)8CFU)/day, day 11 Salmonella gavage 1.0 × 106CFU/mouse
The method comprises the following specific steps:
(1) activating and subculturing lactobacillus pentosus AT6 and lactobacillus rhamnosus LGG, culturing AT 37 deg.C for 18 hr, centrifuging, washing with PBS for three times, and resuspending to 5 × 109CFU/mL。
(2) Mice were treated: model group PBS was gavage, prevention group Lactobacillus pentosus AT6 was gavage, control group Lactobacillus rhamnosus LGG was gavage, treated for 10 days.
(3) Salmonella gavage on day 11, 1.0 × 106CFU/mouse。
The liver and spleen bacterial load results are shown in FIG. 4, and the liver bacterial loads of lever-con, lever-LGG, lever-AT 6, spen-con, spen-LGG and spen-AT 6 are 7.7CFU, 0.7CFU, 0CFU, 3.9CFU, 1.6CFU and 0CFU, respectively.
The bacterial load of Ileum, Cecum and Colon contents is shown in figure 5, and LOG values of Ileum-con, Ileum-LGG, Ileum-AT6, Cecum-con, Cecum-LGG, Cecum-AT6, Colon-con, Colon-LGG and Colon-AT6 bacterial loads are respectively 2.58, 1.04, 0.75, 2.85, 1.32, 0.92, 2.37, 0.86 and 0.37.
Example 5: change in mortality of mice following Salmonella infection following intake of Lactobacillus pentosus AT6
The mice are C57BL/6 strain, female, 6-8w, SPF grade, 10 mice/group, and divided into three groups.
A blank control group, namely PBS intragastric for 10 days, 0.1 mL/day, and PBS intragastric on day 11;
model group-PBS gavage 10 days, 0.1 mL/day, 11 days Salmonella gavage 1.0 × 106CFU/mouse；
Prevention group-Lactobacillus pentosus AT6 gavage for 10 days, 0.1mL (5 × 10)8CFU)/day, day 11 Salmonella gavage 1.0 × 106CFU/mouse；
Positive control group-Lactobacillus rhamnosus LGG gavage 10 days, 0.1mL (5 × 10)8CFU)/day, day 11 Salmonella gavage 1.0 × 106CFU/mouse。
The method comprises the following specific steps:
(1) culturing Lactobacillus pentosus AT6 and Lactobacillus rhamnosus LGG AT 37 deg.C for 18h, centrifuging, washing with PBS for three times, and resuspending to 5 × 109CFU/mL。
(2) Mice were treated: and performing gavage by PBS (phosphate buffer solution) in a control group and a model group, performing gavage by Lactobacillus pentosus AT6 in a prevention group, and performing treatment for 10 days in total by using Lactobacillus rhamnosus LGG in a positive control group.
(3) Salmonella infection Salmonella gavage on day 11, 1.0 × 106CFU/mouse。
(4) Mortality observation: mice in each group were observed daily for mortality and recorded.
The results show that: 20 mice in each group died 0 mice in the control group 15 days after the gavage of salmonella, and the mortality rate was 0%; model group (model) mice died 12, with 60% deaths, i.e., 40% survival; the treated group (lactobacillus pentosus AT6) mice died 7, with a mortality rate of 35%, i.e. a survival rate of 65%; control (lactobacillus rhamnosus LGG) mice died 8, with a mortality rate of 40% and a survival rate of 60%. Lactobacillus pentosus AT6 treatment reduced the mortality of mice by salmonella by 25% AT 15 days.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. Lactobacillus pentosus (Lactobacillus pentosus) is preserved in China general microbiological culture Collection center (CGMCC) at 29.3.2017 with the preservation number of CGMCC No.13956, and the preservation address of Beijing province No. 3 of Suzuku No.1 of North Chen West Lu of the sunward province, China academy of sciences.
2. A pharmaceutical composition comprising the Lactobacillus pentosus (Lactobacillus pentosus) of claim 1.
3. The pharmaceutical composition according to claim 2, wherein the content of Lactobacillus pentosus is not less than 1 × 108CFU/g or 1 × 108CFU/mL。
4. The pharmaceutical composition of claim 2 or 3, further comprising a pharmaceutically acceptable carrier.
5. The pharmaceutical composition according to claim 4, wherein the pharmaceutically acceptable carrier is one or more carriers selected from the group consisting of fillers, wetting agents, disintegrants, binders, lubricants and flavoring agents which are generally used pharmaceutically.
6. A method of inhibiting salmonella biomass in vitro, characterized in that lactobacillus pentosus according to claim 1 is added to a liquid, semi-solid or solid containing salmonella.
7. The method according to claim 6, wherein the Lactobacillus pentosus of claim 1 is inoculated into MRS medium, cultured at 35-37 ℃ for 14-36 h, and then added to a Salmonella-containing liquid, solid or semi-solid.
8. The method according to claim 7, wherein the concentration ratio of Lactobacillus pentosus to Salmonella of claim 1 is 1:1 to 100.
9. Food product prepared using the lactobacillus pentosus according to claim 1.
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