CN111088181A - Bifidobacterium breve strain BK55 and application thereof in inhibiting clostridium difficile - Google Patents

Bifidobacterium breve strain BK55 and application thereof in inhibiting clostridium difficile Download PDF

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CN111088181A
CN111088181A CN201911317410.8A CN201911317410A CN111088181A CN 111088181 A CN111088181 A CN 111088181A CN 201911317410 A CN201911317410 A CN 201911317410A CN 111088181 A CN111088181 A CN 111088181A
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clostridium difficile
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/519—Breve
    • C—CHEMISTRY; METALLURGY
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Abstract

The invention provides a Bifidobacterium breve strain BK55 and application thereof in inhibiting Clostridium difficile, and relates to the technical field of probiotic application, wherein the preservation number of the strain BK55 is CGMCC No. 17366. The strain BK55 provided by the invention can tolerate low pH and high bile salt concentration of gastrointestinal tract. The strain Bifidobacterium breve BK55 is co-cultured with Clostridium difficile in a non-contact manner, so that the growth of the Clostridium difficile and the generation of toxins or the death of thalli caused by the leakage of intracellular substances of the Clostridium difficile can be specifically inhibited by inhibiting the growth, the spore generation, the virulence gene expression and the toxin yield of the Clostridium difficile and destroying the cell membrane permeability and the integrity of the Clostridium difficile, and the purpose of preventing, controlling or treating CDI is achieved.

Description

Bifidobacterium breve strain BK55 and application thereof in inhibiting clostridium difficile
Technical Field
The invention belongs to the technical field of probiotic application, and particularly relates to a bifidobacterium breve strain BK55 and application thereof in inhibition of clostridium difficile.
Background
Diarrhea (CDI) caused by Clostridium difficile (Clostridium difficile) accounts for nearly 30% of antibiotic diarrhea, and antibiotic treatment of CDI is the current mainstream therapy, but the problems of damage to intestinal flora and generation of drug-resistant strains are difficult to control. Specific probiotics have certain clinical effects on treating CDI, but the unclear mechanism of action brings difficulties to accurate medication and comprehensive evaluation of the efficacy of the probiotics.
Disclosure of Invention
In view of the above, the present invention aims to provide bifidobacterium breve strain BK55 and its application in inhibiting clostridium difficile, wherein strain BK55 can significantly inhibit growth, spore production, virulence gene expression and toxin production of clostridium difficile, and can also destroy cell membrane permeability and integrity of clostridium difficile, thereby causing cell leakage of intracellular substances of clostridium difficile to cause cell death.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a Bifidobacterium breve (Bifidobacterium breve) strain BK55, wherein the preservation number of the strain BK55 is CGMCC No. 17366.
Preferably, the nucleotide sequence of 16S rRNA of the strain BK55 is shown as SEQ ID No. 1.
The invention also provides application of the strain BK55 in inhibition of Clostridium difficile (Clostridium difficile).
Preferably, the inhibiting comprises: inhibiting growth, spore production, virulence gene expression, toxin production, and disrupting the permeability and integrity of the cell membrane of Clostridium difficile.
Preferably, the virulence genes comprise tcdA and tcdB; the toxin comprises a Clostridium difficile A/B toxin.
The invention also provides application of the strain BK55 in preparation of food, health-care food or medicines for preventing and/or treating diarrhea caused by clostridium difficile.
Preferably, the viable bacteria concentration of the strain BK55 in the food, the health food or the medicine is more than 106CFU/mL or 106CFU/g。
The strain BK55 provided by the invention can tolerate low pH and high bile salt concentration of gastrointestinal tracts, is suitable for gastrointestinal tract environments, inhibits growth, spore production, virulence gene expression and toxin yield of clostridium difficile, and simultaneously destroys cell membrane permeability and integrity of clostridium difficile, so that intracellular substances of clostridium difficile cells leak out to cause cell death, and the risk of occurrence and development of diarrhea caused by clostridium difficile is reduced.
Biological preservation information
Bifidobacterium breve (Bifidobacterium breve) BK55 with the preservation number of CGMCC No.17366, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the concrete address is the institute of microbiology, China academy of sciences, No. 3, West Lu No.1 Homeh, North Jing, the area of rising sun, and the preservation time is 2019, 03 and 20 days.
Drawings
FIG. 1 is a schematic diagram of non-contact co-culture of the strain BK55 and Clostridium difficile.
Detailed Description
The invention provides a Bifidobacterium breve (Bifidobacterium breve) strain BK55, wherein the preservation number of the strain BK55 is CGMCC No. 17366.
The strain BK55 is preferably separated and screened from healthy infant intestinal tracts, is gram-staining positive bacilli, does not generate spores, can grow in an anaerobic environment, has the length of 16S rRNA of the strain BK55 of 849bp, and has a specific nucleotide sequence shown in SEQ ID No. 1. the strain BK55 has stronger activity of tryptophan transpeptidase, β -galactosidase and α -glucosidase, stronger acid resistance and bile salt resistance, and can better survive in a gastrointestinal tract environment.
The invention also provides application of the strain BK55 in inhibition of Clostridium difficile.
The inhibition according to the present invention preferably comprises: inhibiting growth, spore production, virulence gene expression, toxin production, and disrupting the permeability and integrity of the cell membrane of Clostridium difficile. In the present invention, the virulence genes preferably comprise tcdA and tcdB; the toxin preferably comprises a Clostridium difficile A/B toxin. The method can destroy the cell membrane permeability and integrity of clostridium difficile, can cause potassium ions and phosphate to permeate out of cell membranes in large quantity, and causes ATP, nucleic acid and protein substances in cells to leak obviously, thereby causing the death of bacteria.
The invention also provides application of the strain BK55 in preparation of food, health-care food or medicines for preventing and/or treating diarrhea caused by clostridium difficile. The food, health food or medicine takes the strain BK55 as an active ingredient, and the viable bacteria concentration of the strain BK55 is more than 106CFU/mL or 106CFU/g. The invention does not specially limit the dosage form and the preparation method of the product, and the conventional dosage form and the preparation method in the field can be utilized.
The bifidobacterium breve strain BK55 and its use according to the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
Example 1
Bifidobacterium breve BK55 is obtained by directly separating from intestinal tract of healthy infant, and the specific separation and screening process is as follows: healthy infants without diarrhea and other intestinal diseases, breast-fed, and without any antibiotic are selected. 3-5 g of fresh excrement is scraped by a small sterilized rod from a diaper which is just changed by a baby and is put into an aseptic collecting tube, and the aseptic collecting tube is put into an anaerobic box for storage and immediately sent back to a laboratory for bifidobacterium isolation. 50mL of fecal diluent was added to the collection tube, homogenized by shaking, and diluted in 10-fold gradients. Culturing the gradient dilution solution on a bifidobacterium selective medium TPY, selecting a single colony, and performing gram staining, diasterococcus spectroscopy and 16S rRNA gene sequencing identification to obtain the bifidobacterium breve BK 55.
The Bifidobacterium breve BK55 has rod or rod shape, positive gram stain, no spore formation, and growth in strict anaerobic environment. Collecting thallus containing cysteine in MRS culture logarithmic phase, extracting DNA with DNA extraction kit, performing 16S rRNA gene sequencing by Shanghai Senno gene science and technology, and comparing the sequence with GenBank database to identify Bifidobacterium breve (Bifidobacterium breve).
Example 2
After culturing the strain BK55 on a L-MRS solid medium at 37 ℃ for 24h, scraping a single colony on a plate, and performing biochemical reaction detection by using an API 50CH biochemical identification kit, wherein the results are shown in Table 1:
TABLE 1 results of the carbohydrate metabolism profile test of Bifidobacterium breve BK55
Figure BDA0002326217170000031
Figure BDA0002326217170000041
Figure BDA0002326217170000051
Note: in Table 1, "+" indicates positive and "-" indicates negative.
The results showed that it belongs to the species Bifidobacterium, Bifidobacterium breve.
Example 3
The enzymatic activity of the strain BK55 was analyzed 5-fold quantitatively using an API ZYM kit (Merriella, France). According to the kit operation instruction, thallus cells growing in the L-MRS culture solution are collected, and sterile water is used for preparing bacterial suspension for the next enzyme activity analysis. After the thalli and the reagent are added, the color depth of the reagent strip is graded from light to deep from "-", "+ + + +" 4, which shows that the enzyme activity is from weak to strong, and the experimental results are shown in table 2:
TABLE 2 Bifidobacterium breve BK55 API ZYM zymogram test results
Figure BDA0002326217170000052
Figure BDA0002326217170000061
The results in Table 2 show that the strain BK55 has stronger tryptophan transpeptidase, β -galactanase and α -glucosidase activities.
Example 4
Carrying out anaerobic culture on the strain BK55 in an L-MRS liquid culture medium at 37 ℃ for 16 hours, centrifuging at 4 ℃ and 2500rpm for 10min, collecting thalli, washing with Phosphate Buffer Solution (PBS), then suspending in the PBS, taking 1.0ml of cell suspension to 9.0ml of sterile PBS with the pH of 3.0, processing at 37 ℃ for 0 and 3 hours, then carrying out viable count, and calculating the survival rate:
the survival rate is 3h viable count/0 h viable count x 100%.
As can be seen, the survival rate of the strain BK55 after 3h of treatment at pH3.0 is still 92%, which indicates that the strain BK55 has stronger acid resistance.
Example 5
Carrying out anaerobic culture on the strain BK55 in an L-MRS liquid culture medium at 37 ℃ for 16 hours, centrifuging at 4 ℃ and 2500rpm for 10min, collecting thalli, washing with Phosphate Buffer Solution (PBS), then suspending in the PBS, taking 1.0ml of cell suspension to 9.0ml of PBS buffer solution with the concentration of 0.3% (w/v) Bile salt (Bile salts, Merk), carrying out viable count before and after treatment for 3 hours at 37 ℃, and calculating the survival rate:
the survival rate is 3h viable count/0 h viable count x 100%.
As can be seen, the survival rate of the strain BK55 after being treated for 3 hours by 0.3% bile salt is still 95%, which shows that the strain BK55 has stronger bile salt resistance.
Example 6
A24-well cell culture plate with a tranwell chamber (as shown in FIG. 1) was used for non-contact co-culture of a strain BK55 and Clostridium difficile (ATCC 9689), wherein the upper layer of BK55 in FIG. 1 is a Bifidobacterium breve layer, and the lower layer of CD is a Clostridium difficile layer. The effect of the co-culture of strain BK55 for 24h and 48h on the growth and sporulation of clostridium difficile without direct contact with clostridium difficile was examined:
the overnight cultured strain BK55 and Clostridium difficile were adjusted to OD with fresh L-MRS medium and BHI medium, respectively600＝0.5±0.05(5×108CFU/mL). To the bottom layer of a 24-well cell culture plate with transwell chambers was added 1000. mu.L LBHI medium and 50. mu.L C.difficile. Subsequently, the reaction mixture was placed in a transwell chamber, and 50 was added to the chamberMu L of Bifidobacterium breve liquid. Control group 50. mu. L L-MRS liquid medium was added to the transwell chamber. Anaerobic culture at 37 deg.C for 24h or 48 h. The transwell cells were removed and clostridium difficile was counted: respectively sucking 1 mL of clostridium difficile bacterial liquid from the treatment group and the control group, respectively taking 0.5mL of bacterial liquid from the clostridium difficile bacterial liquid, performing gradient dilution, and then coating a flat plate to count bacterial colonies; mixing the remaining 0.5mL of bacterial liquid with 0.5mL of 100% ethanol, shaking for 1h at room temperature to kill vegetative cells, centrifuging the sample for 5min at 12000r/min, collecting thalli, washing twice with PBS (pH 7.4) buffer solution, suspending the thalli in 0.5mL of PBS, mixing the thalli with BHI agar by a gradient dilution method, inverting the mixture, and performing anaerobic culture at 37 ℃ for 72 h; respectively counting to obtain the viable count of the thallus and the spore. Independent experiments were performed in total three times, and the results are shown in table 3:
TABLE 3 Effect of Bifidobacterium breve BK55 on growth and sporulation of Clostridium difficile
Figure BDA0002326217170000071
Figure BDA0002326217170000081
Compared with a control group, the strain BK55 can remarkably inhibit the growth and reduce the spore production of clostridium difficile when being co-cultured for 24 hours under the condition of not directly contacting with the clostridium difficile; the growth and spore production of clostridium difficile were still continuously reduced when the cells were co-cultured for 48 h. It can be seen that the strain BK55 can be co-cultured with Clostridium difficile, and can generate obvious inhibition effect on the growth and sporulation amount of Clostridium difficile without direct contact.
Example 7
The strain BK55 was co-cultured with Clostridium difficile using the method described in example 6. Observing the expression difference of virulence genes tcdA and tcdB in Clostridium difficile under the condition of existence of Bifidobacterium breve BK55 by adopting a fluorescent quantitative PCR method;
the method comprises the following steps: c, extracting Clostridium difficile RNA, performing reverse transcription to obtain cDNA, and storing the synthesized cDNA template at-80 ℃ for later use. The primers designed in Table 4 were used for fluorescent quantitative PCR, and the 16S rRNA gene was used as an internal reference gene.
TABLE 4 fluorescent quantitative PCR primer information
Figure BDA0002326217170000082
The reaction conditions were as follows: 30s at 94 ℃ and 40 cycles later (94 ℃ for 5s, 55 ℃ for 15 s and 72 ℃ for 10 s). According to 2－ΔΔCtThe method relatively quantifies the target gene, and the Ct average value of 16S rRNA (internal reference) is subtracted from the sample average Ct of the tcdA and tcdB gene (delta Ct) to obtain the differential expression multiple of the target gene.
The expression of c difficile virulence genes tcdA and tcdB is affected by strain BK55 as shown in table 5:
TABLE 5 Effect of Strain BK55 on expression of virulence genes of Clostridium difficile tcdA and tcdB
Figure BDA0002326217170000091
It can be seen that when the control group is normalized to 100, the expression levels of clostridium difficile virulence genes tcdA and tcdB are significantly reduced in the experimental group, i.e. after the strain BK55 is co-cultured with clostridium difficile in a non-contact way for 24 hours. Further downregulation after 48 h. The strain BK55 can obviously inhibit the expression of virulence genes of Clostridium difficile.
The strain BK55 was co-cultured with Clostridium difficile using the method described in example 6. The content of toxin A/B in the cell-free supernatant is determined by using a Clostridium difficile A/B toxin kit by adopting an Aldape method. The effect of clostridium difficile toxin production by bifidobacterium breve is shown in table 6:
TABLE 6 Clostridium difficile toxin production results affected by the strain BK55
Figure BDA0002326217170000092
From the viewpoint of toxin production, the toxin production at 24h in the treatment group was significantly lower than that in the control group; the toxin production in the treated group was further reduced at 48h, while the control group was further increased. The strain BK55 was shown to significantly reduce toxin production by Clostridium difficile.
Example 8
By measuring the contents of potassium ions and inorganic phosphate in cells of clostridium difficile co-cultured with the strain BK55, whether the cell membrane permeability of clostridium difficile is remarkably changed and the structure of the membrane is damaged or not is seen.
The method comprises the following steps: the strain BK55 was co-cultured with Clostridium difficile using the method described in example 6. Taking cell suspensions of a control group and a treatment group at 24h and 48h respectively, rapidly performing ice-bath, centrifuging, collecting supernate and determining extracellular K+And inorganic phosphate content. Resuspending the collected precipitated cells in 5% (W/V) trichloroacetic acid, freezing at-20 deg.C overnight, thawing, incubating at 95 deg.C for 10min, centrifuging to remove the precipitated cells, and measuring intracellular K in the supernatant+And inorganic phosphate content. K+The content was measured by inductively coupled plasma emission spectrometer (ICP, ICAP7600, USA) at 767 nm. The inorganic phosphate was measured by Mo-Sb colorimetry, ultraviolet-visible Spectrophotometer (UV Spectrophotometer, Lamda 950, China).
The results of the measurement are shown in Table 7, and the intracellular K of Clostridium difficile co-cultured with the strain BK55+And inorganic phosphate largely leaks out, which indicates that the permeability of the cell membrane has changed remarkably and the cell membrane structure is damaged.
TABLE 7 Effect of the Strain BK55 on the cell Membrane permeability of Clostridium difficile
Figure BDA0002326217170000101
Example 9
Whether the integrity of the cell membrane of clostridium difficile was damaged or not was determined by measuring the ATP content and the ultraviolet absorbing substances inside and outside the cell of clostridium difficile co-cultured with strain BK 55.
The strain BK55 was co-cultured with Clostridium difficile using the method described in example 6. The clostridium difficile thalli of the control group and the treatment group are respectively taken at 24h and 48h for biological fermentationMeasuring ATP content inside and outside cells by light method, using full-wavelength enzyme-labeling instrument and luciferase kit (
Figure BDA0002326217170000102
2.0Kit, Promega, USA). The difficile cell suspensions of the control and treatment groups were taken at 24h and 48h, respectively, the centrifuged supernatants were filtered through a 0.22 μm filter, and the absorbance of the cell-free supernatants, i.e., the Ultraviolet (UV) absorbing material, was measured at 260nm and 280nm using an ultraviolet-visible spectrophotometer, respectively.
The results of the measurement are shown in Table 8, and the ultraviolet absorbing substance (nucleic acid substance (OD) of Clostridium difficile co-cultured with the strain BK55260) And proteinaceous substances (OD)280) Significant leakage indicating that the integrity of the c.difficile cell membrane has been compromised.
TABLE 8 Effect of Bifidobacterium breve BK55 on the integrity of C.difficile cell membranes
Figure BDA0002326217170000111
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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Claims (7)

1. Bifidobacterium breve (Bifidobacterium breve) strain BK55 is characterized in that the preservation number of the strain BK55 is CGMCC No. 17366.
2. The strain BK55 as claimed in claim 1, wherein the nucleotide sequence of 16S rRNA of the strain BK55 is shown as SEQ ID No. 1.
3. Use of the strain BK55 according to claim 1 or 2 for inhibiting Clostridium difficile (Clostridium difficile).
4. Use according to claim 3, wherein the inhibition of Clostridium difficile comprises: inhibiting growth, spore production, virulence gene expression, toxin production, and disrupting the permeability and integrity of the cell membrane of Clostridium difficile.
5. The use of claim 4, wherein said virulence genes comprise tcdA and tcdB; the toxin comprises a Clostridium difficile A/B toxin.
6. Use of the strain BK55 according to claim 1 or 2 for the preparation of a food, health food or pharmaceutical product for the prevention and/or treatment of diarrhea caused by Clostridium difficile.
7. The use according to claim 6, wherein the viable bacteria concentration of the strain BK55 in the food, health food or medicine is more than 106CFU/mL or 106CFU/g。
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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN113005049A (en) * 2020-12-30 2021-06-22 江南大学 Bifidobacterium breve capable of relieving diarrhea and application thereof
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