CN115261251B - Streptococcus thermophilus S869 and application thereof in regulating immunity and intestinal functions - Google Patents
Streptococcus thermophilus S869 and application thereof in regulating immunity and intestinal functions Download PDFInfo
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- CN115261251B CN115261251B CN202210487799.6A CN202210487799A CN115261251B CN 115261251 B CN115261251 B CN 115261251B CN 202210487799 A CN202210487799 A CN 202210487799A CN 115261251 B CN115261251 B CN 115261251B
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Abstract
The invention relates to the technical field of microorganisms, and provides streptococcus thermophilus (Streptococcus thermophilus) S869 which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, wherein the preservation number is CGMCC No.24190. Through the technical scheme, the problems that the immunocompetence difference of the live bacteria and the inactivated bacteria in the existing bacterial strain is large, and the existing bacterial strain cannot be used for adjusting the organism immunity and the intestinal function well at the same time are solved.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to streptococcus thermophilus S869 and application thereof in regulating immunity and intestinal functions.
Background
With the rapid development of economy and the improvement of living standard of residents, more and more people pay attention to sub-health conditions of bodies, and accordingly, the probiotic composition is welcomed because of no side effect on bodies. And the probiotics are scientifically and reasonably used, which is beneficial to the microecological balance and the health of human bodies.
Probiotics are a class of active microorganisms that benefit a host by colonizing the human body to alter the composition of the host's flora at a location. By regulating the immune function of host mucous membrane and system or regulating the balance of flora in intestinal tract, the effect of promoting nutrient absorption and maintaining intestinal health is achieved, so that single microorganism or mixed microorganism with definite composition beneficial to health is produced.
The probiotics have strain specificity in regulating the immunity of organisms, different strains have differences in the capacity of regulating the immunity, and most active probiotics are on the market, and can regulate the immunity of organisms when a certain number of live bacteria are taken in a sufficient quantity. However, active lactic acid bacteria have a certain limitation in regulating immunity, and the action mechanism of the inactivated bacteria for regulating immunity is not clear. The most traditional probiotics such as lactobacillus, bifidobacterium and the like are verified to have the function of regulating the immunity of organisms.
In addition, another large use of probiotics is to regulate intestinal health, and more probiotics currently on the market include: bifidobacterium lactis, bifidobacterium longum, lactobacillus rhamnosus, lactobacillus casei and the like are verified to be capable of regulating intestinal dysfunction, preventing diarrhea and reducing constipation.
While streptococcus thermophilus (Streptococcus thermophilus) is mainly used in the field of yogurt fermentation, and has less research on the probiotic action of streptococcus thermophilus single strains. Therefore, there is a need for more targeted screening of strains with specific physiological activities.
Disclosure of Invention
The invention provides streptococcus thermophilus S869 and application thereof in regulating immunity and intestinal functions, and solves the problems that the immunocompetence difference between live bacteria and inactivated bacteria in the existing strain is large, and the existing strain cannot simultaneously regulate body immunity and intestinal functions.
The technical scheme of the invention is as follows:
streptococcus thermophilus (Streptococcus thermophilus) S869 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the month of 2021 and 24, wherein the preservation address is the North Chen Xili No. 1 and 3 of the Korean area of Beijing city, the preservation number is CGMCC No.24190, and the viability of the biological material is detected as viable by the preservation center.
The invention also provides a streptococcus thermophilus bacterial preparation which contains the streptococcus thermophilus and is a solid or liquid bacterial preparation.
The invention also provides a food containing the streptococcus thermophilus or the streptococcus thermophilus bacterial preparation.
The invention also provides application of the streptococcus thermophilus S869 in preparing a composition for regulating immunity, wherein the composition comprises food, health-care products, medicines and feeds.
The invention also provides application of the streptococcus thermophilus S869 in preparing a composition for promoting proliferation and endocytosis of RAW264.7 macrophages, wherein the composition comprises food, health care products, medicines and feeds.
The invention also provides application of the streptococcus thermophilus S869 in preparing a composition for regulating intestinal functions, wherein the composition comprises food, health-care products, medicines and feeds.
The invention also provides application of the streptococcus thermophilus S869 in preparing a composition for preventing and repairing intestinal epithelial cell injury caused by ETEC, wherein the composition comprises food, health products, medicines and feeds.
The invention also provides application of the streptococcus thermophilus S869 in preparation of a composition for improving the expression level of the cell mucin MUC2 and MUC5AC genes, wherein the composition comprises food, health care products, medicines and feeds.
As a further technical scheme, the composition comprises one or more of streptococcus thermophilus S869 viable bacteria, inactivated bacteria and metabolites.
As a further technical scheme, the formulation of the composition comprises any one of powder, granules, emulsion and tablets.
The invention has the beneficial effects that:
1. the streptococcus thermophilus S869 can produce extracellular polysaccharide with high yield, and the activity and the inactivation type streptococcus thermophilus S869 can promote proliferation and phagocytic capacity of RAW264.7 macrophages and can stimulate cells to secrete cytokines such as TNF-alpha, IL-6, IL-10 and the like; the enhancement of cytokines TNF-alpha and IL-6 can excite immune defense mechanisms, but excessive secretion can cause immune disorder and cause immune imbalance; anti-inflammatory factor IL-10 can inhibit the induction synthesis and secretion of inflammatory factor, and can protect organism. The active and inactivated streptococcus thermophilus S869 strain can improve the immunity of organisms and has extremely high application value.
2. The streptococcus thermophilus S869 has the capability of repairing intestinal epithelial cell injury caused by ETEC, improves the expression level of cell mucin MUC2 and MUC5AC genes, and can repair and prevent intestinal mucosa barrier injury caused by ETEC. S869 can be used in a diarrhea composition for preventing and treating abnormal neurotransmitter expression and enterotoxigenic escherichia coli infection, and the S869 has good effects in repairing intestinal epithelial cell injury and intestinal mucosa barrier.
Drawings
The invention will be described in further detail with reference to the drawings and the detailed description.
FIG. 1 is a glucose standard curve of an exopolysaccharide experiment;
FIG. 2 is a graph showing comparison of extracellular polysaccharide yields of Streptococcus thermophilus S869 and MRS medium;
FIG. 3 is a graph showing comparison of cell proliferation in Streptococcus thermophilus S869, lactobacillus rhamnosus LGG, DMEM medium;
FIG. 4 is a graph showing comparison of the phagocytosis of Streptococcus thermophilus S869, lactobacillus rhamnosus LGG and DMEM medium cells;
FIG. 5 is the effect of experimental strains on mouse body mass;
FIG. 6 is the effect of experimental strains on the expression of intestinal epithelial cell mucin MUC2 mRNA;
FIG. 7 is the effect of experimental strains on the expression of the intestinal epithelial cell mucin MUC5AC mRNA;
FIG. 8 is an effect of experimental strains on intestinal epithelial cell serotonin transporter SERT mRNA expression;
FIG. 9 shows the effect of experimental strains on intestinal epithelial cell proliferation.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Streptococcus thermophilus (Streptococcus thermophilus) S869 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 24190 in the 12 th month of 2021.
Experimental example 1
The experimental steps are as follows:
(1) Screening of extracellular polysaccharide-producing Strain
The S869 streptococcus thermophilus was streaked (2 replicates) on MRS solid medium to obtain isolated and purified strains of S869 streptococcus thermophilus, then cultured anaerobically at 37℃for 48h, and the colony characteristics were observed and recorded. The bacterial colony is lightly touched by using a sterile toothpick, is lightly pulled outwards and is controlled to vertically leave in 2 seconds, so that continuous wiredrawing is formed on the surface of a culture medium, 6 bacterial colonies are repeated, 2 parallel tests are carried out on each bacterial colony, the maximum length (mm) of the wiredrawing of the bacterial colony is measured, the result is expressed as an average value plus or minus standard deviation, and the bacterial strain with the wiredrawing length of more than 40mm is screened. The results show that the colony wiredrawing length of the test strain streptococcus thermophilus S869 is above 40 mm.
(2) Extraction of extracellular polysaccharide
Activating the strain, transferring the strain to an MRS liquid culture medium, culturing for 48 hours at 37 ℃, taking 10mL of culture solution, carrying out water bath at 95 ℃ for 5 minutes to deactivate enzyme, centrifuging, taking supernatant, taking 5mL of cooled supernatant, adding 2.5mL of 12% trichloroacetic acid, standing for half an hour, centrifuging after stirring, placing the supernatant into a centrifuge tube, adding 95% ethanol with the volume of three times that of the supernatant, precipitating with ethanol at 4 ℃ for overnight, centrifuging to obtain precipitate, namely extracellular polysaccharide, adding 10mL of pure water, dialyzing in a dialysis bag for 2 days to obtain extracellular crude polysaccharide water solution, and changing water every 8 hours in the dialysis process.
(3) Determination of extracellular polysaccharide content by sulfuric acid-phenol method
Taking 11 25mL test tubes with plugs, respectively weighing 100 mug/mL glucose standard solution 0, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL and 1mL, respectively adding pure water to 1mL, adding 1mL of phenol solution with mass concentration of 6%, shaking uniformly, adding 5mL of concentrated sulfuric acid, shaking uniformly again, standing for 30min at room temperature, measuring absorbance at 490nm of an enzyme-labeled instrument wavelength, drawing a glucose standard curve by taking the mass concentration of glucose as an abscissa and the absorbance as an ordinate, and drawing a glucose standard curve y=0.005 x-0.01, R as shown in figure 1 2 ＝0.998。
1mL of the above crude extracellular polysaccharide aqueous solution is added into a test tube with a plug, 1mL of MRS culture medium is taken as a control, then 1mL of phenol solution with mass concentration of 6% is respectively added, 5mL of concentrated sulfuric acid is added after shaking uniformly, shaking is performed again, standing is performed for 30 minutes at room temperature, absorbance is measured at 490nm of the wavelength of an enzyme-labeled instrument, and the extracellular polysaccharide content is calculated according to a glucose standard curve, as shown in FIG. 2.
Experimental results:
as shown in Table 1, the yield of the extracellular polysaccharide with MRS as a control was (304.2.+ -. 0.416) μg/mL; the extracellular polysaccharide yield of S869 was (317.2.+ -. 1.217) μg/mL for Streptococcus thermophilus with significant differences (P < 0.001). S869 increased the cellular polysaccharide yield by 4.3% compared to MRS. The results show that S869 Streptococcus thermophilus has the ability to produce high extracellular polysaccharide.
TABLE 1 Extracellular Polysaccharide (EPS) yield of lactic acid bacteria
| Group of | Control group (MRS culture medium) | Streptococcus thermophilus S869 |
| EPS yield (μg/mL) | 304.2±0.416 | 317.2±1.217 |
Experimental example 2
The experimental steps are as follows:
(1) Cell culture
Resuscitating RAW264.7 cells, placing into a culture flask containing DMEM complete culture solution, and incubating in an incubator at 37deg.C under 5% CO 2 And after the cells grow well and the density reaches 80%, carrying out passage for 3 times.
(2) Preparation of bacterial suspension
The live and inactivated Streptococcus thermophilus S869 bacterial suspensions and the live and inactivated Lactobacillus rhamnosus LGG bacterial suspensions were prepared as follows.
(1) Inoculating lactobacillus into MRS liquid culture medium, activating for 3 generations, culturing at 37deg.C for 18h, centrifuging at 4deg.C for 5min at 5000r/min, and collecting thallus;
(2) by usingWashing the collected thalli with PBS for 1 time, centrifuging to remove supernatant, centrifuging at 4deg.C and 5000r/min for 5min, re-suspending the thalli with DMEM culture solution containing 10% foetal calf serum, gradient diluting the re-suspended thalli 1mL with 0.9% physiological saline, detecting bacterial concentration with flow cytometry, and regulating bacterial concentration to 1.5X10 8 CFU/mL, the viable bacteria suspension is obtained.
(3) Washing the collected thalli with PBS for 1 time, centrifuging to remove supernatant, centrifuging at 4deg.C and 5000r/min for 5min, re-suspending the thalli obtained by centrifuging with PBS, inactivating in water bath at 80deg.C for 30min, centrifuging again to remove supernatant, re-suspending the precipitate with DMEM culture solution containing 10% foetal calf serum, gradient diluting the re-suspended 1mL of bacterial solution with 0.9% physiological saline, detecting bacterial concentration with flow cytometry, and adjusting bacterial concentration to 1.5X10 × 8 CFU/mL to obtain the inactivated bacterial suspension.
(3) Cell proliferation assay
The cells after 3 generations of RAW264.7 obtained in the step (1) are dyed by 0.4% trypan blue dyeing liquid, 40 mu L of cell suspension and 40 mu L of 0.4% trypan blue solution are evenly mixed at a ratio of 1:1, 20 mu L of cell suspension is counted by an automatic cell counter, the cells are diluted, and the density of the cells is 1.5 multiplied by 10 in a 96-well plate 5 Inoculating 100. Mu.L of cell suspension per well at 37℃and 5% CO 2 Incubate overnight in incubator.
Removing supernatant after cell adhesion, adding 100 μl of cell culture solution (DMEM culture medium containing 10% foetal calf serum) into each hole, adding 100 μl of the live bacterial suspension or inactivated bacterial suspension in step (2), respectively, and repeating the blank control group with 100 μl of cell culture solution 5 times, wherein the live bacterial group and cells are cultured at 37deg.C and 5% CO 2 The cells were incubated under the same conditions for 24h with the inactivated bacterial groups.
The CCK-8 method for detecting the activity and proliferation of cells comprises the following specific operations: mu.L of CCK-8 solution was added to each well at 37℃with 5% CO 2 Incubate for 1h and determine the OD of absorbance at a wavelength of 450 nm.
Cell proliferation index and proliferation rate were calculated according to the following formulas:
cell proliferation index= (As-Ac)/Ac;
proliferation rate = cell proliferation index x 100%;
wherein: as is the OD value of the experimental group, ac is the OD value of the blank group.
(4) Phagocytosis experiment
RAW264.7 cells in logarithmic phase were stained with 0.4% trypan blue staining solution, 40. Mu.L of cell suspension was mixed with 40. Mu.L of 0.4% trypan blue solution, and after 3 minutes, 20. Mu.L was counted with an automatic cytometer and diluted to adjust the concentration to 1.5X10 5 100. Mu.L of cell suspension was added to each well of the 96-well plate at 37℃with 5% CO 2 Incubate overnight in incubator.
Removing the culture solution, adding 100 mu L of cell culture solution (DMEM culture medium containing 10% fetal calf serum) into each hole, adding 100 mu L of the live bacterial suspension or the inactivated bacterial suspension in the step (2) respectively, wherein each group comprises 5 compound holes, the blank control group comprises the DMEM culture medium containing 10% fetal calf serum, the live bacterial group and cells are incubated for 4 hours, and the inactivated bacterial group and macrophages are incubated for 24 hours.
The neutral red kit detects the phagocytic effect of cells, and comprises the following specific operations: after the incubation, the supernatant from the 96-well plate was aspirated, washed 2 times with PBS, 200. Mu.L of cell culture medium and 20. Mu.L of neutral red dye were added, and incubated in a cell incubator for 2 hours. The cell culture solution containing the neutral red dye solution was removed, washed 1 time with PBS, and 200. Mu.L of neutral red detection lysate was added to each well to lyse in chamber Wen Yaochuang for 10min. OD values were measured at 540nm wavelength, and cell proliferation index and proliferation rate were calculated according to the following formulas:
cytophagy index = As/Ac;
phagocytic rate = phagocytic index of cells x 100%;
wherein: as represents the OD value of the experimental group, ac represents the OD value of the blank group
(5) ELISA method for detecting cytokine content in supernatant
Cell culture and preparation of bacterial suspension were performed as described above, and 2mL concentration was 2X 10 5 Cells were seeded in 24-well plates at 37℃with 5% CO 2 Culturing overnight in incubator, removing supernatant, adding 2mL DMEM medium containing 10% foetal calf serum. The method comprises the steps of utilizing LPS to mediate cells to generate inflammatory cells, setting six different groups, namely a negative control group, a positive control group, an LGG group, an S869 group, a prevention group and a treatment group, wherein the concentration of the LPS is 1 mug/mL, repeating the steps of each group for 5 times, and incubating the six different groups with the cells, wherein the specific steps are as follows:
(1) negative control group (DMEM)
Adding 100 mu L/hole DMEM into a 24-hole plate, incubating in a cell incubator, incubating a first negative control group for 4 hours, and incubating a second negative control group for 24 hours;
(2) positive control group (LPS)
LPS is added into a 24-hole plate to make the final concentration of the LPS be 1 mug/mL, the LPS is incubated in a cell incubator, the first positive control group is incubated for 4 hours, and the second positive control group is incubated for 24 hours;
(3) LGG group
Adding 100 mu L/lactobacillus rhamnosus LGG viable bacteria suspension or inactivated bacteria suspension into a 24-hole plate, incubating in a cell incubator, incubating a viable bacteria group for 4 hours, and incubating an inactivated bacteria group for 24 hours;
(4) s869 group
Adding 100 mu L/Kong Shire streptococcus S869 viable bacteria suspension or inactivated bacteria suspension into a 24-hole plate, incubating in a cell incubator, incubating a viable bacteria group for 4 hours, and incubating an inactivated bacteria group for 24 hours;
(5) prevention group (S869/LPS)
Adding 100 mu L/hole of streptococcus thermophilus S869 viable bacteria suspension or inactivated bacteria suspension into a 24-hole plate, incubating in a cell incubator, incubating a viable bacteria group for 4 hours, and incubating an inactivated bacteria group for 12 hours; then adding LPS (final concentration is 1 mug/mL), incubating the live bacteria for 4 hours, and incubating the inactivated bacteria for 12 hours;
(6) treatment group (LPS/S869)
LPS (final concentration is 1 mu g/mL) is firstly added into a 24-hole plate, the mixture is incubated in a cell culture box, a live bacterial group is firstly incubated for 4 hours, and an inactivated bacterial group is firstly incubated for 12 hours; then adding 100 mu L/hole of streptococcus thermophilus S869 viable bacteria suspension or inactivated bacteria suspension, incubating the viable bacteria group for 4 hours, and incubating the inactivated bacteria group for 12 hours;
after the incubation is finished, collecting cell culture supernatants of each group, centrifuging at 1000r/min for 10min to remove particles and polymers, respectively subpackaging into sterile centrifuge tubes, and preserving at low temperature.
The amounts of TNF- α, IL-6, IL-10 cytokines secreted in the supernatants of the upper groups were determined according to ELISA kit instructions.
The amount of expression of cytokine mRNA levels was determined by fluorescent quantitative qPCR.
Experimental results:
(1) Cell proliferation results
As shown in FIG. 3, compared with the control group, the live bacteria and the inactivated bacteria of S869 of the streptococcus thermophilus have better proliferation effect on macrophages. Compared with lactobacillus rhamnosus LGG, in the living bacteria, the proliferation effect of streptococcus thermophilus S869 on macrophages is lower than that of lactobacillus rhamnosus LGG; in the inactivated bacteria, the proliferation capacity of the inactivated streptococcus thermophilus S869 on macrophages is higher than that of the inactivated lactobacillus rhamnosus LGG, and the inactivated streptococcus thermophilus S869 has a remarkable difference (P is less than 0.05).
(2) Phagocytosis results of cells
As shown in fig. 4, compared with the control group, both the live bacteria and the inactivated bacteria of streptococcus thermophilus S869 can greatly promote the phagocytic effect of macrophages; in viable bacteria, the phagocytic capacity of streptococcus thermophilus S869 on promoting macrophages is higher than that of lactobacillus rhamnosus LGG, and the difference is significant (P < 0.05); among the inactivated bacteria, the inactivated streptococcus thermophilus S869 can obviously promote phagocytosis of macrophages, and the phagocytosis effect is obviously higher than that of lactobacillus rhamnosus LGG, and has extremely obvious difference (P is less than 0.001).
(3) ELISA method for detecting cytokine content
TABLE 2 Effect of viable bacteria on cytokine secretion
Note that: "-" means that the cytokine secretion is less than the detection limit; "+" represents secretion amount of 15.625-125pg/mL; "++" represents a secretion amount of 125-500pg/mL; "+". ++'s representing secretion an amount of 500-1000pg/mL; "+". ++'s representing secretion an amount greater than 1000pg/mL;
as shown in Table 2, both the S869 viable bacteria and the LGG viable bacteria promote secretion of cytokines TNF- α, IL-6 and IL-10 by co-incubating with cells, and the S869 viable bacteria promote secretion of IL-10 by cells more than the LGG viable bacteria, which indicates that the S869 viable bacteria are normally able to activate macrophages and up-regulate secretion of cytokines TNF- α, IL-6 and IL-10, producing a physiological inflammatory response, and enhancing the immune ability of the organism. When LPS induces cells to form inflammatory cells, compared with a positive control group, the prevention group and the treatment group of the S869 viable bacteria have no difference in TNF-alpha and IL-6 values, but the secretion of the anti-inflammatory factor IL-10 is increased, which indicates that after inflammation occurs, the S869 viable bacteria can promote the secretion of the anti-inflammatory factor IL-10, thereby regulating the inflammatory reaction of cells and further improving the immunity of organisms.
TABLE 3 Effect of inactivated bacteria on cytokine secretion
Note that: "-" means that the cytokine secretion is less than the detection limit; "+" represents secretion amount of 15.625-125pg/mL; "++" represents a secretion amount of 125-500pg/mL; "+". ++'s representing secretion an amount of 500-1000pg/mL; "+". ++'s representing secretion an amount greater than 1000pg/mL;
as shown in Table 3, both the inactivated S869 group and the inactivated LGG group promote secretion of cytokines TNF- α, IL-6 and IL-10 by incubating with cells, and the S869 inactivated bacteria promote secretion of IL-10 by cells more than the LGG inactivated bacteria, which indicates that the S869 inactivated bacteria normally activate macrophages and up-regulate secretion of cytokines TNF- α, IL-6 and IL-10, resulting in physiological inflammatory response, and improving immunity of the organism. When LPS induces cells to form inflammatory cells, compared with the positive control two groups, the TNF-alpha and IL-6 values of the inactivated S869 prevention group and the treatment group are not different, but the secretion of the anti-inflammatory factor IL-10 is increased, which indicates that after inflammation occurs, the S869 inactivated bacteria can promote the secretion of the anti-inflammatory factor IL-10, thereby regulating the inflammatory reaction of cells and further improving the immunity of organisms.
In conclusion, the co-incubation of the activated and deactivated streptococcus thermophilus S869 bacterial suspensions with macrophages activates megaloblastic cells and up-regulates secretion of cytokines TNF-alpha, IL-6 and IL-10, and the ability of the activated and deactivated S869 bacteria to secrete IL-10 is higher than that of the activated and deactivated LGG bacteria; when LPS induces cells to form inflammatory cells, the values of pro-inflammatory factors TNF-alpha and IL-6 are not different, but the secretion of anti-inflammatory factors IL-10 is increased, which indicates that the S869 viable bacteria and the inactivated bacteria can increase the secretion of the anti-inflammatory factors IL-10 after the inflammation occurs, thereby regulating the inflammatory reaction of the cells and further improving the immunity of the organism.
Example 3
The experimental steps are as follows:
live streptococcus thermophilus S869 bacterial powder, killed streptococcus thermophilus S869 bacterial powder, 40 male SPF grade BABL/c healthy mice (purchased from Beijing velon LiHua Co., ltd.).
The feeding environment of 40 mice is kept at (23+/-2) DEG C, the relative humidity is (50+/-10)%, and 12 hours of illumination is carried out for 12 hours of night alternation. After the mice were adaptively bred for 1 week, 40 mice were randomly divided into 4 groups of 10 mice each, which were respectively a blank group, a model group, a live streptococcus thermophilus S869 bacteria group, and an inactivated streptococcus thermophilus S869 bacteria group. The remaining 3 groups, except the blank group, were subjected to continuous 3-day subcutaneous injection of cyclophosphamide (80 mg/kg) to establish an immunosuppressive mouse model. After molding, each group was continuously perfused with a dose of 0.lmL/(10 g.d) for 4 weeks, and the blank group and the model group were given sterile physiological saline, and the live Streptococcus thermophilus S869 group and the inactivated Streptococcus thermophilus S869 group were 1×10 8 CFU/day (obtained by diluting live bacteria powder and inactivated bacteria powder of streptococcus thermophilus S869 with normal saline). At the end of the experiment, the corresponding index was detected.
Experimental results:
(1) Variation in body constitution of mice
Mice were weighed periodically weekly, changes in body mass were recorded, and the amount of lavage was adjusted according to body mass. The change in body mass of mice during the experiment is shown in FIG. 5. As shown in the figure, compared with the blank group, the growth of immunosuppressive mice (model group, live streptococcus thermophilus S869 bacterial group and inactivated streptococcus thermophilus S869 bacterial group) is obviously inhibited, the weight growth is slow, and the fact that immunosuppressive mice are successfully modeled proves that cyclophosphamide can inhibit appetite of the mice, influence absorption and further inhibit the body mass growth of the mice. The quality growth rate of the live bacteria group and the inactivated bacteria group of the streptococcus thermophilus S869 is obviously faster than that of the model group by intervening the lactic acid bacteria for 4 weeks of gastric lavage, and the live bacteria group and the inactivated bacteria group of the streptococcus thermophilus S869 can promote the quality growth of the bodies, wherein the quality growth rate of the live bacteria group of the streptococcus thermophilus S869 is higher, and the quality growth rate of the inactivated bacteria group of the streptococcus thermophilus S869 is slower.
(2) Index of viscera
After 4 weeks of stomach irrigation, the mice were weighed, the eyeballs were taken for blood, thymus, spleen, liver and kidney were taken after the cervical vertebrae were sacrificed, and the surrounding tissues were peeled off cleanly and weighed.
Organ index calculation formula: organ index= (wet weight of mouse organ/body mass) ×100%
TABLE 4 spleen index and thymus index results
Note that: containing x indicates that the model group has significant differences (P < 0.05) from the other groups
As can be seen from the data in table 4, the spleen index and thymus index of mice in the model group were significantly lower than those in the other groups (blank group, S869 viable bacteria group and S869 inactivated bacteria group); compared with the model group, the spleen index and thymus index of the streptococcus thermophilus S869 live bacteria group and the inactivated bacteria group are obviously improved by intervening the lactobacillus for 4 weeks after gastric lavage, wherein the spleen index and thymus index of the streptococcus thermophilus S869 live bacteria group mice are higher than those of the streptococcus thermophilus S869 inactivated bacteria group.
(3) Number of white blood cells in blood
After 4 weeks of gastric lavage, the mice orbit was bled, 20 μl was aspirated and placed in a dilution of 380 μl white blood cells and shaken well. Dripping the leucocyte suspension into a counting tank according to the erythrocyte counting method, standing for 3min, and counting after leucocytes sink. White blood cells are characterized by round shape, transparent pulp, purple black nucleus and slightly refractive. The number of white blood cells = 500 total number of 5 large cells, and the detection results are shown in table 5.
TABLE 5 results of leukocyte count in blood
Note that: containing × indicates that the model group has significant differences from the other groups (P < 0.01)
From the data in Table 5, the number of leukocytes in the blood of mice in the model group was significantly lower than that in the other groups (blank group, S869 live bacteria group and S869 inactivated bacteria group), indicating that administration of cyclophosphamide resulted in a significant decrease in the number of leukocytes. Compared with the model group, after taking the streptococcus thermophilus S869 live bacteria and the inactivated bacteria, the number of the white blood cells in blood can be obviously increased, the damage of an organism immune system can be reduced, and the organism immunity can be enhanced.
(4) Cytokine content in serum
After 4 weeks of gastric lavage, 0.5-1.0 mL of eyeball blood is taken from each mouse and placed in a 1.5mL centrifuge tube, after standing for 1h, the mixture is centrifuged at 4 ℃ for 3000r/min and 15min to fully separate out serum, and the serum is transferred to another centrifuge tube for low-temperature preservation and to be measured. The content of TNF-a, IL-6 and IL-10 in the serum of mice was determined by ELISA according to the kit instructions. The measurement results are shown in Table 6.
TABLE 6 determination of cytokine content in serum
Note that: containing x indicates that the model group has significant differences (P < 0.05) from the other groups
As can be seen from the data in Table 6, the serum levels of TNF-a and IL-6 were higher in the mice in the model group than in the blank group, and the IL-10 level was lower than in the blank group; indicating that cyclophosphamide affects the relevant cytokine levels in mouse serum. After 4 weeks of intervention with live and inactivated streptococcus thermophilus S869, the TNF-a content in serum was not significantly different compared to the model group; but the content of inflammatory factor IL-6 is obviously reduced, and the content of anti-inflammatory factor IL-10 is obviously increased. It is shown that the live bacteria and the inactivated bacteria of the streptococcus thermophilus S869 can improve the immunity of organisms by regulating the content of cytokines in serum.
Experimental example 4
The experimental strains are S869L1, S869L2, S869L3, S869D1, S869D2, S869D3 and S869S, wherein
S869L1 is high-concentration S869 viable bacteria;
S869L2 is a medium-concentration S869 viable bacteria;
S869L3 is low-concentration S869 viable bacteria;
S869D1 is high-concentration S869 inactivated bacteria;
S869D2 is medium concentration S869 inactivated bacteria;
S869D3 is low-concentration S869 inactivated bacteria;
S869S is a metabolite.
The experimental steps are as follows:
(1) S869 regulates HT-29 cell mucin expression in a simulated state of fermentation outside an intestinal flora
Adult healthy feces were diluted 1:10 with anaerobic physiological saline to prepare a fecal suspension, and 500 μl of fecal suspension was inoculated into each fermentation vial. (Each fermentation vial contained 5mL of YCFA medium having a composition of 1g/L arabinogalactan, 2g/L pectin, 1g/L xylan, 3g/L starch, 0.4g/L glucose, 3g/L yeast extract, 3g/L peptone, 0.8g/L NaCl, 0.5g/L KH) 2 PO 4 、0.5g/L K 2 HPO 4 、0.4g/L CaCl 2 .2H 2 O、0.08g/L MgSO 4 .7H 2 O, 0.01g/L chlorhexidine, and 0.3. Mu.g/L vitamin mixture was added
Preparation of probiotic groups:
intervention group (corresponding to S869/L/D/S in the accompanying drawings): inoculating 100 mu L of each experimental strain into a fermentation small bottle, and culturing for 24 hours in a 37 ℃ incubator;
treatment group (corresponding to E+S869/L/D/S in the accompanying drawings): 100. Mu.L of enterotoxigenic Escherichia coli (ETEC) broth was inoculated into the above fermentation vial to give a final concentration of 10 7 Culturing the thalli/mL in a 37 ℃ incubator for 12 hours, then inoculating 100 mu L of each experimental strain into a fermentation vial, and co-culturing in the 37 ℃ incubator for 12 hours;
preventive group (corresponding to S869/L/D/S+E in the accompanying drawings): inoculating 100 mu L of each experimental strain into a fermentation small bottle, culturing for 12 hours in a culture box at 37 ℃, then inoculating 100 mu L of ETEC bacterial liquid into the fermentation small bottle, and co-culturing for 12 hours in a culture box at 37 ℃;
control group (corresponding to control in the drawing): adding 100 mu L of anaerobic physiological saline, and culturing for 24 hours in a 37 ℃ incubator; taking fermentation liquor after the culture is finished, centrifuging for 2min at 12000r/min, filtering, sterilizing and preserving the supernatant by using a 0.22 mu m filter membrane to obtain sterile fermentation supernatant;
ETEC group: 100. Mu.L of ETEC bacteria solution was inoculated into a fermentation vial to a final concentration of about 10 7 Culturing the thalli/mL in an incubator at 37 ℃ for 24 hours;
after resuscitating HT-29 cells, the cells are recovered in 5% CO 2 Culturing at 37deg.C in incubator, collecting 3 rd generation HT-29 cells, adjusting cell concentration, and adjusting cell density to 2.5X10 5 Inoculating 2mL of each cell/mL into 24-well plate, and inoculating 5% CO at 37deg.C 2 Incubate in incubator for 24h. After cell attachment, the supernatant was discarded, 2mL MyCoy's 5A medium (10% serum) was added to each well, and 200. Mu.L of the supernatant of the probiotic group obtained in the batch culture outside the intestinal flora was added, 200. Mu.L MyCoy's 5A medium (10% serum) was added to the control group, 3 replicates of each group, and the culture was performed at 37℃and 5% CO 2 Incubating for 24h under the condition, and collecting cells of each well.
(2) RNA level verification
(1) RNA is extracted by adopting an RNA Easy Fast animal tissue/cell total RNA extraction kit (centrifugal column type) DP451, and the integrity, purity and concentration of the RNA are detected.
(2) Reverse transcription
First strand cDNA was synthesized using a rapid reverse transcription kit according to the procedure described.
The resulting cDNA was used in subsequent experiments, or stored at low temperature.
(3) Fluorescent quantitative PCR detection of mucin MUC2, MUC5AC gene expression
Primer design: the housekeeping gene adopts 18SrRNA and GAPDH
| Primer name | Sequence (5 'to 3') |
| MUC2-F | GACCCGCACTATGTCACCTT |
| MUC2-R | GGACAGGACACCTTGTCGTT |
| MUC5AC-F | CCAGCTCTGTGGCTTACTCC |
| MUC5AC-R | TCGGAGGTGGATATTGAAGG |
| 18SrRNA-F | TGTGATGCCCTTAGATGTCC |
| 18SrRNA-R | GATAGTCAAGTTCGACCGTC |
| GAPDH-F | CCCTTCATTGACCTCAACTACATGG |
| GAPDH-R | CATGGTGGTGAAGACGCCAG |
Program setting: 95 ℃ for 15min;40 cycles: 95℃for 10s,60℃for 30s and 72℃for 30s; melting curve: the temperature is 65-95 ℃ and the temperature is kept at 0.5 ℃ in each step for 5s.
Experimental results:
the intestinal mucosa barrier has the capability of protecting intestinal health, plays an important role in constipation and diarrhea, and the effect of probiotics on the expression of intestinal epithelial cell mucin MUC2 and MUC5AC mRNA is shown in FIG. 6 and FIG. 7. In the intervention group, the high-concentration S869 thalli improves the expression of cell mucin MUC2 mRNA, has the effects of improving the intestinal lubricity and relieving constipation; the S869 metabolite in the treatment group improves the expression level of MUC2 and MUC5AC genes, and has better capability of improving the expression level of intestinal epithelial cell mucin genes, thereby repairing the intestinal mucosa barrier damage caused by ETEC. The S869 thalli and the inactivated thalli in the prevention group improve the expression level of the cell mucin genes, thereby preventing the intestinal mucosa barrier from being damaged caused by ETEC.
In conclusion, the S869 thalli plays a better role in increasing the intestinal canal lubrication degree and promoting defecation; the S869 metabolite has the capability of repairing the intestinal mucosa barrier damage caused by ETEC, and the S869 thallus and the inactivated thallus have the capability of preventing the intestinal mucosa barrier damage caused by ETEC.
Experimental example 5
And detecting the serotonin transporter gene SERT expression after co-culturing thalli and fermentation supernatant with HT-29 cells, wherein the treatment mode is the same as that of experiment example 4.
Primer design: the housekeeping gene adopts 18SrRNA and GAPDH
Program setting: 95 ℃ for 15min;40 cycles: 95℃for 10s,60℃for 30s and 72℃for 30s; melting curve: the temperature is 65-95 ℃ and the temperature is kept at 0.5 ℃ in each step for 5s.
Experimental results:
the effect of the experimental strain on the expression of the serotonin transporter SERT mRNA in intestinal epithelial cells is shown in fig. 8, the expression level of the SERT mRNA in cells is reduced in an intervention group, a treatment group and a prevention group, the effect of the metabolite of S869 on the reduction of the expression level of the SERT mRNA in cells is best in the intervention group, the effect of the metabolite of S869 in a low-concentration live bacteria group is best in the treatment group, the effect of the metabolite group is best in the prevention group, and the effect of the metabolite group is shown to be the best, so that S869 has the effects of inhibiting intestinal motility and preventing diarrhea symptoms caused by ETEC, wherein the comprehensive performance of the metabolite on the prevention and treatment of diarrhea symptoms caused by ETEC is the best.
Experimental example 6
The experimental steps are as follows:
measuring cell proliferation rate by CCK-8 kit, specifically collecting HT-29 cells after culturing for 2d of 3 rd generation, and determining density of 10 5 Each cell/mL was seeded in 96-well plates with 100. Mu.L per well at 37℃and 5% CO 2 Incubate in incubator for 24h. After cell attachment, the supernatant was discarded, 100. Mu.L MyCoy's 5A medium was added to each well, and 10. Mu.L samples of the sterilized in vitro fermentation supernatant of intestinal flora were added (see experimental example 4 for details), 5 replicates per group, at 37℃and 5% CO 2 Incubate under conditions for 12h. 10. Mu.L of CCK-8 solution was added to each well, incubated in a cell incubator for 1 hour, OD was measured at a wavelength of 450nm, and cell proliferation index was calculated. Cell proliferation index= (As-Ac)/Ac, where As represents experimental OD value and Ac represents blank OD value.
As shown in FIG. 9, none of the S869 supernatants exhibited a cell proliferation effect. In the intervention group, the medium concentration S869 inactivated bacteria shows a certain cell proliferation effect. In the treatment group, the proliferation capacity of the S869 inactivated bacteria cells is reduced along with the reduction of the concentration, and the high-concentration S869 inactivated bacteria cells have better capacity of repairing intestinal epithelial cell injury caused by ETEC. In the prevention group, the proliferation capacity of the S869 bacterial cells tends to decrease and then increase with the decrease of the concentration, and the low-concentration S869 bacterial cells have better capacity of preventing intestinal epithelial cell injury caused by ETEC.
Table 8 improvement of diarrhea by S869
S869 is useful in a diarrhea composition for preventing abnormal neurotransmitter expression and enterotoxigenic E.coli infection. In addition, S869 has good effect in repairing intestinal epithelial cell injury and repairing intestinal mucosa barrier.
| Experimental strains | Cell proliferation | Intestinal mucosa barrier | Regulation of neurotransmitters |
| S869L1 | - | ++ | - |
| S869L2 | - | - | - |
| S869L3 | - | - | - |
| S869D1 | - | - | - |
| S869D2 | + | - | - |
| S869D3 | - | - | - |
Mid-cell proliferation-means cell proliferation rate <0%; + represents a cell proliferation rate of 0-5%; ++ means cell proliferation rate 5-10%; ++ represents cells proliferation rate >10%; intestinal mucosal barrier and neurotransmitter regulation-representing mucin or neurotransmitter transporter gene expression fold <1; + represents a fold 1-1.5 expression of mucin or neurotransmitter transporter genes; ++ means that the fold expression of mucin or neurotransmitter transporter genes is 1.5-2; ++ represents mucin protein or neurotransmitters transporter gene expression fold >2; in-regulated aquaporins-representing aquaporin gene expression fold <1; + represents that the expression multiple of aquaporin gene is 0.5-1; ++ means aquaporin gene expression fold 0.4-0.5; ++ represents aquaporins gene expression fold <0.1;
TABLE 10 development results of diarrhea relieving function
Note that: mid-cell proliferation-means cell proliferation rate <0%; + represents a cell proliferation rate of 0-5%; ++ means cell proliferation rate 5-10%; ++ represents cells proliferation rate >10%; intestinal mucosa barrier and in-regulated aquaporins-representing mucin or neurotransmitter transporter gene expression fold <1; + represents a fold 1-1.5 expression of mucin or neurotransmitter transporter genes; ++ means that the fold expression of mucin or neurotransmitter transporter genes is 1.5-2; ++ represents mucin protein or neurotransmitters transporter gene expression fold >2; neurotransmitter-expression fold <1; + represents that the expression multiple of aquaporin gene is 0.5-1; ++ means that the aquaporin gene expression fold number is 0.4-0.5; ++ represents aquaporins gene expression fold <0.1;
s869 can be used in a diarrhea composition for preventing and treating abnormal neurotransmitter expression and enterotoxigenic escherichia coli infection, and the S869 has good effects in repairing intestinal epithelial cell injury and intestinal mucosa barrier.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (3)
1. Streptococcus thermophilus @Streptococcus thermophilus) S869, wherein the culture medium is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.24190.
2. A Streptococcus thermophilus preparation comprising the Streptococcus thermophilus of claim 1 in a solid or liquid state.
3. A food product comprising the streptococcus thermophilus of claim 1 or the streptococcus thermophilus bacterial preparation of claim 2.
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