CN117660244A - Composite probiotics for improving ulcerative colitis and application thereof - Google Patents

Composite probiotics for improving ulcerative colitis and application thereof Download PDF

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Publication number
CN117660244A
CN117660244A CN202311656425.3A CN202311656425A CN117660244A CN 117660244 A CN117660244 A CN 117660244A CN 202311656425 A CN202311656425 A CN 202311656425A CN 117660244 A CN117660244 A CN 117660244A
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strain
ulcerative colitis
probiotic
improving
lactobacillus
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肖传兴
李源涛
黄钻元
徐炜
张帮周
何剑全
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Shanghai Chengge Biotechnology Co ltd
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Shanghai Chengge Biotechnology Co ltd
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract

The invention relates to a compound probiotics for improving ulcerative colitis and application thereof, which consists of a fermented lactobacillus mucilaginosus (Limosilactobacillus fermentum) TG017 strain, a lactobacillus paracasei (Lacticaseibacillus paracasei) TG006 strain, a lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) TG021 strain and a lactobacillus plantarum (Lactiplantibacillus plantarum) TG008 strain. The four can be mutually matched and promoted to jointly exert the remarkable effect of improving ulcerative colitis, and the specific expression is as follows: delaying the weight loss of the colonitis mice; decreasing DAI score in colitis mice; improving colon length shortening in colitis mice; reduces the level of pro-inflammatory cytokines IL-1 beta and TNF-alpha and increases the anti-inflammatory cytokine IL-10.

Description

Composite probiotics for improving ulcerative colitis and application thereof
Technical Field
The invention belongs to the technical field of probiotics, relates to composite probiotics for improving ulcerative colitis and application thereof, and in particular relates to composite probiotics for improving ulcerative colitis, a probiotic agent for improving ulcerative colitis and application thereof in preparing products for preventing, relieving or treating inflammatory bowel diseases.
Background
Inflammatory Bowel Disease (IBD) is a chronic inflammatory disease of the intestinal tract, and the symptoms typically present in patients are diarrhea, abdominal pain, bloody stool, and weight loss. Commonly used anti-IBD drugs are mainly aminosalicylic acid derivatives, including sulfasalazine (SASP) and 5-aminosalicylic acid (also known as mesalamine, 5-ASA). Drugs commonly used to treat IBD often must be taken over a long period of time and have certain side effects. Modulation of microbiota by administration of live bacteria (probiotics) may be an effective method to prevent and even cure IBD. Some probiotics may have positive relief of enteritis symptoms, which is attracting attention of many researchers. Many probiotics exhibit a variety of beneficial effects, such as inhibiting the growth of pathogens, increasing the levels of beneficial metabolites in the gut, modulating the host immune system and gut flora.
For example, CN114908022A discloses a Paramycolatopsis strain, a culture method and application thereof. The Paramycolatopsis strain source is separated from human excrement, and the preservation number is CCTCC NO: m2022645. The strain has good therapeutic effect on Inflammatory Bowel Disease (IBD) induced by Dextran Sodium Sulfate (DSS). In a dextran sodium sulfate-induced inflammatory bowel disease mouse model, the strain can slow down weight loss of mice, increase colorectal length and relieve loose stool and hematochezia phenomena caused by inflammatory bowel disease in mice. The strain can reduce histology and inflammation score of inflammatory bowel disease mice, and has good effect of treating inflammatory bowel disease.
For example, CN116218735a discloses a bacteroides simplex strain, a culture method thereof and application in preparing medicines for treating inflammatory bowel disease. The strain is separated from intestinal tracts of healthy human bodies and preserved in China center for type culture collection of microorganisms, and the preservation number is CCTCC NO: m20221994. The strain can avoid damage to the integrity of colorectal mucosa, repair the existing intestinal mucosa damage, further improve the condition of loose stool and hematochezia, relieve the rapid weight reduction caused by colorectal damage, and finally realize the treatment of inflammatory bowel disease.
However, since the influence of the complex probiotics on inflammatory bowel diseases, particularly ulcerative colitis and its intestinal mucosa immunity is more complex, effective strategies for preventing or treating inflammatory bowel diseases using the complex probiotics are relatively few, and thus it is very significant to develop a safe and effective complex probiotic strategy for preventing or treating inflammatory bowel diseases.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a compound probiotics for improving ulcerative colitis and application thereof, in particular to a compound probiotics for improving ulcerative colitis, a probiotic agent for improving ulcerative colitis and application thereof in preparing products for preventing, relieving or treating inflammatory bowel diseases.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a composite probiotic for improving ulcerative colitis, wherein the composite probiotic for improving ulcerative colitis comprises a compound probiotic with a preservation number of CCTCC NO: lactobacillus mucilaginosus (Limosilactobacillus fermentum) TG017 strain of M2023513, lactobacillus paracasei (Lacticaseibacillus paracasei) TG006 strain with the preservation number of CGMCC No.25452, and CCTCCNO with the preservation number of CCTCCNO: lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) TG021 strain of M2023486 and a accession number cctccc NO: lactobacillus plantarum (Lactiplantibacillus plantarum) TG008 strain of M20221458.
The invention creatively develops a strategy for preventing, improving or treating inflammatory bowel diseases, especially ulcerative colitis, and also develops a novel probiotic compound mode, namely, four strains of lactobacillus fermentum (Limosilactobacillus fermentum) TG017 strain, lactobacillus paracasei (Lacticaseibacillus paracasei) TG006 strain, lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) TG021 strain and lactobacillus plantarum (Lactiplantibacillus plantarum) TG008 strain are compounded and combined, and the four strains can be matched and mutually promoted to jointly play a remarkable effect of improving the ulcerative colitis, which is specifically expressed as follows: (1) delay weight loss in colitis mice; (2) decreasing DAI score in colitis mice; (3) improving colon length shortening in colitis mice; (4) Reduces the level of pro-inflammatory cytokines IL-1 beta and TNF-alpha and increases the anti-inflammatory cytokine IL-10.
Therefore, the compound probiotics have good prospect in preparing products for preventing, relieving or treating inflammatory bowel diseases, especially ulcerative colitis. In addition, lactobacillus mucilaginosus, lactobacillus paracasei, lactobacillus rhamnosus and lactobacillus plantarum are all probiotics, and have high safety and are not easy to generate resistance when being used for preparing products for preventing, relieving or treating inflammatory bowel diseases, especially ulcerative colitis.
Preferably, the ratio of the number of the viable bacteria of the TG017 strain, the TG006 strain, the TG021 strain and the TG008 strain is (1-5): 1-5.
Based on the potential interaction relationship among the TG017 strain, the TG006 strain, the TG021 strain and the TG008 strain, the invention also finds that when four strains are compounded according to the specific viable bacteria count ratio, the effect of the four strains on improving ulcerative colitis is more remarkable.
The specific point values in the above (1-5) may be selected from 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, etc., and other specific point values in the numerical range may be selected, which will not be described herein.
In a second aspect, the invention provides a probiotic for improving ulcerative colitis, the strain in the probiotic comprising the complex probiotic for improving ulcerative colitis according to the first aspect.
Preferably, the viable bacteria content of the TG017 strain, the TG006 strain, the TG021 strain and the TG008 strain in the probiotics is not less than 1 multiplied by 10 9 CFU/g or 1X 10 9 CFU/mL, e.g. 5X 10 9 CFU/g、1×10 10 CFU/g(CFU/mL)、5×10 10 CFU/g(CFU/mL)、1×10 11 CFU/g(CFU/mL)、5×10 11 CFU/g(CFU/mL)、1×10 12 CFU/g(CFU/mL)、5×10 12 CFU/g(CFU/mL)、1×10 13 CFU/g (CFU/mL), etc., other specific values within the numerical range may be selected, and will not be described in detail herein.
Preferably, the formulation of the probiotic agent comprises freeze-dried powder, capsules, tablets or granules.
The formulation of the probiotics related to the invention is not limited, and comprises the most commonly used freeze-dried powder, or further prepared capsules, tablets or granules. The lyophilized powder can be prepared by the following method:
activating the TG017 strain, the TG006 strain, the TG021 strain or the TG008 strain, and then respectively inoculating the activated strains into a culture medium for culture to obtain a culture solution; centrifuging the culture solution to obtain thalli; re-suspending the thalli by using a freeze-drying protective agent to obtain re-suspension; freeze-drying the heavy suspension, and mixing according to a proportion to obtain the finished product.
Preferably, the medium includes BHIS medium or MRS medium.
Preferably, the time of the cultivation is 18-30 hours.
Preferably, the centrifugation is carried out at 4000-6000rpm for 5-15min at 2-6deg.C.
Preferably, the lyophilization is by vacuum freezing.
Preferably, the probiotic agent further comprises a protective agent and/or a functional auxiliary agent.
Preferably, the protective agent comprises any one or a combination of at least two of skim milk, gelatin, dextrin, acacia, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.
Preferably, the functional auxiliary agent comprises any one or a combination of at least two of fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, isomalto-oligosaccharide, soy oligosaccharide, inulin, spirulina, arthrospira, coriolus versicolor polysaccharide, stachyose, polydextrose, alpha-lactalbumin or lactoferrin.
In a third aspect, the present invention provides the use of a complex probiotic for ameliorating ulcerative colitis as described in the first aspect or a probiotic as described in the second aspect in the manufacture of a product for preventing, alleviating or treating inflammatory bowel disease.
Preferably, the inflammatory bowel disease comprises ulcerative colitis.
Compared with the prior art, the invention has the following beneficial effects:
the invention creatively develops a strategy for preventing, improving or treating inflammatory bowel diseases, especially ulcerative colitis, and also develops a novel probiotic compound mode, namely, four strains of lactobacillus fermentum (Limosilactobacillus fermentum) TG017 strain, lactobacillus paracasei (Lacticaseibacillus paracasei) TG006 strain, lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) TG021 strain and lactobacillus plantarum (Lactiplantibacillus plantarum) TG008 strain are compounded and combined, and the four strains can be matched and mutually promoted to jointly play a remarkable effect of improving the ulcerative colitis, which is specifically expressed as follows: (1) delay weight loss in colitis mice; (2) decreasing DAI score in colitis mice; (3) improving colon length shortening in colitis mice; (4) Reduces the level of pro-inflammatory cytokines IL-1 beta and TNF-alpha and increases the anti-inflammatory cytokine IL-10.
Drawings
FIG. 1 is a flow chart of an animal experiment design of the present invention;
FIG. 2 is a graph of survival for each group of mice;
FIG. 3 is a graph of body weight statistics for each group of mice;
FIG. 4 is a plot of DAI score for each group of mice;
FIG. 5 is a colon sample anatomy of each group of mice;
FIG. 6 is a graph of colon length statistics for each group of mice;
FIG. 7 is a graph showing statistical results of TNF- α content in colon tissue of each group of mice;
FIG. 8 is a graph showing statistical results of IL-1β content in colon tissues of mice in each group;
FIG. 9 is a graph showing statistics of IL-10 content in colon tissue of each group of mice.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Peptone, beef extract, yeast extract, glucose, sodium acetate, tween 80, dipotassium hydrogen phosphate, diammonium citrate, magnesium sulfate heptahydrate, manganese sulfate monohydrate referred to in the following examples were purchased from national pharmaceutical chemicals company.
The following examples relate to the following media:
MRS Medium (g/L): 10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 5g of sodium acetate, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 2g of diammonium citrate, 0.1g of magnesium sulfate heptahydrate and 0.05g of manganese sulfate monohydrate.
The lactobacillus fermentum TG017 strain according to the following examples was classified and named as lactobacillus fermentum Limosilactobacillus fermentum, the collection unit was China center for type culture Collection, the collection time was 2023, 4 and 10 days, and the collection number was cctccc NO: m2023513, address: chinese, university of martial arts, martial arts.
The strain of Lactobacillus paracasei TG006 according to the following examples is classified and named as Lactobacillus paracasei Lacticaseibacillus paracasei, and has a preservation unit of China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) of 2022, 8 and 1, a preservation number of CGMCC No.25452, and an address of: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
The lactobacillus rhamnosus TG021 strain related to the following examples is classified and named as lactobacillus rhamnosus Lacticaseibacillus rhamnosus, the preservation unit is China center for type culture Collection, the preservation time is 2023, 4 months and 6 days, and the preservation number is cctccc NO: m2023486, address: chinese, university of martial arts, martial arts.
The lactobacillus plantarum TG008 strain related to the following examples is classified and named as lactobacillus plantarum Lactiplantibacillus plantarum, the preservation unit is China center for type culture Collection, the preservation time is 2022, 9 and 20 days, and the preservation number is CCTCC NO: m20221458, address: chinese, university of martial arts, martial arts.
Preparation of bacterial suspensions as referred to in the examples below: inoculating the required strain into an MRS liquid culture medium, culturing for 24 hours at 37 ℃ for activation, picking 3-5 single colonies, respectively inoculating into 10mL of the MRS liquid culture medium, and culturing for 24 hours at 37 ℃ to obtain bacterial liquid; centrifuging the bacterial liquid at 5100rpm at 4deg.C for 10min, collecting the lower bacterial mud, adding physiological saline for resuspension, centrifuging, washing, and repeating twice. Finally, adding proper amount of physiological saline to resuspend the bacterial mud, diluting the bacterial liquid, and measuring the OD by using a micro spectrophotometer 600 Value and according to OD 600 The dilution factor is calculated to adjust the bacterial liquid viable count of each strain to 5 multiplied by 10 9 CFU/mL。
Examples
The present example explores the effect of the composite probiotics of the present invention on improving ulcerative colitis mice models, including the effects on mice survival, body weight, disease activity index score, colon length, colon tissue inflammatory cytokine levels:
(1) Test animals:
SPF grade C57BL/6J mice (23-26 g), male, 8-9 weeks old, purchased from Guangdong Kangdong Biotechnology Co., ltd., license number SCXK (Guangdong) 2020-0054.
(2) Establishment of DSS-induced mouse ulcerative colitis model:
43C 57BL/6J mice were randomly divided into 4 groups after 7 days of adaptive feeding, respectively normal group (CON, n=9), model group (MOD, n=12), positive drug mesalamine group (5-ASA, 150mg/kg, n=11), complex probiotic group (MIX 4, n=11). The mice of each group were free to drink sterilized water containing 2% DSS for 7 consecutive days, except the CON group mice were free to drink sterilized water. After DSS molding, mice of each group were perfused with the corresponding test subjects for 7 days. The animal experiment design flow is shown in figure 1. Wherein the test substances of the compound probiotics group (MIX 4) are TG017 bacterial suspension, TG006 bacterial suspension, TG021 bacterial suspension and TG008 bacterial suspension, the ratio of viable bacteria number is 1:1:1:1, and the total administration dosage is 5×10 9 CFU/mL, each mouse was lavaged with 0.2mL.
(3) Mice survival, body weight, and disease activity index score:
the appearance, behavior, activity, mental state, etc. of the mice were observed daily, and the food intake, water intake, weight change, stool characteristics and hematochezia level of the mice were recorded on time, and Disease Activity Index (DAI) scores were performed according to the following table criteria.
DAI score= (weight loss score + fecal trait score + hematochezia degree score)/3.
Test results: as shown in fig. 2, the survival curves of the mice in each group show that after DSS induction, the survival rate of the mice in the MOD group is 83.3% and the survival rate of the mice in the 5-ASA group is 81.8%, and the survival rate of the mice can be improved to 100% by administration of the compound probiotic (MIX 4), which indicates that the compound probiotic can significantly improve the survival rate of the ulcerative colitis mouse model.
The statistical results of the body weights of the mice in each group are shown in FIG. 3, and it can be seen from the graph that the body weights of the mice in each group are significantly reduced after DSS induction. Compared with MOD group, the mice have increased weight after MIX4 and 5-ASA administration, which shows that the compound probiotics related to the invention can significantly improve the weight of ulcerative colitis mice model.
As shown in fig. 4, the DAI score of each group of mice is obviously increased after DSS induction, and the DAI score of MOD group of mice can be obviously reduced (P < 0.05) after MIX4 treatment, which indicates that the compound probiotics according to the invention can obviously reduce the DAI score of the ulcerative colitis mouse model.
(4) Colon length of mice, colon tissue inflammatory cytokines:
after the test, each group of mice was dissected, a colon sample was taken as shown in fig. 5, and the colon length of each group was counted, and the colon length was reflected in the severity of inflammation of the mice, and as a result, as shown in fig. 6, the colon of the MOD group of mice was significantly shortened as compared with the CON group of mice. The MIX4 group was able to improve colon shortening in the ulcerative colitis mouse model compared to the MOD group. The invention relates to a composite probiotics which can improve colon shortening of ulcerative colitis mouse model.
A suitable amount of colon sample is weighed and crushed by ultrasound according to the physiological saline with the proportion of 1:9 (w/v). The sonicated sample was centrifuged at 10000r/min at 4℃for 10min and the supernatant was collected. The total protein content of the supernatant was measured as background correction of the inflammatory factor content according to the instructions of BCA protein concentration kit. In addition, the amounts of inflammatory cytokines TNF- α, IL-1β and IL-10 in the supernatants were determined according to the corresponding ELISA kit protocol. The results are shown in FIG. 7, FIG. 8 and FIG. 9, respectively, and it is evident from the graph that the levels of TNF-. Alpha.and IL-1β in colon tissue of MOD mice are significantly increased (P < 0.05) and IL-10 levels are decreased compared with CON mice, indicating that the MOD mice develop a significant inflammatory response. Compared with MOD group, the colon tissue TNF-alpha and IL-1β levels were significantly reduced (P < 0.05) and IL-10 levels were elevated in MIX4 group mice. The above shows that the compound probiotics provided by the invention has obvious inhibition effect on mouse inflammatory cytokines, and the effect is better than that of a 5-ASA group.
In conclusion, the compound probiotics related to the invention have excellent effect of improving ulcerative colitis, and the preparation of the compound probiotics into products for preventing, relieving or treating ulcerative colitis has prospect.
The applicant states that the technical solution of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, i.e. it does not mean that the present invention must be implemented by the above embodiments. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims (10)

1. The composite probiotics for improving ulcerative colitis is characterized in that the composite probiotics for improving ulcerative colitis are prepared from the following components with the preservation number of CCTCC NO: lactobacillus mucilaginosus (Limosilactobacillus fermentum) TG017 strain of M2023513, lactobacillus paracasei (Lacticaseibacillus paracasei) TG006 strain with the preservation number of CGMCC No.25452, and CCTCC NO: lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) TG021 strain of M2023486 and a accession number cctccc NO: lactobacillus plantarum (Lactiplantibacillus plantarum) TG008 strain of M20221458.
2. The composite probiotic for improving ulcerative colitis according to claim 1, wherein the ratio of the viable count of TG017 strain, TG006 strain, TG021 strain and TG008 strain is (1-5): 1-5.
3. A probiotic for improving ulcerative colitis, wherein the strain in the probiotic comprises the complex probiotic for improving ulcerative colitis according to claim 1 or 2.
4. The probiotic agent for improving ulcerative colitis according to claim 3, wherein the viable bacteria content of each of the TG017 strain, TG006 strain, TG021 strain and TG008 strain in the probiotic agent is not less than 1 x 10 9 CFU/g or 1X 10 9 CFU/mL。
5. The probiotic for ameliorating ulcerative colitis according to claim 3 or 4, wherein the formulation of the probiotic comprises a lyophilized powder, a capsule, a tablet or a granule.
6. The probiotic agent for improving ulcerative colitis according to claim 3 or 4, characterized in that it further comprises a protective agent and/or a functional auxiliary agent.
7. The probiotic for ameliorating ulcerative colitis according to claim 6, wherein the protective agent comprises any one or a combination of at least two of skim milk, gelatin, dextrin, acacia, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.
8. The probiotic for ameliorating ulcerative colitis according to claim 6, wherein the functional aid comprises any one or a combination of at least two of fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, isomalto-oligosaccharides, soy oligosaccharides, inulin, spirulina, arthrospira, coriolus versicolor polysaccharides, stachyose, polydextrose, alpha-lactalbumin or lactoferrin.
9. Use of a complex probiotic for ameliorating ulcerative colitis according to claim 1 or 2 or of a probiotic according to any of claims 3-8 for the preparation of a product for preventing, alleviating or treating inflammatory bowel disease.
10. The use of claim 9, wherein the inflammatory bowel disease comprises ulcerative colitis.
CN202311656425.3A 2023-12-05 2023-12-05 Composite probiotics for improving ulcerative colitis and application thereof Pending CN117660244A (en)

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Application Number Priority Date Filing Date Title
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