CN117187117A - Probiotic composition for relieving constipation and regulating intestinal flora disorder and application thereof - Google Patents

Probiotic composition for relieving constipation and regulating intestinal flora disorder and application thereof Download PDF

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CN117187117A
CN117187117A CN202311028895.5A CN202311028895A CN117187117A CN 117187117 A CN117187117 A CN 117187117A CN 202311028895 A CN202311028895 A CN 202311028895A CN 117187117 A CN117187117 A CN 117187117A
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constipation
probiotic composition
serum
mice
content
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赵阿丹
郭新梅
朱迪凡
张晨玥
彭灿
吴忠坤
刘学聪
王云
何协勋
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Jinqiao Biotechnology Co ltd
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Abstract

The invention discloses a probiotic composition for relieving constipation and regulating intestinal flora disorder and application thereof, and belongs to the field of microorganisms. The bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 probiotic composition provided by the invention has the effects of regulating intestinal flora, obviously improving apparent pathological indexes of constipation and improving intestinal peristalsis; compared with lactobacillus rhamnosus LGG, the probiotic composition provided by the invention can better improve the quantity of beneficial bacteria (bifidobacteria and lactobacillus) of mice with dysbacteriosis, improve the water content of excrement of constipation mice, obviously shorten the time for the mice with constipation to discharge head and black stool, improve the MTL content in serum, reduce the VIP content in serum, and have more obvious promotion effect on small intestine peristalsis, so that a more personalized treatment scheme can be adopted for constipation patients in the future, and the probiotic composition has a very considerable application prospect.

Description

Probiotic composition for relieving constipation and regulating intestinal flora disorder and application thereof
Technical Field
The invention relates to a probiotic composition for relieving constipation and regulating intestinal flora disorder and application thereof, and belongs to the field of microorganisms.
Background
Constipation is a gastrointestinal dysfunction disease with higher incidence and wider coverage group, and the gastrointestinal dynamics group of the department of digestive diseases of the China society issues a file of Chinese chronic constipation expert consensus (2019) in 2019, wherein data show that about five millions of adults in China have constipation puzzles, which are equivalent to 4.0% -10.0% of prevalence. The prevalence of constipation also increases year by year with age, with prevalence of > 70 years old reaching 23.0% and > 80 years old reaching 38.0%, and up to 80% of patients requiring long-term care. Constipation causes dysbacteriosis in the intestinal tract in addition to clinical symptoms such as difficult defecation, low defecation frequency, dry and hard feces and the like. Constipation can change the composition of intestinal microbiota, increase the permeability of the intestine to pathogens and endotoxins, and promote oxidative and inflammatory reactions. Several studies have found increased oxidative damage and decreased antioxidant response in rats and chronically constipation children. In addition, serum inflammatory cytokine levels are positively correlated with chronic constipation. In addition, it was found that interleukin-6 and IL-12 levels were higher in constipation children and IL-1 beta and tumor necrosis factor levels were unchanged compared to healthy controls.
At present, drugs remain the main way to treat constipation, but more or less have some toxic and side effects. In recent years, intestinal flora has become a research hotspot in the scientific research field, and research on diet, correlation between intestinal flora and diseases is important, so that constipation relief through dietary supplementation of probiotics is possible. Probiotics have a number of probiotic properties as a viable microorganism that when ingested in sufficient quantities can produce numerous positive benefits to the host. Most of the probiotic preparations in the market at present are composite probiotic preparations. There are studies showing that intestinal constipation symptoms can be more effectively alleviated if multi-strain (lactobacillus acidophilus, lactobacillus paracasei, lactococcus lactis, bifidobacterium lactis and bifidobacterium bifidum) probiotic products are taken for a long period of time. Researchers can relieve constipation by using composite probiotic powder (bifidobacterium lactis V9, lactobacillus casei Zhang and lactobacillus plantarum P9), and the composite probiotic powder can play a role in relieving constipation of mice by promoting intestinal peristalsis and adjusting intestinal flora structure. And, there have been studied that a complex probiotic formula (bifidobacterium lactis CP-9, bifidobacterium infantis BLI-02, bifidobacterium breve BV-889, lactobacillus rhamnosus MP-108, streptococcus thermophilus SY-66) containing 5 probiotics is mixed at 1.5X10 9 The viable bacteria of the CFU count gastric lavage mice for 15 days, and the result shows that the probiotic compound formula has the effects of improving constipation of the mice, relieving intestinal mucosa injury and relieving diarrhea symptoms.
The probiotic preparation for relieving constipation in the prior art is mostly composed of 4-5 probiotics, and the number of viable bacteria in the probiotic composition is required to be 10 in order to achieve the effect of relieving constipation 9 CFU is even higher, and takes at least 14 days to achieve constipation relieving effect, and has the advantages of multiple probiotic species, large viable count and long onset time. There is a need for a probiotic composition profileThe probiotic composition for relieving constipation has the advantages of single probiotic composition, less required viable bacteria and short acting time, so as to obtain a probiotic formula with low cost and good effect.
Disclosure of Invention
It is a first object of the present invention to provide a probiotic composition comprising cells of bifidobacterium infantis (bifidobacterium infantis) BLI-02, bifidobacterium animalis (Bifidobacterium animalis) BB-115 and lactobacillus rhamnosus (Lactobacillus rhamnosus) MP108 and/or extracellular metabolites thereof.
In one embodiment, the probiotic composition has a ratio of numbers of cells of bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115, and lactobacillus rhamnosus MP108 of 1:1:1.
in one embodiment, the number of cells in the cell is not less than 4.1X10 8 CFU/g or 4.1X10 8 CFU/mL。
In one embodiment, the cell is a living cell or a dead cell.
In one embodiment, the probiotic composition is a bacterial powder or bacterial suspension.
In one embodiment, the probiotic composition further comprises a protective agent comprising one or more of skim milk powder, maltodextrin, trehalose, and lactose.
The invention also provides application of the probiotic composition in preparation of medicines for relieving constipation.
In one embodiment, the pharmaceutical product further comprises a pharmaceutical carrier and/or pharmaceutical excipients, the pharmaceutical carrier comprising one or more of fillers, binders, wetting agents, disintegrants, lubricants, binders commonly used in medicine.
In one embodiment, the pharmaceutical product is in the form of a granule, capsule, tablet, pill or oral liquid.
The invention also provides application of the probiotic composition in preparation of food or health care products for relaxing bowel.
In one embodiment, the food product comprises a beverage, a soft candy, a tabletted candy, a snack food, etc., and the dairy product comprises fermented milk, flavored fermented milk, fermented milk beverage, cream, cheese, milk-containing beverage, or milk powder; the bean product comprises soymilk or soymilk powder; the fruit and vegetable product comprises a fruit and vegetable product prepared by taking at least one of Chinese cabbage, white radish, cucumber, beet, yellow peach or waxberry product as a raw material.
In one embodiment, the food product is a fermented food product, including a solid food product, a liquid food product, or a semi-solid food product.
In one embodiment, the food product is a beverage or snack containing a composition of bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115, and lactobacillus rhamnosus MP108 as described above, or a microbial preparation as described above.
In one embodiment, the bowel relaxing includes, but is not limited to, at least one of the following effects:
(a) The diversity of intestinal flora is increased, and the number of beneficial bacteria bifidobacteria and lactobacillus in feces is obviously increased;
(b) Improving the water content of the excrement and improving the small intestine propulsion rate/full intestinal peristalsis;
(c) Up-regulating motilin content in serum and down-regulating vasoactive intestinal peptide content in serum;
(d) Reducing the level of interleukin-1, interleukin-6 and interleukin-8 in serum;
(e) The content of short chain fatty acid in the excrement is obviously improved;
(f) Regulating intestinal flora and relieving constipation.
Advantageous effects
The multi-strain formula probiotics (bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP 108) can remarkably improve the quantity of bifidobacteria and lactobacillus in the mouse feces, and the quantity is improved by 1-2 orders of magnitude compared with a control group or before intervention; the water content of the excrement of a constipation mouse and the propulsion rate of the small intestine can be obviously improved, and the first granule of excrement discharge time is reduced, compared with a model group, the water content of the excrement is improved by 8.15 percent after intervention, the propulsion rate of the small intestine is improved by 13.15 percent, and the first granule of excrement discharge time is shortened by 58.6 minutes (50.0 percent); effectively regulating the content of excitatory and inhibitory gastrointestinal active peptides in serum, remarkably improving the content of MTL in serum of a constipation mouse, improving 81.85pg/mL (namely improving by 43.3%), remarkably reducing the content of VIP in serum of the constipation mouse, reducing by 53.34pg/mL (namely reducing by 23.2%), facilitating intestinal propulsion and achieving the final purpose of recovering the health level; can significantly down-regulate the levels of pro-inflammatory cytokines IL-1 (down-regulated 57.76pg/mL, namely 23.6%), IL-6 (down-regulated 28.01pg/mL, namely 33.5%), IL-8 (down-regulated 27.93pg/mL, namely 22.7%) in the serum of a constipation mouse, improve the inflammatory response of the constipation mouse and further relieve constipation; can increase the content of acetic acid (39.86 mu mol/g, namely 2.6 times) and isovaleric acid (0.7338 mu mol/g, namely 6.4 times) in the feces, has remarkable effect and promotes the health of mice. Therefore, the invention can be regarded as a medicine for relieving or treating constipation, can be applied to medicines or some fermented foods and functional foods, even beverages, soft sweets, pressed candies, snacks and the like, thereby widely playing the roles and having very valuable application prospects.
Drawings
Fig. 1: the experimental flow of bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 formula probiotics on constipation-relieving animals induced by loperamide.
Fig. 2: schematic of the effect of bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 on the intestinal microbiota of mice before and after the intervention of the probiotics.
Fig. 3: schematic representation of changes in apparent indicators of constipation relief in mice induced with loperamide to produce constipation by bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115, and lactobacillus rhamnosus MP108 formula probiotics.
Fig. 4: the change of neurotransmitter content in serum of mice with loperamide induced constipation after intervention of bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 formula probiotics is shown.
Fig. 5: the change of inflammatory factor content in serum of mice with constipation induced by loperamide after intervention of bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 formula probiotics is shown.
Fig. 6: the change of short chain fatty acid in the constipation-inducing mouse feces of loperamide after the intervention of the formula probiotics of Bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and Lactobacillus rhamnosus MP 108.
Wherein, P <0.05, P <0.01, P <0.001, P <0.0001, compared to constipation model group.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
Male C57BL/6J mice referred to in the examples below were purchased from Jiangsu Jiugang Biotech Co.
The following examples relate to the deposited numbers of the strains as follows:
a multi-strain formula probiotic comprising bifidobacterium infantis (bifidobacterium infantis) BLI-02, bifidobacterium animalis (Bifidobacterium animalis) BB-115 and lactobacillus rhamnosus (Lactobacillus rhamnosus) MP108, said bifidobacterium infantis BLI-02 having a accession number of CGMCC No. 15212; the preservation number of the bifidobacterium animalis (Bifidobacterium animalis) BB-115 is CGMCC No. 21840; said lactobacillus rhamnosus (Lactobacillus rhamnosus) MP108 has been disclosed in patents CN102604854B and CN103436461B under the accession number DSM24229.
The strain of Lactobacillus rhamnosus LGG according to the following examples is disclosed in article Lactobacillus rhamnosus GG supernatant promotes intestinal mucin production through regulating 5-HT 4 R and gut microbiota.Food&Function,2022,13(23):12144-12155》。
The following examples relate to the following media:
MRS liquid medium: 10g of beef extract; 10g of tryptone; 5g of yeast powder; glucose 20g; 5g of anhydrous sodium acetate; mgSO (MgSO) 4 ·7H 2 O 0.1g；MnSO 4 ·H 2 O0.05 g; 2g of diammonium hydrogen citrate; k (K) 2 HPO 4 ·3H 2 O2.6 g; tween 80 1ml; l-cysteine hydrochloride 0.8g; adjusting the pH to 6.8+/-0.2; constant volume to 1L. Autoclaving at 115℃for 20min.
MRS solid medium: 2% agar powder is added on the basis of MRS liquid culture medium.
Preparation of a stock solution of Bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and Lactobacillus rhamnosus MP108 in the following examples
Inoculating Bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and Lactobacillus rhamnosus MP108 into MRS solid culture medium respectively, culturing at 37deg.C for 72 hr to obtain single colony, inoculating the prepared single colony into MRS liquid culture medium, and culturing at 37deg.C for 24 hr for activation;
the bacterial liquid after 3 generations of activation is inoculated into 1L MRS liquid culture medium with an inoculum size of 2% (v/v), and the bacterial liquid is cultured for 24 hours at 37 ℃ in an anaerobic incubator after shaking and mixing. Centrifuging at 8000g/min and 4deg.C for 15min, removing supernatant, cleaning with sterile physiological saline containing 0.05% -0.1% L-cysteine hydrochloride for 2 times, centrifuging under the same conditions, removing supernatant, re-suspending with 30% glycerol to obtain pre-gastric juice, and freezing at-80deg.C for one week.
Before animal experiments are carried out, the frozen bacterial liquid in the refrigerator is taken out, centrifuged for 5min at 6000r/min, washed twice with sterile physiological saline, the bacterial liquid is resuspended with 10% skim milk, and after shaking evenly, the number of viable bacteria is measured by a flat plate pouring method after initial and one week of frozen storage.
Experimental results: the initial viable count is 4.1X10 8 The order of magnitude of viable bacteria does not change after 1 week in CFU/mL, which indicates that the bacteria liquid can not influence the experiment after freezing and storing, and can be used for animal experiments.
Preparation of Bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and Lactobacillus rhamnosus MP108 powders as referred to in the examples below
The preparation method comprises the steps of taking the prepared bacterial liquid of the bifidobacterium infantis BLI-02, the bifidobacterium animalis BB-115 and the lactobacillus rhamnosus MP108, freeze-drying for 10-24 hours at the temperature of between minus 40 ℃ and minus 60 ℃ and the vacuum degree of 0.05-0.01mbar, then drying for 30 hours at the temperature of 25-35 ℃ to obtain freeze-dried bacterial powder, crushing and sieving the freeze-dried bacterial powder, and then mixing the freeze-dried bacterial powder with maltodextrin to obtain the prepared bacterial powder of the bifidobacterium infantis BLI-02, the bifidobacterium animalis BB-115 and the lactobacillus rhamnosus MP 108.
The detection method involved in the following examples is as follows:
the detection method for regulating intestinal flora of mice comprises the following steps:
the experimental design and the related index determination are carried out by referring to the method for checking and evaluating the functions of health food (2022 edition) which is helpful for adjusting the intestinal flora. The 2-3 feces in the anus of the mice on day 0 and day 28 of the intervention are respectively aseptically taken and placed in an asepsis centrifuge tube for standby. The intestinal flora was detected according to the following detection method: firstly, weighing wet weight of excrement, then carrying out serial dilution by 10 times, selecting dilution of colony number at 30-300, inoculating on a corresponding culture medium for culturing, identifying and counting colony according to colony morphology, gram staining microscopic examination, biochemical reaction and the like, calculating the bacterial numbers of bifidobacteria, lactobacillus, enterobacteria, enterococcus and clostridium perfringens in each gram of wet excrement, and taking logarithm (lg CFU/g) as a result for statistics.
Neurotransmitters in mouse serum are detected as follows:
measurement of constipation-associated gastrointestinal modulator content in mouse serum using ELISA kit: motilin (MTL), gastrin (Gas), substance P (SP), vasoactive Intestinal Peptide (VIP), vasoactive intestinal peptide (SS), casein Peptide (PYY), acetylcholine (Ach), and serotonin (5-HT), and the above method is performed according to the specification.
The inflammatory factor levels in the serum of mice were measured as follows:
measuring the content of constipation-associated inflammatory factors in the serum of the mice by using an ELISA kit: interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), transforming growth factor beta (TGF-beta), and the methods of operation described above are performed as desired in the specification.
The method for detecting the concentration of short-chain fatty acids in feces according to the following examples comprises the following steps:
the cecal content collected before the end of the experiment was freeze-dried, the dry weight of the cecal content was calculated and frozen at-80 ℃. The specific method comprises the following steps:
weigh 20mg of feces, re-suspend with 500. Mu.L of saturated NaCL solution, add 20. Mu.L of 10% H 2 SO 4 A solution; adding 1000 μl of anhydrous diethyl ether, shaking uniformly, extracting fatty acid, and centrifuging at 12000rpm at 4deg.C for 15min; taking the upper diethyl ether phase, adding 0.25g of anhydrous Na 2 SO 4 Drying; standing for 30min, centrifuging at 12000rpm and 4deg.C for 5min to obtain upper diethyl ether phase, and determining short chain fatty acid content in mouse lyophilized feces by GC-MS. Rtx-Wax column (column length 30m, inner diameter 25 μm) was used; the carrier gas is He, and the flow rate is 2mL/min; the sample injection volume is 1 mu L, the temperature is increased to 140 ℃ according to 7.5 ℃/min, then the temperature is increased to 200 ℃ according to 60 ℃/min, and the ionization temperature is 20 ℃ after 3 min; the analysis adopts a full scanning mode, and standard curves are measured by an external standard method, so that the concentration of various short-chain fatty acids is calculated.
Example 1: modulation of intestinal flora by bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 compositions
The method comprises the following specific steps:
(1) Preparation of Bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and Lactobacillus rhamnosus MP108 powder
Taking out the standby bacterial liquid of the bifidobacterium infantis BLI-02, the bifidobacterium animalis BB-115 and the lactobacillus rhamnosus MP108 in a refrigerator at the temperature of minus 80 ℃, respectively streaking in an MRS solid culture medium, culturing for 48 hours at the temperature of 37 ℃, respectively picking single bacterial colonies in the MRS liquid culture medium, and culturing for 24 hours at the temperature of 37 ℃ to obtain seed liquid;
inoculating the prepared seed solutions into new MRS liquid culture medium respectively with inoculum size of 2% (v/v), culturing at 37deg.C for 24 hr, and culturing again in the same manner to obtain fermentation liquid of Bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and Lactobacillus rhamnosus MP 108;
then centrifuging the prepared fermentation liquids of Bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and Lactobacillus rhamnosus MP108 at 6000r/min and 4deg.C for 5min, re-suspending with 10% (w/v) skimmed milk, adding malt paste, and refining to obtain lyophilized powder with viable count of Bifidobacterium infantis BLI-02 of 1.0X10 9 ～1.0×10 11 CFU/g, viable count of bifidobacterium animalis BB-115 is 1.0X10 9 ～1.0×10 11 CFU/g, live bacteria number of lactobacillus rhamnosus MP108 is 1.0X10 9 ～1.0×10 11 CFU/g。
(2) Preparation of lactobacillus rhamnosus LGG bacterial powder
The preparation method is the same as in the above (1).
(3) Modulation of intestinal disorders in mice by bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 composition powders
Re-suspending the lyophilized powder obtained in step (1) in sterile physiological saline to obtain combined bacterial suspension, wherein the viable count of the bifidobacterium infantis BLI-02 in the bacterial suspension is 1.4X10 8 ～1.5×10 8 CFU/ml, viable count of bifidobacterium animalis BB-115 is 1.4X10 8 ～1.5×10 8 CFU/ml, live bacteria number of lactobacillus rhamnosus MP108 is 1.4X10 8 ～1.5×10 8 CFU/ml。
The steps are as follows(2) The obtained freeze-dried bacterial powder is resuspended in sterile physiological saline to prepare LGG bacterial suspension, wherein the viable count of lactobacillus rhamnosus LGG in the bacterial suspension is 4.1X10% 8 ～4.5×10 8 CFU/ml。
(4) 30 healthy male C57BL/6J mice at 6 weeks of age are taken and adapted to the environment for 1 week, and randomly divided into 3 groups: control group (normal intestinal flora), lactobacillus rhamnosus LGG intervention group (LGG group), combined probiotics (ratio of bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 is 1:1:1), intervention group (LBM group), each group containing 10 mice. On days 8-28, the control group was filled with 0.2ml of sterile physiological saline once per day, and the LGG group and LBM group were filled with 0.2ml of bacterial suspension once per day, the total dose of the strain filled with stomach was 4.1X10% 8 CFU/mL, 9 points on each day early begin lavage. During the test period, the mice were fed with a small mouse quasi-feed, and the feces were collected weekly and stored in a-80 ℃ refrigerator as soon as possible for later use. After the experiment is finished, 100 mg/(kg.bw) ketamine is injected into the abdominal cavity of the mice, eyeballs are taken out for blood collection after anesthesia is finished, and then cervical vertebrae are removed for killing the mice. The blood is centrifuged for 10min at 4 ℃ and 3000 Xg, and the serum is packaged and frozen at-80 ℃ for standby. The specific experimental groups and animal experimental procedures are shown in Table 1 and FIG. 1, respectively.
Table 1 experimental animal groups
The experimental design and the related index determination are carried out by referring to the method for checking and evaluating the functions of health food (2022 edition) which is helpful for adjusting the intestinal flora. Feces on day 28 were collected under sterile conditions for later use. The intestinal flora was detected according to the following detection method: firstly, weighing wet weight of excrement in two different states, then carrying out 10-time serial dilution, selecting dilution of colony number at 30-300, inoculating on a corresponding culture medium for culturing, identifying and counting colony according to colony morphology, gram staining microscopic examination, biochemical reaction and the like, calculating the bacterial number of bifidobacterium, lactobacillus, enterobacter, enterococcus and clostridium perfringens in each gram of wet excrement, and taking logarithm (lg CFU/g) as a result for statistics.
After 21 days of intervention, there was no statistical difference in the numbers of bifidobacteria, lactobacilli, enterobacteria, enterococci, clostridium perfringens in the intestinal microbiota of the mice of the blank group (fig. 2). After 21 days of oral administration of different multi-strain probiotics, the LGG group and the LBM group have significantly increased bifidobacterium and lactobacillus amounts (P < 0.0001) in the feces of mice compared with the LGG group and the LBM group before intervention, and the intestinal bacilli, the enterococci and the clostridium perfringens are improved by 1-2 orders of magnitude without significant changes. Wherein, the LBM group significantly increased the bifidobacterium number in the mouse feces compared with the LGG group. Comparison between the experimental group and the control group after bacterial intervention shows that the results are the same as the results. The result accords with the experiment result positive of the method for testing and evaluating the functions of health food (2022 edition). Thus, bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 formula probiotics have a good effect in restoring the number of beneficial bacteria (bifidobacteria, lactobacillus) in the intestinal flora disturbed mouse faeces.
Example 2: relief of loperamide-induced constipation-related symptoms from bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 compositions
The method comprises the following specific steps:
(1) 40 healthy male C57BL/6J mice of 6 weeks of age are taken and adapted to the environment for 1 week, and randomly divided into 4 groups: control, model (constipation), lactobacillus rhamnosus LGG intervention (LGG), combined probiotics (bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP 108) intervention (LBM), each group containing 10 mice. On days 8-14, a constipation model is built, the control group is filled with gastric sterile physiological saline, the other groups are filled with gastric sterile physiological saline of 10 mg/(kg.bw) loperamide hydrochloride, after 1h, the control group is filled with gastric sterile physiological saline, the model group is filled with gastric sterile physiological saline, and the gastric administration doses of bacteria corresponding to the bacterial intervention group are all 0.2mL. During the test period, the mice were fed with a small mouse quasi-feed, and the feces were collected weekly and stored in a-80 ℃ refrigerator as soon as possible for later use. After the experiment is finished, 100 mg/(kg.bw) ketamine is injected into the abdominal cavity of the mice, eyeballs are taken out for blood collection after anesthesia is finished, and then cervical vertebrae are removed for killing the mice. The blood is centrifuged for 10min at 4 ℃ and 3000 Xg, and the serum is packaged and frozen at-80 ℃ for standby. The specific experimental groups and animal experimental procedures are shown in Table 2 and FIG. 1, respectively.
The grouping and treatment methods of experimental animals (bowel relaxing) are shown in Table 2:
TABLE 2 grouping of experimental animals (bowel relaxing)
(2) Mouse faeces were collected weekly for the detection of faecal moisture content. The method comprises the following steps: putting the mice after the completion of stomach filling into a clean cage box filled with filter paper, collecting fresh faeces, weighing, freeze-drying, and calculating the water content of the faeces according to the formula (1):
(3) On experiment day 14, a mixed solution of loperamide hydrochloride (10 mg/(kg. Bw)) and activated carbon was filled in the stomach in an amount of 0.2mL, and the time period from the time of filling the stomach with ink to the time of discharging the first black stool was recorded as the first black stool discharge time. Treatment groups exceeding the time to first black stool in the last mouse of the model group showed no efficacy, and each treatment group was compared with the model group to demonstrate differences in constipation relief for each group.
(4) The mice are fasted for 12 hours before dissection, water is not forbidden, on 15 days, all groups of mice except the normal group are irrigated with 0.2mL of a mixed solution of loperamide hydrochloride and activated carbon, the normal group is irrigated with a mixed solution of sterile normal saline and activated carbon, the mice are killed after 30 minutes, the intestinal canal from the pylorus to the cecum is cut after the abdominal cavity of the mice is opened, the intestinal canal is pulled into a straight line, the length of the intestinal canal is measured to be the total length of the small intestine, the length from the pylorus to the front edge of ink is the front propulsion length of the activated carbon solution, and the small intestine propulsion rate is calculated according to the formula (2):
the experimental results of the fecal water content, the first granule discharge and the black stool time and the small intestine propulsion rate are shown in fig. 3, and as can be seen from fig. 3, the gastric lavage LGG and LBM can significantly improve the fecal water content and the small intestine propulsion rate of the constipation mice and significantly shorten the first granule discharge and black stool time (P < 0.05) compared with the constipation model group. Wherein, compared with the model group, the water content of the feces after the intervention of the LBM group is improved by 8.15 percent, the propulsion rate of the small intestine is improved by 13.15 percent, and the first granule discharge and black feces time is shortened by 50.0 percent (58.6 min) which is equivalent to that of the control group; compared with the LGG group, the LBM group has more obvious effects on the fecal water content (the LGG group only increases by 6.95%, the fecal water content of the LBM group is 1.02 times that of the LGG group) and the first granule discharge and blackish stool time (the LGG is only shortened by 26.6% (31.9 min), and the LBM group has more obvious effects on the small intestine than the LGG group in the first granule discharge and blackish stool time which is shortened by 30.55%. Thus, bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and Lactobacillus rhamnosus MP108 have good effects in restoring the symptoms of constipation, dryness and slow gastrointestinal motility in mice.
In general, bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 formula probiotics have a good constipation relieving effect.
Example 3: the composition of Bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and Lactobacillus rhamnosus MP108 can significantly up-regulate Motilin (MTL) level in serum of constipation mice and reduce Vasoactive Intestinal Peptide (VIP) content in serum
The method comprises the following specific steps:
(1) The grouping, modeling and processing method of the C57BL/6J mice are the same as in example 2.
(2) After the mice were sacrificed on day 15, the collected blood of the mice was allowed to stand for 2 hours, and centrifuged at 3000 Xg for 15 minutes to obtain serum, and the concentrations of Motilin (MTL), gastrin (Gas), substance P (SP), vasoactive Intestinal Peptide (VIP), vasoactive intestinal peptide (SS), tyrosin (PYY), acetylcholine (Ach), and serotonin (5-HT) in the serum were calculated from standard curves by performing experiments according to the specification using the kit for measuring the concentration of Motilin (MTL), gastrin (Gas), substance P (SP), vasoactive Intestinal Peptide (VIP), vasoactive intestinal peptide (SS), tyrosin (PYY), acetylcholine (Ach), and serotonin (5-HT).
As shown in fig. 4, as compared with the control group, the serum of the model mice after loperamide hydrochloride treatment has significantly reduced content of excitatory neurotransmitter MTL (P < 0.05), while the serum Gas, SP, ach level has no significant change, the serum of the mice has significantly increased content of inhibitory neurotransmitter VIP and PYY, and the SS and 5-HT levels have no significant difference, as shown in fig. 4. Indicating that constipation caused by loperamide hydrochloride may be associated with abnormal levels of MTL, VIP, PYY in serum. When LBM was administered, the MTL levels in serum were significantly increased (P < 0.001) and VIP levels were significantly down-regulated (P < 0.05) to return to normal levels in the LGG and LBM-intervention groups compared to the model group. Wherein, compared to the model group, the MTL content in serum after LBM intervention was increased by 81.85pg/mL (43.3%), the LGG content was increased by 80.31pg/mL (42.5%), the VIP content in serum after LBM intervention was decreased by 53.34pg/mL (23.2%), and the LGG content was decreased by 46.14pg/mL (20.1%). In addition, LBM significantly reduced 5-HT levels, down-regulated 46.08ng/mL (42.5%), while LGG group was not significantly different. The MTL in the serum of the LBM group is obviously up-regulated, and the MTL is a polypeptide consisting of 22 amino acids, is distributed in all small intestines, and can promote and influence gastrointestinal motility and transportation of water and electrolyte by the gastrointestinal tract, so that the shrinkage of the stomach and intestinal peristalsis are promoted, and the higher the content of MTL in the serum is, the stronger the promoting effect of the MTL on the shrinkage of the stomach and the intestinal peristalsis is. LBM regulates gastrointestinal neurotransmitter secretion by restoring serum MTL, VIP levels, thereby alleviating constipation.
Example 4: the composition of Bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and Lactobacillus rhamnosus MP108 can significantly down regulate the levels of proinflammatory cytokines interleukin-1 (IL-1), interleukin-6 (IL-6) and interleukin-8 (IL-8) in serum of constipation mice
The method comprises the following specific steps:
(1) The grouping, modeling and processing method of the C57BL/6J mice are the same as in example 2.
(2) After the mice were sacrificed on day 15, the collected blood of the mice was allowed to stand for 2 hours, and centrifuged at 3000 Xg for 15 minutes to obtain serum, and the concentrations of interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), transforming growth factor beta (TGF-beta) and transforming growth factor beta (TGF-beta) in the serum were calculated from standard curves by performing experiments according to the specification using interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta) detection kits.
As shown in FIG. 5, the serum levels of proinflammatory cytokines IL-1, IL-6 and IL-8 in the serum of the constipation mice induced by loperamide hydrochloride are significantly increased (P < 0.05) compared with the normal group, and the content changes of the proinflammatory cytokines IL-10 and TGF-beta are not statistically different in the model group compared with the control group. It is demonstrated that the construction of constipation model using loperamide hydrochloride is associated with abnormal levels of pro-inflammatory cytokines IL-1, IL-6, IL-8 in serum. Comparison to the model group found that all probiotic treated groups were able to significantly down-regulate IL-6, IL-8 levels in serum of constipation mice (P < 0.05), wherein LBM and LGG treated groups significantly reduced the content of IL-1 in serum (P < 0.01), wherein IL-1 down-regulated 57.76pg/mL (23.6%), IL-6 down-regulated 28.01pg/mL (33.5%), IL-8 down-regulated 27.93pg/mL (22.7%) after LBM intervention compared to the model group; while LGG group IL-1 down-regulated 50.73pg/mL (20.7%), IL-6 down-regulated 18.72pg/mL (22.4%), and IL-8 did not differ significantly. In our results, while there was no significant difference in IL-10 and TGF- β levels in the blank versus model, there was a significant up-regulation of IL-10 levels in the LBM versus model (no significant difference in LGG) and there was a trend of down-regulation of TGF- β levels versus model, but no significant difference. In conclusion, the LBM is used for reducing the content of proinflammatory cytokines in serum after intervention, improving the level of inflammatory factors in serum of a constipation mouse, and further relieving constipation.
Therefore, the bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 formula probiotics can reduce the inflammatory response of the constipation mice by reducing the content of IL-1, IL-6 and IL-8 in serum, thereby effectively relieving constipation.
Example 5: composition of Bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and Lactobacillus rhamnosus MP108 for increasing short chain fatty acid content in constipation mouse feces
The method comprises the following specific steps:
(1) The grouping, modeling and processing method of the C57BL/6J mice are the same as in example 2.
(2) The cecal content collected before the end of the experiment was freeze-dried and the short chain fatty acid content in the freeze-dried cecal content of mice was determined by GC-MS. Thus, the concentration of various short chain fatty acids was calculated.
The experimental results are shown in fig. 6, and after molding with loperamide, the amounts of all short chain fatty acids in the feces of the mice in the model group were down-regulated, wherein the amounts of acetic acid, propionic acid, butyric acid and isovaleric acid were significantly reduced compared with the control group. After bacterial interference, the acetic acid content of all probiotics treatment groups is obviously increased (P is less than 0.05) compared with a model group, and the content of isobutyric acid and isovaleric acid in constipation mouse faeces is obviously increased (P is less than 0.05) by an LBM treatment group, wherein compared with the model group, the acetic acid content in the faeces of the LBM treatment group is increased by 39.86 mu mol/g, the acetic acid content in the LGG group is only increased by 31.77 mu mol/g, and the acetic acid content in the faeces of the LBM treatment group is 1.2 times that of the LGG group; the isovaleric acid content in the feces of the LBM treatment group is increased by 0.7338 mu mol/g, the isovaleric acid content of the LGG group is increased by 0.5107 mu mol/g, and the isovaleric acid content in the feces of the LBM treatment group is 1.4 times that of the LGG group. In addition, the butyric acid content in the feces of the LBM treatment group is increased by 6.031 mu mol/g, the butyric acid content in the LGG group is increased by 2.167 mu mol/g, and the butyric acid content in the feces of the LBM treatment group is 1.4 times that in the LGG group; the isobutyric acid content in the feces of the LBM treatment group is increased by 1.114 mu mol/g, while the isobutyric acid content in the LGG group is increased by 0.6913 mu mol/g, and the isobutyric acid content in the feces of the LBM treatment group is 1.4 times that in the LGG group.
Thus, bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 formula probiotics have an important role in increasing the Short Chain Fatty Acid (SCFAs) content in faeces, mainly in relieving constipation by increasing the content of acetic acid, butyric acid, isobutyric acid and isovaleric acid in faeces. The short chain fatty acid can reduce the pH value of the intestinal tract, promote the absorption of calcium and magnesium ions in the intestinal tract, inhibit the infection of harmful bacteria, stimulate the peristalsis of the intestinal tract and promote the health of mice.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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 (10)

1. A probiotic composition, characterized in that it comprises cells of bifidobacterium infantis (Bifidobacterium infantis) BLI-02, bifidobacterium animalis (Bifidobacterium animalis) BB-115 and lactobacillus rhamnosus (Lactobacillus rhamnosus) MP108 and/or extracellular metabolites thereof.
2. The probiotic composition according to claim 1, characterized in that the ratio of the number of cells of bifidobacterium infantis BLI-02, bifidobacterium animalis BB-115 and lactobacillus rhamnosus MP108 in said cells is 1:1:1.
3. the probiotic composition according to claim 2, characterized in that the number of cells of the cells is not less than 4.1 x 10 8 CFU/g or 4.1X10 8 CFU/mL。
4. A probiotic composition according to claim 3, wherein said cells are living or dead cells.
5. The probiotic composition according to claim 4, characterized in that the probiotic composition is a bacterial powder or a bacterial suspension.
6. The probiotic composition according to claims 1 to 5, further comprising a protective agent comprising one or more of skim milk powder, maltodextrin, trehalose, lactose.
7. Use of a probiotic composition according to any one of claims 1 to 6 for the preparation of a medicament for alleviating constipation.
8. The use according to claim 7, wherein the medicament further comprises a pharmaceutical carrier and/or pharmaceutical excipients, the pharmaceutical carrier comprising one or more of fillers, binders, wetting agents, disintegrants, lubricants, binders commonly used in medicine.
9. Use of a probiotic composition according to any one of claims 1 to 6 for the preparation of a food or health product for relaxing bowel, said food comprising a beverage, a soft candy, a tabletted candy or a snack containing a probiotic formulation.
10. The use according to claim 9, wherein the modulation of intestinal flora and relaxing bowel comprises any of the following:
(a) The diversity of intestinal flora is increased, and the number of beneficial bacteria bifidobacteria and lactobacillus in feces is increased;
(b) Improving the water content of the excrement and improving the small intestine propulsion rate/full intestinal peristalsis;
(c) Up-regulating motilin content in serum and down-regulating vasoactive intestinal peptide content in serum;
(d) Reducing the level of interleukin-1, interleukin-6 and interleukin-8 in serum;
(e) The content of short-chain fatty acid in the excrement is improved;
(f) Regulating intestinal flora and relieving constipation.
CN202311028895.5A 2023-08-16 2023-08-16 Probiotic composition for relieving constipation and regulating intestinal flora disorder and application thereof Pending CN117187117A (en)

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