CN117736943B - Composite microbial agent for improving irritable bowel syndrome and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of biological agents, in particular to a composite microbial inoculum for improving irritable bowel syndrome, a preparation method and application thereof. A compound bacterial agent for improving irritable bowel syndrome comprises bifidobacterium bifidum (Bifidobacterium bifidum) JYBB-163, bifidobacterium lactis (Bifidobacterium lactis) JYBR-390 and lactococcus lactis (Lactococcus lactis) JYLL-60. The bifidobacterium bifidum JYBB-163, the bifidobacterium lactis JYBR-390 and the lactococcus lactis JYLL-60 in the composite microbial agent for improving the irritable bowel syndrome can be synergistic, so that the problems of weight reduction, diarrhea and stool non-shaping caused by the diarrhea type irritable bowel syndrome are remarkably improved, and the composite microbial agent has excellent effects compared with other microbial agents.
Description
Technical Field
The invention relates to the technical field of biological agents, in particular to a composite microbial inoculum for improving irritable bowel syndrome, a preparation method and application thereof.
Background
Irritable bowel syndrome (Irritable bowel syndrome, IBS) is an intestinal stress reaction syndrome mainly represented by abdominal pain, abdominal discomfort, altered bowel habits or altered stool traits. In recent years, it has been considered as a global functional disease with the highest incidence of human beings. Irritable bowel syndrome belongs to a multifactorial disease, the etiology and pathophysiology mechanisms are complex, and the morbidity factors comprise dysbacteriosis of intestinal tracts, overgrowth of small intestinal bacteria, abnormal intestinal motility, intestinal hypersensitivity, abnormal brain-intestinal axis regulation and the like. Diarrhea type irritable bowel syndrome (IBS-D) is the subtype with highest intestinal incidence rate in China, belongs to typical diseases causing dysbacteriosis of intestinal flora, and changes the number and the variety of the intestinal flora obviously. IBS has a great influence on individuals, delays and repeats the course of the disease, seriously affects the work, study and life of patients, reduces the life quality of patients, and causes great psychological and economic burden. Therefore, active search for effective IBS treatment is of great significance.
At present, traditional Chinese medicine treatment comprises modes of medication, acupuncture treatment, massage, fumigation and the like, and is mainly improved from the whole function of a patient. Because of the complex mechanism of IBS-D, traditional Chinese medicine treatment is generally costly and has a long course of treatment, and patients may be suspected of being effective. The Western medicine treatment method mainly comprises the steps of medicine treatment, including ion channel regulator, osmotic laxative and 5-hydroxytryptamine receptor agonist, and the medicine treatment is easy to have side effects and causes other adverse symptoms. When the probiotic product is used for the disease, corresponding strains are only supplemented for specific physiological symptoms expressed by IBS-D patients, for example, the saccharomyces boulardii which is conventionally used for diarrhea is supplemented for improving diarrhea, the mental problems caused by IBS-D are not emphasized, and the effect of improving IBS-D is limited.
Disclosure of Invention
Aiming at the technical problems of long time, easy side effect and limited effect of the existing IBS-D treatment method, the invention provides a composite microbial inoculum for improving irritable bowel syndrome, and a preparation method and application thereof. The composite bacterial agent for improving the irritable bowel syndrome provided by the invention has the advantages that the bacterial strains in the composite bacterial agent for improving the irritable bowel syndrome have synergistic effect, and the prepared product for improving the diarrhea type irritable bowel syndrome not only can improve physiological problems such as diarrhea and abdominal pain caused by IBS-D, but also can relieve mental problems such as anxiety and depression caused by IBS-D.
In a first aspect, the present invention provides a complex microbial agent for improving irritable bowel syndrome, comprising bifidobacterium bifidum (Bifidobacterium bifidum) JYBB-163, bifidobacterium lactis (Bifidobacterium lactis) JYBR-390, and lactococcus lactis (Lactococcus lactis) JYLL-60; the bifidobacterium bifidum JYBB-163 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in 7 months and 8 days in 2019, and the preservation address is North Star Xili No.1, 3 in the Korean region of Beijing, and the preservation number is CGMCC No.18091; bifidobacterium lactis JYBR-390 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 7 th month 8 of 2019, and has a preservation address of North Star Xili No.1, 3 in the Korean region of Beijing, and a preservation number of CGMCC No.18093; lactococcus JYLL-60 has been deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 9 months and 2 days in 2021, and has a deposit address of North Star Xili No.1, 3 in the Korean region of Beijing, and a deposit number of CGMCC No.23353.
The inventor finds that bifidobacterium bifidum JYBB-163 can improve the semen quality and fertility of men in the previous study, bifidobacterium lactis JYBR-390 has constipation treatment effect, lactococcus lactis JYLL-60 can improve immunity, and unexpectedly finds that a composite microbial inoculum combining the three bacteria can prepare an IBS-D product and has good effect of relieving physiological problems and mental problems caused by IBS-D when the three bacteria are deeply studied.
Further, the total colony count of the composite bacterial agent for improving the irritable bowel syndrome is 1.0X10 7cfu/g-1.0×109 cfu/g.
Further, the complex bacterial agent for improving the irritable bowel syndrome exists in the form of bacterial suspension.
Further, the composite microbial agent for improving the irritable bowel syndrome also comprises maltodextrin.
In a second aspect, the invention provides a preparation method of a composite microbial inoculum for improving irritable bowel syndrome, which comprises the following specific steps:
inoculating bifidobacterium bifidum JYBB-163, bifidobacterium lactis JYBR-390 and lactococcus lactis JYLL-60 into MRS liquid culture medium respectively for culture, centrifugally collecting, washing, re-suspending in maltodextrin aqueous solution to obtain bacterial suspension, and freeze-drying to obtain bifidobacterium bifidum JYBB-163 bacterial powder, bifidobacterium lactis JYBR-390 bacterial powder and lactococcus lactis JYLL-60 bacterial powder respectively; and mixing the bifidobacterium bifidum JYBB-163 bacterial powder, the bifidobacterium lactis JYBR-390 bacterial powder and the lactococcus lactis JYLL-60 bacterial powder with equal mass after the same multiple of gradient dilution respectively, so as to obtain the composite bacterial agent for improving the irritable bowel syndrome.
In a third aspect, the invention provides an application of a composite microbial inoculum for improving irritable bowel syndrome in preparing a product for improving diarrhea type irritable bowel syndrome.
The product for improving diarrhea type irritable bowel syndrome is a medicine for improving diarrhea type irritable bowel syndrome.
Further, the complex bacterial agent for improving irritable bowel syndrome is used for improving physiological problems and/or mental problems caused by diarrhea type irritable bowel syndrome.
Further, improving physiological problems includes improving weight loss, diarrhea and/or stool non-formation problems, and improving mental problems includes reducing depression and/or anxiety levels.
The invention has the beneficial effects that:
The invention provides a composite microbial inoculum for improving irritable bowel syndrome, a preparation method and application thereof. The bifidobacterium bifidum JYBB-163, the bifidobacterium lactis JYBR-390 and the lactococcus lactis JYLL-60 in the composite microbial agent for improving the irritable bowel syndrome can be synergistic, so that the problems of weight reduction, diarrhea and stool non-shaping caused by the diarrhea type irritable bowel syndrome are remarkably improved, and compared with other microbial agents, the composite microbial agent has excellent effects; experiments prove that the composite microbial agent for improving the irritable bowel syndrome has the advantages that the composite microbial agent for improving the problems of weight reduction, diarrhea and stool non-shaping caused by IBS-D is not superior in effect because the composite microbial agent for improving the problems of weight reduction, diarrhea and stool non-shaping, which are formed by bifidobacterium bifidum, bifidobacterium animalis and lactococcus lactis is composed of bifidobacterium CICC10395, bifidobacterium animalis CICC6250 and lactococcus lactis CICC 6246. In addition, the compound bacterial agent for improving irritable bowel syndrome provided by the invention can also improve anxiety, depression mental problems, visceral pain sensitivity problems and pain stimulation problems caused by IBS-D, and can improve the life quality of IBS-D patients.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the description of the embodiments or the prior art will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
FIG. 1 is a graph showing the weight change of rats in each group, ** p < 0.01 in comparison with the control group, and ## p < 0.01 in comparison with the model group.
FIG. 2 is a control of travel in open field experiments for IBSmixes groups of rats, * P < 0.05 compared to the control group, and # P < 0.05 compared to the model group.
FIG. 3 is a control of the concentration of sugar taken in a sugar water bias experiment for IBSmixes groups of rats, * P < 0.05 compared to the control group, and # P < 0.05 compared to the model group.
Detailed Description
In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in 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 those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Bifidobacteria JYBB-163 of the following examples and comparative examples were deposited at the China general microbiological culture Collection center, having a deposit number of CGMCC No.18091, in the area North Star, kogyo, of Beijing, and were disclosed in the Chinese patent application CN 116103207A; bifidobacterium lactis JYBR-390 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 7 months 8 days of 2019, and has a preservation address of North Star Xili No.1, 3 in the Korean region of Beijing, and a preservation number of CGMCC No.18093, and is disclosed in a Chinese patent application No. CN 113969253A; lactococcus JYLL-60 was deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 9 months and 2 days of 2021, and has a deposit address of North Star, west Liu No.1, 3, and a deposit number of CGMCC No.23353, which are disclosed in Chinese patent No. 114381411A, and are commercially available as bifidobacterium bifidus CICC10395, bifidobacterium animalis CICC6250 and lactococcus lactis CICC 6246.
Example 1 Complex microbial inoculant for improving irritable bowel syndrome
(1) Preparation of MRS liquid medium:
Mixing peptone 10g, beef powder 5g, sodium acetate trihydrate 5g, dipotassium phosphate heptahydrate 2g, tween 80 1mL, manganese sulfate tetrahydrate 0.05g, triammonium citrate 2g, glucose 20g, magnesium sulfate heptahydrate 0.2g and distilled water 1000mL, adjusting pH to 6.8, stirring uniformly, and sterilizing at 121deg.C and 0.1MPa for 20 min.
(2) Preparing bacterial powder:
Inoculating the preserved bifidobacterium bifidum JYBB-163, bifidobacterium lactis JYBR-390 and lactococcus lactis JYLL-60 into MRS liquid culture medium according to 2vol% respectively, culturing for 24 hours at 37 ℃, centrifugally collecting thalli at 4000r/min, washing with sterile physiological saline, and suspending the thalli in 15wt% maltodextrin aqueous solution to obtain bacterial suspension, wherein the bacterial concentration in the bacterial suspension is 1.5X10 10 cfu/mL; after freeze-drying the bacterial suspension, bacterial powders of 1.0X10 11 cfu/g of the three bacteria are respectively obtained.
(3) Preparing a microbial inoculum:
bifidobacterium bifidum JYBB-163 microbial inoculum: 1.0X10 11 cfu/g bifidobacterium bifidum JYBB-163 bacterial powder, accurately weighing 1.0g bacterial powder, carrying out 10-time gradient dilution with sterile water, and obtaining 1.0X10 7 cfu/g bifidobacterium bifidum JYBB-163 bacterial agent after 10- 4 times dilution;
Bifidobacterium lactis JYBR-390 inoculant: 1.0X10 11 cfu/g of bifidobacterium lactis JYBR-390 bacterial powder, accurately weighing 1.0g of bacterial powder, carrying out 10-time gradient dilution by using sterile water, and obtaining 1.0X10 7 cfu/g of bifidobacterium lactis JYBR-390 bacterial agent after 10- 4 times dilution;
Lactobacillus JYLL-60 microbial inoculum: 1.0X10 11 cfu/g lactococcus lactis JYLL-60 bacterial powder, accurately weighing 1.0g bacterial powder, carrying out 10-time gradient dilution with sterile water, and obtaining 1.0X10 7 cfu/g lactococcus lactis JYLL-60 bacterial agent after 10- 4 times dilution.
(4) Preparing a composite microbial agent for improving irritable bowel syndrome:
The bifidobacterium bifidum JYBB-163 microbial inoculum, the bifidobacterium lactis JYBR-390 microbial inoculum and the lactococcus lactis JYLL-60 microbial inoculum in the step (3) are mixed according to the weight ratio of 1:1:1 to obtain the composite microbial inoculum for improving the irritable bowel syndrome, wherein the composite microbial inoculum is 1.0X10 7 cfu/g.
Example 2 Complex microbial inoculant for improving irritable bowel syndrome
The same as in steps (1) and (2) of example 1;
(3) Preparing a microbial inoculum:
Bifidobacterium bifidum JYBB-163 microbial inoculum: 1.0X10 11 cfu/g bifidobacterium bifidum JYBB-163 bacterial powder, accurately weighing 1.0g bacterial powder, carrying out 10-time gradient dilution with sterile water, and obtaining 1.0X10 8 cfu/g bifidobacterium bifidum JYBB-163 bacterial agent after 10- 3 times dilution;
Bifidobacterium lactis JYBR-390 inoculant: 1.0X10 11 cfu/g of bifidobacterium lactis JYBR-390 bacterial powder, accurately weighing 1.0g of bacterial powder, carrying out 10-time gradient dilution by using sterile water, and obtaining 1.0X10 8 cfu/g of bifidobacterium lactis JYBR-390 bacterial agent after 10- 3 times dilution;
lactobacillus JYLL-60 microbial inoculum: 1.0X10 11 cfu/g lactococcus lactis JYLL-60 bacterial powder, accurately weighing 1.0g bacterial powder, carrying out 10-time gradient dilution with sterile water, and obtaining 1.0X10 8 cfu/g lactococcus lactis JYLL-60 bacterial agent after 10- 3 times dilution.
(4) Preparing a composite microbial agent for improving irritable bowel syndrome:
the bifidobacterium bifidum JYBB-163 microbial inoculum, the bifidobacterium lactis JYBR-390 microbial inoculum and the lactococcus lactis JYLL-60 microbial inoculum in the step (3) are mixed according to the weight ratio of 1:1:1 to obtain the composite microbial inoculum for improving the irritable bowel syndrome, wherein the composite microbial inoculum is 1.0X10 8 cfu/g.
Example 3 Complex microbial inoculants for improving irritable bowel syndrome
The same as in steps (1) and (2) of example 1;
(3) Preparing a microbial inoculum:
Bifidobacterium bifidum JYBB-163 microbial inoculum: 1.0X10 11 cfu/g bifidobacterium bifidum JYBB-163 bacterial powder, accurately weighing 1.0g bacterial powder, carrying out 10-time gradient dilution with sterile water, and obtaining 1.0X10 9 cfu/g bifidobacterium bifidum JYBB-163 bacterial agent after 10- 2 times dilution;
Bifidobacterium lactis JYBR-390 inoculant: 1.0X10 11 cfu/g of bifidobacterium lactis JYBR-390 bacterial powder, accurately weighing 1.0g of bacterial powder, carrying out 10-time gradient dilution by using sterile water, and obtaining 1.0X10 9 cfu/g of bifidobacterium lactis JYBR-390 bacterial agent after 10- 2 times dilution;
Lactobacillus JYLL-60 microbial inoculum: 1.0X10 11 cfu/g lactococcus lactis JYLL-60 bacterial powder, accurately weighing 1.0g bacterial powder, carrying out 10-time gradient dilution with sterile water, and obtaining 1.0X10 9 cfu/g lactococcus lactis JYLL-60 bacterial agent after 10- 2 times dilution.
(4) Preparing a composite microbial agent for improving irritable bowel syndrome:
The bifidobacterium bifidum JYBB-163 microbial inoculum, the bifidobacterium lactis JYBR-390 microbial inoculum and the lactococcus lactis JYLL-60 microbial inoculum in the step (3) are mixed according to the weight ratio of 1:1:1 to obtain the composite microbial inoculum for improving the irritable bowel syndrome, wherein the composite microbial inoculum is 1.0X10 9 cfu/g.
Comparative example 1 bifidobacterium bifidum JYBB-163 microbial inoculum
Bifidobacterium bifidum JYBB-163 microbial inoculum obtained in step (3) of example 1.
Comparative example 2 bifidobacterium lactis JYBR-390 inoculant
The bifidobacterium lactis JYBR-390 strain obtained in the step (3) of example 1.
Comparative example 3 lactococcus lactis JYLL-60 microbial inoculum
Lactococcus lactis JYLL-60 bacterial preparation obtained in step (3) of example 1.
Comparative example 4 two-bacterium composite microbial agent No. 1
The bifidobacterium bifidum JYBB-163 microbial inoculum and the bifidobacterium lactis JYBR-390 microbial inoculum obtained in the step (3) of the example 1 are uniformly mixed according to the weight ratio of 1:1, and the two-bacteria composite microbial inoculum No.1 with the weight of 1.0x 7 cfu/g can be obtained.
Comparative example 5 two-bacterium composite microbial agent No. 2
The bifidobacterium lactis JYBR-390 microbial agent and the lactococcus lactis JYLL-60 microbial agent obtained in the step (3) of the example 1 are uniformly mixed according to the weight ratio of 1:1, and the two-bacteria composite microbial agent No. 2 with the concentration of 1.0x 7 cfu/g can be obtained.
Comparative example 6 two-bacterium composite microbial agent No. 3
The bifidobacterium bifidum JYBB-163 microbial inoculum obtained in the step (3) of the example 1 and the lactococcus lactis JYLL-60 microbial inoculum are uniformly mixed according to the weight ratio of 1:1, and the two-bacteria composite microbial inoculum No. 3 with the weight of 1.0X10 7 cfu/g can be obtained.
Comparative example 7 comparative composite microbial agent
1.0X10 11 cfu/g of bacterial powder prepared from bifidobacterium bifidum CICC10395, bifidobacterium animalis CICC6250 and lactococcus lactis CICC6246 is weighed respectively, 1.0g is diluted by 10 times of weight gradient with sterile water, 1.0X10 7 cfu/g of bacterial agents of three strains are obtained respectively after 10 times of dilution by 4 times, and the weight ratio of the bacterial agents is 1:1:1 to obtain the comparative composite microbial inoculum with the concentration of 1.0X10 7 cfu/g.
Experimental example 1 Effect of Complex microbial inoculant for improving irritable bowel syndrome on diarrhea type irritable bowel syndrome
1. Preparation of laboratory mice
100 SPF-grade male SD rats (180+ -20 g) were first adaptively fed for one week, and the feed type was a normal feed (purchased from Australian corporation of Beijing), and then randomly divided into 10 groups, and the remaining rats were subjected to molding treatment except for the control group.
2. Preparation of diarrhea type irritable bowel syndrome rat model
Preparing senna leaf leaching liquid: taking 600g of senna leaves and water: material = 4:1, mixing and boiling for 30min, and filtering; repeatedly boiling for 3 times, mixing the leaching solution, concentrating to 1L solution by reduced pressure distillation, and refrigerating for use, wherein the concentration is 0.6 g/mL.
Preparation of a rat model of irritable bowel syndrome: the stomach is irrigated according to the senna leachates with the weight of 3.0g/kg of the rat, the four limbs of the rat are restrained and the tail is clamped for stimulation after 1h, and the stimulation lasts for 14 days once a day.
3. Experimental grouping
Control group: 10 normal rats were fed with normal feed for 2 weeks, 1g distilled water was fed to the stomach 1 time a day.
Model group: 10 model rats were fed with normal feed for 2 weeks, and 1g distilled water was infused with stomach every day.
JYBB-163 groups: 10 model rats were fed with normal feed for 2 weeks and 1g of bifidobacterium bifidum JYBB-163 of comparative example 1 was fed 1 time per day.
JYBR-390 groups: 10 model rats were fed with normal feed for 2 weeks and 1g of bifidobacterium bifidum JYBR-390 of comparative example 2 were fed 1 time per day.
JYLL-60 groups: 10 model rats were fed with normal feed for 2 weeks and 1g of the lactococcus lactis JYLL-60 inoculum of comparative example 3 was fed 1 time per day.
Two bacteria group 1:10 model rats were fed with normal feed for 2 weeks, 1g of the two-bacteria complex microbial inoculum No.1 of comparative example 4 was fed 1 time per day.
Two bacteria group No. 2: 10 model rats were fed with normal feed for 2 weeks, 1g of the two-bacteria compound microbial inoculum No.2 of comparative example 5 was fed 1 time per day.
Two bacteria group 3: 10 model rats were fed with normal feed for 2 weeks, 1g of the two-bacteria complex microbial inoculum No. 3 of comparative example 6 was fed 1 time per day.
IBSmixes groups: 10 model rats were fed with normal feed for 2 weeks, 1g per day of the composite microbial inoculum of example 1 for improving irritable bowel syndrome.
Comparing the composite bacterial group: 10 model rats were fed with normal feed for 2 weeks, 1 time per day with 1g of the comparative example 7 comparative composite microbial inoculum.
4. Weight changes before and after the experiment
The weight records of the rats before, before and after the modeling are shown in figure 1, and the weight of the rats before and after the modeling is obviously lower than that of the rats in the control group, which indicates that the modeling is successful; whereas the rats in example 1 IBSmixes had significantly higher body weight than the model group, there was no significant difference from the control group. Meanwhile, the weight of rats in IBSmixes groups is obviously higher than that of rats in other 7 groups, which proves that the compound bacterial agent for improving the irritable bowel syndrome has an improvement effect on the weight reduction caused by the irritable bowel syndrome and is obviously superior to that of the comparative compound bacterial agent.
5. Fecal character grading before and after experiment
Rats before and after the experiment were placed in a cage, filter paper was placed at the bottom layer of the cage, fecal characteristics of the rats within 4 hours were recorded and scored according to a classification table (table 1), and after the grade was determined, the score was made to the decimal point. The scoring results are shown in Table 2, the stool characters of the model mice are pasty and the stool of the water sample is most, and the score is obviously higher than that of the control group, which indicates that the modeling is successful. After the experiment is finished, JYBB-163 groups, JYLL-60 groups have no significant difference from the control of the model group, and other experimental groups have significant differences. However, only IBSmixes groups in the experimental group have no obvious difference from the control group, which indicates that the composite microbial inoculum for improving the irritable bowel syndrome has good effect on improving the fecal characteristics and is superior to other groups.
TABLE 1 fecal trait grading scale
TABLE 2 fecal character grading (minutes, x+ -s) of rats
| Grouping | Before the experiment | After the experiment |
| Control group | 3.67±0.52 | 3.58±0.59 |
| Model group | 6.24±0.68* | 6.15±0.48* |
| JYBB-163 groups | 6.35±0.59* | 5.87±0.54* |
| JYBR-390 groups | 6.41±0.61* | 5.24±0.61*# |
| JYLL-60 groups | 6.38±0.54* | 5.76±0.64* |
| Group 1 of two bacteria | 6.32±0.67* | 5.14±0.53*# |
| Group 1 of two bacteria | 6.29±0.38* | 4.86±0.60*# |
| Group 1 of two bacteria | 6.34±0.67* | 4.75±0.54*# |
| IBSmixes group | 6.42±0.66* | 3.65±0.41# |
| Contrast composite bacterial group | 6.34±0.56* | 5.18±0.56*# |
Note that: * P < 0.05 compared with the control group; # P < 0.05 compared to model group.
6. Fecal moisture content before and after the experiment
The feces of the above rats were collected, the feces weight (wet weight) was measured, and then dried in an oven at 105℃for 4 hours, and the dry weight was measured to calculate the feces water content. The measurement results are shown in Table 3, and the fecal water content of the rats in the model group is obviously higher than that of the rats in the control group, which indicates that the model formation is successful. After the experiment, the water content of the excrement is obviously reduced in the 3 two bacterial groups, IBSmixes group and comparative compound bacterial group compared with the model group, wherein the IBSmixes group is not obviously changed in the water content of the excrement compared with the control group. The composite microbial inoculum for improving the irritable bowel syndrome has obvious improvement effect on the aspect of too high fecal water content caused by the irritable bowel, and is superior to other groups.
TABLE 3 moisture content of rat feces (%, x+ -s)
Note that: * P < 0.05 compared with the control group; # P < 0.05 compared to model group.
Experimental example 2 Effect of Complex microbial inoculant for improving irritable bowel syndrome on mental problems caused by diarrhea type irritable bowel syndrome
1. Open field experiment
The control group, the model group and the IBSmixes group which are subjected to the experiment in the experimental example 1 are subjected to open field experiment, rats in each group are firstly adapted for 30min before the experiment, the experimental room is ensured to be dark, the experimental equipment and the space are ensured to have no peculiar smell, and the rats are cleaned by alcohol before the experiment. Room temperature was maintained at 20 ℃, and a field of 100cm (length) ×100cm (width) ×40cm (height) was divided into 25 grids at the computer analysis system side using an animal behavior recording analyzer. The total course of the rats in open field and the central area (central 9 grids) course were recorded within 10 min. The recorded results are shown in fig. 2, and compared with a control group, the model group mice have obviously reduced walking total distance and central area walking distance, which indicates that the modeling is successful; the IBSmixes group had a significantly increased distance travelled compared to the model group and no significant difference compared to the control group. The composite bacterial agent for improving the irritable bowel syndrome has an effect of improving the mental problems caused by the irritable bowel syndrome, and can reduce the anxiety level of experimental animals.
2. Sweet water preference experiment
The three groups of rats subjected to the open field experiment were subjected to a syrup preference experiment, each group of rats was adapted to the experimental environment prior to the experiment, and mice in the feeder were continuously exposed to two conventional water bottles for 48 hours, one containing 1% sucrose solution (wt/vol, in g/mL) and the other containing distilled water. All rats were also given food ad libitum. After this, rats were transferred to the chamber of the sugar water preference device for 24 hours of device adaptation. Thereafter, the rats were deprived of food and water for 24 hours. All rats were given a sugar water preference test of 1% sugar cane sugar water (wt/vol, units g/mL) and a tube of conventional water for 12 hours immediately after the end of deprivation. Each tube was weighed before and after testing. Calculated syrup drinking concentration = syrup volume/(syrup volume + distilled water volume) x 1%. The results are shown in fig. 3, and the sugar water intake concentration of the model group is obviously reduced compared with that of the control group; there was no significant difference in sugar water intake concentration between IBSmixes and control groups. The composite bacterial agent for improving the irritable bowel syndrome has an effect of improving mental problems caused by irritable bowel, and can reduce the depression level of experimental animals.
Experimental example 3 Effect of Complex microbial inoculant for improving irritable bowel syndrome on pain stimulus caused by diarrhea type irritable bowel syndrome
1. Abdominal recoil reflection (AWR) experiment
AWR scoring was performed on the control, model, IBSmixes groups after the experiment in example 2, as follows:
The rats were restricted from moving forward and backward after 24h of fasting before the experiment.
The syringe is connected with a sphygmomanometer, and scales of the syringe under the conditions of 20mmHg, 40mmHg, 60mmHg and 80mmHg of the sphygmomanometer are measured and marked.
The injector is connected with the catheter and is used for injecting the gas of the score line respectively.
After lubricating the catheter, the catheter is inserted into the anus of a rat for 6cm, the tail end of the rat is fixed by using an adhesive tape for 1cm outside the anus, the movement of the rat is limited, and after the catheter is stabilized, gas is injected to observe the reaction of the abdominal wall of the rat to the stimulus.
The gas injection was repeated 3 times for 20s each, 5min each, and the results were averaged by the AWR scoring criteria (table 4).
Table 4 AWR scoring criteria
Table 5 comparison of AWR scores (score, x.+ -. S) after each group of rats was tested
| Grouping | 20mmHg | 40mmHg | 60mmHg | 80mmHg |
| Control group | 0.36±0.49 | 0.92±0.23 | 1.64±0.31 | 2.61±0.53 |
| Model group | 1.31±0.43** | 2.76±0.54** | 3.36±0.74** | 3.76±0.85** |
| IBSmixes group | 0.37±0.46## | 0.88±0.41## | 1.67±0.42## | 2.58±0.63## |
Note that: ** P < 0.01 compared with the control group; ## P < 0.01 compared with the model group
As can be seen from table 5, the model group scores were significantly higher than the control group and IBSmixes group; compared with a control group, the IBSmixes groups have no obvious difference in scores, which indicates that the compound bacterial agent for improving irritable bowel syndrome has an improving effect on the problem of visceral pain sensitivity caused by irritable bowel.
2. Neuropeptide SP detection assay
Neuropeptides SP are associated with pain sensation, and the change of pain sensation degree caused by intestinal abnormality of rats is judged by detecting the neuropeptides SP content at different positions. Mainly detecting the SP content in serum and colon, and judging the peripheral sensitization degree; and detecting the SP content in the sea horse, and judging the central sensitization degree.
(1) Serum samples: after the rats after the end of the AWR experiment were fed normally for another day, 10% (wt/vol, unit was g/mL) chloral hydrate was prepared and anesthetized according to the injection amount of 0.4mL/100g of the body weight of the rats. After confirming that the rat is in an anesthetic state, cutting off the thoracic cavity, taking blood from the apex of the heart, standing the collected blood at room temperature for 2 hours, and centrifuging for 10min at a rotating speed of 3000r/min. And taking the supernatant after centrifugation by a pipetting gun into a 1.5mL freezing tube, and storing in a refrigerator for later use.
(2) Hippocampal samples: after the rat breaks the head, the cranium is opened, the hippocampal tissues of the left and right hemispheres of the brain are rapidly peeled off, and the rat is put into a refrigerator for preservation and measurement.
(3) Colon sample: the abdominal cavity of the rat is cut, the distal colon is taken to be about 2cm, and the rat is put into a refrigerator for storage for standby.
Thawing the sample, washing, sucking excessive liquid with filter paper, cutting, homogenizing, pulverizing tissue, centrifuging, collecting supernatant, and determining SP content in rat serum, colon and Hippocampus tissue according to ELISA kit (purchased from Shanghai-associated Mich.) specification.
TABLE 6 comparison of SP content at different sites (ng/mL, x.+ -. S) for each group of rats
Note that: ** P < 0.01 compared with the control group; ## P < 0.01 compared with the model group
As can be seen from Table 6, the SP content of the model group was significantly higher than that of the control group and IBSmixes groups, indicating that the pain level was relatively strong in rats of the model group. The SP content of IBSmixes groups is not obviously different from that of a control group, so that the compound bacterial agent for improving the irritable bowel syndrome has an effect of relieving pain caused by irritable bowel.
Although the present invention has been described in detail by way of preferred embodiments with reference to the accompanying drawings, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (8)
1. The composite microbial agent for improving the irritable bowel syndrome is characterized by comprising bifidobacterium bifidum (Bifidobacterium bifidum) JYBB-163, bifidobacterium lactis (Bifidobacterium lactis) JYBR-390 and lactococcus lactis (Lactococcus lactis) JYLL-60, wherein the preservation number of the bifidobacterium bifidum JYBB-163 is CGMCC No.18091; the preservation number of the bifidobacterium lactis JYBR-390 is CGMCC NO.18093; the preservation number of the lactococcus lactis JYLL-60 is CGMCC No.23353.
2. The complex bacterial agent for improving irritable bowel syndrome according to claim 1, characterized in that the total colony count of the complex bacterial agent for improving irritable bowel syndrome is 1.0 x 10 7cfu/g-1.0×109 cfu/g.
3. The complex bacterial agent for improving irritable bowel syndrome according to claim 1, wherein the complex bacterial agent for improving irritable bowel syndrome is present in the form of bacterial suspension.
4. The complex bacterial agent for improving irritable bowel syndrome according to claim 1, characterized in that the complex bacterial agent for improving irritable bowel syndrome further comprises maltodextrin.
5. A method for preparing the composite microbial inoculum for improving irritable bowel syndrome according to claim 1, which is characterized by comprising the following specific steps:
inoculating bifidobacterium bifidum JYBB-163, bifidobacterium lactis JYBR-390 and lactococcus lactis JYLL-60 into MRS liquid culture medium respectively for culture, centrifugally collecting, washing, re-suspending in maltodextrin aqueous solution to obtain bacterial suspension, and freeze-drying to obtain bifidobacterium bifidum JYBB-163 bacterial powder, bifidobacterium lactis JYBR-390 bacterial powder and lactococcus lactis JYLL-60 bacterial powder respectively; and mixing the bifidobacterium bifidum JYBB-163 bacterial powder, the bifidobacterium lactis JYBR-390 bacterial powder and the lactococcus lactis JYLL-60 bacterial powder with equal mass after the same multiple of gradient dilution respectively, so as to obtain the composite bacterial agent for improving the irritable bowel syndrome.
6. Use of the complex bacterial agent for improving irritable bowel syndrome according to claim 1 in the preparation of a medicament for improving diarrhea type irritable bowel syndrome.
7. The use according to claim 6, wherein the complex bacterial agent for improving irritable bowel syndrome is used for improving physiological and/or mental problems caused by diarrhea-type irritable bowel syndrome.
8. The use according to claim 7, wherein improving physiological problems comprises improving problems of weight loss, diarrhea and/or stool misshaping, and improving mental problems comprises reducing depression levels and/or anxiety levels.
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