CN114231443A - Lactobacillus plantarum complex bacteria and application thereof in preparation of complex probiotics for relieving intestinal inflammation or treating ulcerative colitis - Google Patents
Lactobacillus plantarum complex bacteria and application thereof in preparation of complex probiotics for relieving intestinal inflammation or treating ulcerative colitis Download PDFInfo
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Abstract
The invention discloses a lactobacillus plantarum complex bacterium and application thereof in preparation of complex probiotics for relieving intestinal inflammation or treating ulcerative colitis, and belongs to the technical field of microorganisms. The lactobacillus plantarum complex bacteria comprise lactobacillus plantarum DPUL-F50 and lactobacillus plantarum Y44, wherein the strain preservation number of the DPUL-F50 is CCTCCNO: m2021838; the strain preservation number of Y44 is CCTCC NO: and M2018558. The lactobacillus compound can obviously inhibit the growth of escherichia coli and staphylococcus aureus; promoting the expression of anti-inflammatory cytokines; can relieve the shortening of colon length of colitis mice, regulate intestinal microbiota of colitis mice, increase microbiota diversity, and reduce relative abundance of harmful flora.
Description
Technical Field
The invention relates to a lactobacillus plantarum complex bacterium and application thereof in preparation of complex probiotics for relieving intestinal inflammation or treating ulcerative colitis, belonging to the technical field of microorganisms.
Background
Inflammatory Bowel Diseases (IBDs) are a chronic inflammatory relapsing disease, and Crohn's Disease (CD) and Ulcerative Colitis (UC) are the major types of inflammatory bowel disease. The clinical symptoms of inflammatory bowel disease are typically: weight loss, abdominal pain, diarrhea, and gastrointestinal bleeding. Inflammatory bowel disease is generally closely related to genetics, epigenetics and the environment. In particular, environmental factors, including dietary consumption of food, lack of exercise, and smoking, play the most important role in the development and progression of colorectal cancer. The incidence of IBD is increasing around the world, and current methods of IBD treatment include the use of anti-inflammatory steroids or immunosuppressive agents to reduce inflammation, and surgical removal of the damaged intestinal tract to control the symptoms. In addition, there may be side effects such as allergic reactions, fever, liver problems, etc. when using anti-inflammatory drugs.
Probiotics are living microorganisms and ingestion of a certain amount will produce beneficial effects on the host. The probiotic bacteria can promote the intestinal tract to produce innate immune substances, such as mucin secreted by goblet cells. In another aspect, probiotics modulate the adaptive immune response of the gut, including inducing the production of cytokines by intestinal epithelial cells and macrophage polarization, playing a key role in inflammatory responses, and being induced to synthesize and secrete inflammatory mediators and proinflammatory cytokines/chemokines. In addition, the probiotics can regulate the intestinal microbial ecological balance, increase the intestinal microbial diversity and enable the intestinal flora to tend to be normalized. The probiotics colonize in intestinal tract to generate various compounds such as organic acid and the like in the metabolic process, thereby improving the ulcerative colitis. Thus, ingestion of certain lactobacillus bacteria may help the host relieve ulcerative colitis.
Compared with single strain probiotics, the compound of different probiotics has synergistic effect on the aspect of probiotic function and is the development trend of probiotic products.
Disclosure of Invention
The invention provides a Lactobacillus plantarum complex bacterium, which comprises Lactobacillus plantarum (Lactobacillus plantarum) DPUL-F50 and Lactobacillus plantarum (Lactobacillus plantarum) Y44, wherein the Lactobacillus plantarum (Lactobacillus plantarum) Y44 is preserved in China center for type culture collection (CCTCC NO): m2018558. Lactobacillus plantarum (Lactobacillus plantarum) DPUL-F50 has been preserved in China Center for Type Culture Collection (CCTCC) at 7/8/2021, and the strain preservation number is CCTCC NO: m2021838.
The invention also aims to provide application of the Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 composite bacteria in preparation of composite probiotics for relieving intestinal inflammation or treating ulcerative colitis.
Further, in the above technical solution, the relieving the intestinal inflammation includes inhibiting the growth of pathogenic microorganisms.
Further, in the above technical solution, the pathogenic microorganisms include escherichia coli and staphylococcus aureus.
Furthermore, in the technical scheme, the relieving the intestinal inflammation comprises inhibiting inflammatory markers such as NO, ROS, TNF-alpha and iNOs generated by macrophages induced by LPS, and promoting the expression of anti-inflammatory cytokines IL-10 and antioxidase SOD, CAT and GPx 1.
Further, in the above technical solution, the treatment of ulcerative colitis includes alleviation of weight loss, colon shortening and colon histological changes.
Further, in the above technical scheme, the treatment of ulcerative colitis includes regulating humoral immunity and cellular immunity, increasing diversity of intestinal flora, and reducing abundance of harmful flora in the intestinal flora.
Drawings
FIG. 1 shows the inhibition effect of Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and their complexes on the growth of Escherichia coli and Staphylococcus aureus.
FIG. 2 shows the effect of Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and their complexes on LPS-induced production of inflammatory factors by RAW264.7 cells.
FIG. 3 shows the effect of Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and their complexes on LPS-induced production of iNOs, SOD, CAT and GPx1 by RAW264.7 cells.
FIG. 4 shows the effect of Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and their complexes on the body weight of colitis mice.
FIG. 5 shows the effect of Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and their complexes on colon length in colitis mice.
FIG. 6 shows the effect of Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and their complexes on colonic histological changes in colitis mice.
FIG. 7 shows the effect of Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and their complexes on the abundance of intestinal microorganisms.
FIG. 8 is the effect of Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and their complexes on the levels of the phylum and family of enteritidis mice, Bar chart is a heat map at the family level; b1 represents the normal group, B2 represents the model group, B3 represents the Y44 group, B4 represents the DPUL-F50 group; b5 represents the lactobacillus mixture group, with different lower case letters between groups representing significant differences (p < 0.05).
Detailed Description
The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.
EXAMPLE 1 Strain culture
The Lactobacillus plantarum Y44 and Lactobacillus plantarum DPUL-F50 are cultured and activated in an MRS culture medium, and the specific steps are as follows:
a. the strain culture method comprises the following steps:
(1) MRS Medium preparation
Weighing the raw materials according to the proportion of 20g/L glucose, 10g/L peptone, 5g/L yeast extract, 10g/L beef extract, 1mL tween-80, 2g/L dipotassium hydrogen phosphate, 2g/L diammonium citrate, 5g/L sodium acetate, 0.58g/L magnesium sulfate heptahydrate and 0.25g/L manganese sulfate tetrahydrate, uniformly mixing, dissolving the obtained mixture in deionized water, and sterilizing the obtained solution at the temperature of 121 ℃ for 20min to obtain the MRS culture medium.
(2) Cultivation of the Strain
Lactobacillus plantarum Y44 and Lactobacillus plantarum DPUL-F50 are streaked and inoculated in the culture medium containing 2% agar in the step (1), anaerobic culture is carried out for 48h to 72h at the temperature of 37 ℃, single colonies are picked up and inoculated in an MRS liquid culture medium, and culture is carried out for 18h at the temperature of 37 ℃.
b. Screening and purifying lactobacillus plantarum DPUL-F50
Adding 0.5mL of Xinjiang yogurt into 4.5mL of normal saline, mixing to obtain original bacterial liquid, diluting the original bacterial liquid to 10% with normal saline-4，10-5And 10-6And (3) coating, after culturing for 48 hours on an MRS plate, selecting colonies with different forms, carrying out secondary streaking until microscopic examination is carried out after single colony is detected, finally inoculating the single colony into a liquid culture medium for liquid culture, and carrying out strain preservation on the cultured strains.
c. Strain preservation
The strain is preserved in China center for type culture Collection (CCTCC for short, address: Wuhan city Wuchang Lojia mountain, China center for type culture Collection, post code 430072). The strain preservation number of the Lactobacillus plantarum (Lactobacillus plantarum) Y44 is CCTCC NO: m2018558, with a preservation date of 2018, 8, 21, and a classified name of Lactobacillus plantarum (Lactobacillus plantarum), and the strain name is Y44. The strain preservation number of the Lactobacillus plantarum (Lactobacillus plantarum) DPUL-F50 is CCTCC NO: m2021838, with a preservation date of 2021, 7/8, was classified and named Lactobacillus plantarum (Lactobacillus plantarum), and the strain name was DPUL-F50.
EXAMPLE 2 preparation of a Lactobacillus mixture
Lactobacillus plantarum Y44 and Lactobacillus plantarum DPUL-F50 (hereinafter, Y44 and DPUL-F50) were inoculated into MRS medium and activated 3 times, respectively. The activated strain is inoculated in MRS culture solution in an inoculum size of 2% (v/v) and cultured for 18h at 37 ℃. Mixing Lactobacillus plantarum (Lactobacillus plantarum) Y44 and Lactobacillus plantarum (Lactobacillus plantarum) DPUL-F50 according to the weight ratio of 1:1 in an inoculum size of 2% (v/v) was inoculated into MRS culture medium and cultured at 37 ℃ for 18 h. Centrifuging (5000rmp, 5min) to obtain supernatant for subsequent bacteriostatic test, collecting activated strain, washing with 0.85% physiological saline for 3 times, resuspending in 0.85% physiological saline, and adjusting the concentration of Y44, DPUL-F50, and complex of Y44 and DPUL-F50 (hereinafter referred to as Mix) (1:1) to 3 × 108CFU/mL。
Example 3 Effect of Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and their complexes on Escherichia coli and Staphylococcus aureus
An oxford cup double-layer plate diffusion method is adopted for carrying out an antibacterial experiment, 15mL of 2% (m/v) agar is poured into a culture dish to form an agar culture medium, and two oxford cups (the diameter is 8.00mm) are uniformly placed after the agar is solidified. Inoculating the coliform or staphylococcus aureus suspension into 20mL LB solid culture medium cooled to 45-55 ℃ according to the inoculation amount of 2% (v/v), fully and uniformly mixing, and pouring the mixture above the agar layer. After the mixture is naturally solidified, taking out the Oxford cup along the vertical direction by using sterile tweezers, and respectively adding 100 mu L of culture supernatant of single lactobacillus or mixed lactobacillus into the hole. After the plate was incubated in a constant temperature incubator at 37 ℃ for 24 hours, the inhibition zone diameter measured with a vernier caliper was expressed as an average value. As can be seen from FIG. 1, the culture supernatant of the Lactobacillus mixture had a more significant inhibitory effect on the growth of Escherichia coli and Staphylococcus aureus than that of the individual strains of Lactobacillus.
Example 4 modulation of macrophage immune Activity by Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and complexes thereof
1. Cell culture
Murine macrophage (RAW264.7) cell line was purchased from the cell bank of the chinese academy of sciences (shanghai, china). RAW264.7 cell lines were cultured in Dulbecco's modified Eagle's Medium (DMEM, Hyclone, Logan, UT) supplemented with 10% (v/v) inactivated (56 ℃, 30min) fetal bovine serum (Bioindestries, Beit Haemek, Israel), 100U/mL penicillin and 100mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO) containing 5% CO at 37 ℃2Cultured in an incubator.
2. Effect of Lactobacillus plantarum (Lactobacillus plantarum) DPUL-F50 and Lactobacillus plantarum (Lactobacillus plantarum) Y44 and complexes thereof on LPS-induced production of inflammatory factors by RAW264.7 cells
Adjusting RAW264.7 macrophage concentration to 2 × 105cell/mL, inoculating in 24-well cell culture plate, culturing for 24 hr, discarding supernatant, inoculating 1mL 3 × 10 per well8Cells were treated with CFU/mL of Lactobacillus plantarum or Lactobacillus mixture for 2h, the supernatant was discarded after the end of the incubation to wash the cells with PBS 3 times, followed by addition of 1mL of LPS (1. mu.g/mL) to induce the cells for 24h, and the amounts of NO, IL-10 and TNF-. alpha.in the supernatant were determined and the intracellular Reactive Oxygen Species (ROS) was determined. The results show that LPS stimulation of RAW264.7 cells resulted in upregulation of NO, ROS and TNF- α expression compared to the blank, and that pretreatment of RAW264.7 cells with a mixture of lactobacilli reversed this change and upregulated IL-10 alleviated inflammation, as shown in figure 2.
3. Lactobacillus plantarum (Lactobacillus plantarum) DPUL-F50 and Lactobacillus plantarum (Lactobacillus plantarum) Y44 and their complex influence on the production of iNOs, CAT, SOD and GPx1 by inhibiting LPS-induced RAW264.7 cells
Cells were seeded in 6-well cell culture plates at a density of 3X 10 per well5And (4) cells. After overnight culture. Then theTreating with single strain lactobacillus or lactobacillus mixture for 2 h. Exposure to LPS for 30 min. Cells were collected and lysed in lysis buffer, and the protein content of the cell lysate was then determined and the expression level of the relevant protein was detected by immunoblot analysis.
The results are shown in figure 3, LPS-induced macrophages resulted in upregulation of iNOs expression compared to the blank. The mixed lactobacillus pretreatment of RAW264.7 clearly down-regulated the expression of iNOs, and up-regulated the expression of CAT, SOD and GPx 1. This indicates that the lactobacillus mixture can promote the expression of antioxidant enzymes CAT, SOD and GPx1 to reduce inflammation by inhibiting LPS induced RAW264.7 to produce iNOs.
Example 5 alleviation of inflammation in colitis mice by Lactobacillus plantarum (Lactobacillus plantarum) DPUL-F50 and Lactobacillus plantarum (Lactobacillus plantarum) Y44 and complexes thereof
1. Grouping animals
Male C57BL/6 mice, six weeks old, were acclimatized for one week and randomized into groups of 8 mice each. The normal group (Control) mice were gavaged daily with normal saline and allowed to drink normal drinking water after 7 days, the Model group (Model) mice were gavaged daily with normal saline and allowed to drink 3% dextran sodium sulfate aqueous solution (DSS) after 7 days, and the Y44 group (Y44) was gavaged daily at 0.75X 108CFU/mL Lactobacillus plantarum Y44, free 3% dextran sulfate aqueous solution (DSS) after 7 days, and DPUL-F50 group (DPUL-F50) gavage 0.75X 10 daily8CFU/mL Lactobacillus plantarum DPUL-F50, free 3% dextran sulfate aqueous solution (DSS) after 7 days, Lactobacillus mixture group (Mix) gavage 0.75X 10 daily8A mixture (1:1) of CFU/mL Lactobacillus plantarum Y44 and Lactobacillus plantarum DPUL-F50 was consumed ad libitum after 7 days in a 3% aqueous solution of Dextran Sulfate Sodium (DSS) for a total of 3 weeks.
2. Effect of Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and complexes thereof on mouse body weight and colon length
Mice were sacrificed 21 days later and the colon of the mice was removed for length measurement by recording the weight change of the mice each day from the start of the experiment. The results are shown in fig. 4 and 5, and show that the lactobacillus mixture can reduce the weight loss and the colon length shortening of the colitis mice.
3. Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and compound thereof for improving colon histological lesion of DSS (DSS colitis) mice
After sacrifice, the colons of mice were removed, fixed with 4% paraformaldehyde, embedded, sectioned and H & E stained. As shown in FIG. 6, compared with the model group, the large-area edema in the colon of the mice perfused with the lactobacillus mixture was remarkably disappeared, and the degree of the crypt structure damage was remarkably less. This indicates that the lactobacillus plantarum complex gastric lavage mice have a remission effect on colitis.
4. Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and compound thereof for regulating cellular immunity and relieving DSS colitis
Eyeball blood sampling is carried out before the mice are killed, and the expression levels of IL-8, IL-10, IL-4 and TNF-alpha in serum are detected by an ELISA kit method. Experimental results Table 1 shows that compared with model mice, the Lactobacillus complex obviously up-regulates the expression levels of anti-inflammatory cytokines IL-10 and IL-4 and down-regulates the expression levels of proinflammatory cytokines IL-8 and IL-1 beta. This indicates that lactobacillus complex intervention in mice can cause colitis in mice by cellular immune-moderation of DSS.
TABLE 1
5. Lactobacillus plantarum DPUL-F50 and Lactobacillus plantarum Y44 and compound thereof for regulating humoral immunity and relieving DSS colitis
Eyeball blood sampling is carried out before the mice are killed, and expression levels of LgM and LgG in serum are detected by an ELISA kit method. Experimental results table 1 shows that LgM and LgG expression levels were significantly up-regulated after lactobacillus complex intervention in mice compared to the model group. These results indicate that lactobacillus complex intervention in mice can induce colitis in mice by humoral immune relief DSS.
6. Functional analysis for regulating intestinal flora of DSS (DSS colitis) mice by Lactobacillus plantarum (Lactobacillus plantarum) DPUL-F50 and Lactobacillus plantarum (Lactobacillus plantarum) Y44 and compound thereof
According to
The soil DNA kit (Omega Bio-tek, Norcross, GA, U.S.) instructions for total DNA extraction of microbial communities from mouse feces and sequencing using the Miseq PE300 platform from Illumina (Megaji Biopharmaceutical technology, Inc., Shanghai). The raw data is uploaded to the NCBI SRA database.
The results show that the lactobacillus complex increases intestinal microbial diversity in DSS-induced colitis mice (fig. 7) and reduces the content of harmful bacteria (fig. 8) compared to the colitis mouse group (Model). The lactobacillus mixture is proved to be capable of reducing the relative abundance of harmful flora in intestinal tracts of enteritis mice.
SEQUENCE LISTING
<110> university of Dalian Industrial university
<120> lactobacillus plantarum complex bacterium and compound benefit thereof in preparation of medicines for relieving intestinal inflammation or treating ulcerative colitis
Application in bacteria
<130> 2021
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1546
<212> DNA
<213> Lactobacillus plantarum DPUL-F50 (Lactobacillus plantarum DPUL-F50)
<400> 1
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Claims (7)
1. The Lactobacillus plantarum compound bacteria are characterized by comprising Lactobacillus plantarum (Lactobacillus plantarum) DPUL-F50 and Lactobacillus plantarum (Lactobacillus plantarum) Y44, wherein the Lactobacillus plantarum DPUL-F50 is preserved in China center for type culture collection (CCTCC NO): m2021838; the Lactobacillus plantarum Y44 is preserved in the China center for type culture collection in 2018, 8, 21 and with the strain preservation number of CCTCC NO: m2018558.
2. Use of a lactobacillus plantarum complex bacterium according to claim 1 for the preparation of a complex probiotic for alleviating symptoms of intestinal inflammation or for the treatment of ulcerative colitis.
3. The use of claim 2, wherein the relieving intestinal inflammation comprises inhibiting the growth of pathogenic microorganisms.
4. Use according to claim 2, wherein said pathogenic microorganisms comprise escherichia coli and staphylococcus aureus.
5. The use according to claim 2, wherein said alleviating intestinal inflammation comprises inhibiting LPS-induced macrophage production of NO, ROS, TNF-a and iNOs inflammatory markers, promoting expression of the anti-inflammatory cytokines IL-10 and the antioxidant enzymes SOD, CAT and GPx 1.
6. The use of claim 2, wherein the treatment of ulcerative colitis comprises slowing weight loss, colon shortening, and colon tissue lesions.
7. The use of claim 2, wherein the treatment of ulcerative colitis comprises modulation of humoral and cellular immunity, increased diversity of the gut flora, and down-regulation of the abundance of harmful flora in the gut flora.
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| CN115612637A (en) * | 2022-07-26 | 2023-01-17 | 山西省生物研究院有限公司 | Lactobacillus plantarum and application thereof |
| CN116024131A (en) * | 2022-12-23 | 2023-04-28 | 深圳保时健生物工程有限公司 | Lactobacillus plantarum strain GOLDGUT-LP101 and application thereof |
| CN116103193A (en) * | 2022-11-15 | 2023-05-12 | 沈阳农业大学 | Probiotic powder and application thereof in relieving colonitis |
| CN116694503A (en) * | 2023-03-27 | 2023-09-05 | 上海华聿康生物科技有限公司 | Lactobacillus plantarum Lp-HZ55 with bowel relaxing and immunity improving functions |
| WO2024056027A1 (en) * | 2022-09-15 | 2024-03-21 | 上海理工大学 | Use of ldh gene-containing lactobacillus plantarum in preparing product for relieving enteritis |
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