CN114621910A - High-flux bacteria adhesion model based on mouse intestinal tissue and construction method and application thereof - Google Patents
High-flux bacteria adhesion model based on mouse intestinal tissue and construction method and application thereof Download PDFInfo
- Publication number
- CN114621910A CN114621910A CN202210103431.5A CN202210103431A CN114621910A CN 114621910 A CN114621910 A CN 114621910A CN 202210103431 A CN202210103431 A CN 202210103431A CN 114621910 A CN114621910 A CN 114621910A
- Authority
- CN
- China
- Prior art keywords
- intestinal
- intestinal tissue
- adhesion
- mouse
- bacterial
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0679—Cells of the gastro-intestinal tract
-
- 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
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/23—Gastro-intestinal tract cells
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/70—Non-animal cells
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a high-throughput bacterial adhesion model based on mouse intestinal tissues and a construction method and application thereof, and relates to the technical field of bacterial adhesion model construction. The method comprises the following steps: 1, preparing bacterial liquid; 2 dissecting a mouse; 3 taking intestinal tissues of the mice in a sterile environment; 4 removing membranes, blood vessels and fat outside the intestinal tissue; 5 cleaning intestinal tissues; 6, intestinal tissue segmentation; 7 inoculating bacterial liquid; and 8, co-culturing the intestinal tissues and the bacterial liquid in a constant-temperature shaking incubator. The model construction method provided by the invention has low operation difficulty and short required time, can truly simulate the real environment in vivo in a short time by adopting intestinal tissue to culture bacteria, clearly reflects the adhesion condition of the bacteria on the real intestinal tissue, and can be used for evaluating the adhesion capability of the bacteria and evaluating the activity of food, health care products and medicines related to the adhesion of the bacteria.
Description
Technical Field
The invention belongs to the field of bacterial adhesion model construction, and particularly relates to a mouse intestinal tissue-based high-throughput bacterial adhesion model and a construction method and application thereof.
Background
The adhesion of bacteria is helpful for the colonization in the intestinal tract, the adhesion colonization of bacteria in the intestinal tract is the basis for the physiological effect of the bacteria, and the adhesion of beneficial bacteria is helpful for maintaining the normal flora structure of the intestinal tract, maintaining the integrity of the form and the function of the intestinal mucosa, enhancing the signal exchange between the bacteria and the intestinal tract cells, inhibiting the colonization of harmful bacteria in the intestinal tract and improving the immunity of organisms. The adhesion of different species varies greatly, and even if the species are the same, the adhesion varies depending on the level of the strain, and the adhesion of the bacteria is strain-specific. The final adhesion results may also vary for the same strain due to the choice of the intestinal site to which the adhesion is to be applied. The adhesion ability of bacteria is often used as an important criterion for evaluating and screening bacteria.
Currently, models for evaluating the adhesion ability of bacteria include cell adhesion models, in vitro mucin adhesion models, and the like. The cell adhesion model is currently the most widely used adhesion capability assessment model. The intestinal cell adhesion model mainly uses CaCo-2 human colon adenocarcinoma cells, HT-29 human colon cancer cells, IPEC-J2 pig small intestine cells and the like, and human or animal intestinal cells are cultured to form a monolayer of cells, thereby representing the in vivo intestinal environment. However, the study of adhesion by using intestinal epithelial cells or intestinal cancer cells does not involve the intestinal epithelial tissue with complex host, the intestinal epithelial tissue not only includes the intestinal epithelial cells, but also contains mucus layer and mucin, the mucus layer composed of highly glycosylated mucin is the main place for adhesion and colonization of bacteria, and the mucin also has important influence on adhesion of bacteria. Therefore, it is difficult to evaluate the adhesion of bacteria truly and effectively by studying the adhesion of bacteria only with intestinal cells as a representative of the intestinal environment in vivo.
The in vitro mucus adhesion model is mainly composed of mucin, which is a constituent of mucus, which covers the surface of intestinal cells to form a mucus barrier. The mucous barrier separates bacteria from the intestinal mucosa, prevents the bacteria from directly contacting the intestinal epithelial cells and defends the invasion of pathogenic microorganisms. The in vitro mucus adhesion model is established by intestinal mucus proteins extracted from host feces and intestinal tracts, and the model considers the adhesion of bacteria to mucus, but ignores the real environment of the mucus in the intestinal tracts and ignores the adhesion competition effect of different intestinal microorganisms to the mucus in the complex environment in vivo. Therefore, merely representing the adhesion of bacteria in studies of intestinal environment in vivo by mucin extracted from feces or intestines still has certain limitations.
And for marvelous and the like, the adhesiveness of the lactobacillus is evaluated according to the surface hydrophobicity, the self-aggregation capability and the adhesion capability with Caco-2 cells of the lactobacillus, and high-adhesiveness strains are screened out. However, the hydrophobicity and self-aggregation ability only indirectly reflect the adhesion of bacteria, and the cell adhesion model only uses intestinal cells to represent the in vivo intestinal environment to study the adhesion of bacteria, so that the adhesion of bacteria is difficult to be evaluated truly and effectively (study on the adhesion and immunoregulation of Marigy. probiotic lactobacillus, university of Jilin, 2013.)
The adhesion of bacteria on intestinal tracts occurs in a complex environment in vivo, and in order to accurately evaluate the intestinal adhesion capability of bacteria, the intestinal tract environment in vivo needs to be more truly simulated.
Disclosure of Invention
In order to overcome the defects of the existing model, the invention aims to provide a high-flux bacterial adhesion model based on mouse intestinal tissues and a construction method and application thereof. The invention uses the isolated intestinal tissue to simulate the in-vivo intestinal environment, constructs the high-flux bacteria adhesion model based on the intestinal tissue of the mouse, has simple culture condition, lower experimental cost and short modeling time, adopts the intestinal tissue of the mouse, can better simulate the in-vivo real environment, and more truly and effectively evaluates the adhesion of bacteria.
The purpose of the invention is realized by the following technical scheme:
the invention provides a method for constructing a high-flux bacterial adhesion model based on mouse intestinal tissues, which comprises the following steps:
(1) preparing bacterial liquid;
(2) dissecting a mouse;
(3) taking intestinal tissues of the mice in a sterile environment;
(4) removing membranes, blood vessels and fat outside the intestinal tissue;
(5) cleaning intestinal tissue;
(6) intestinal tissue segmentation;
(7) inoculating bacterial liquid;
(8) and co-culturing the intestinal tissues and the bacterial liquid in a constant-temperature shaking table incubator.
Further, the bacterial liquid in the step (1) is fresh seed liquid cultured for 10-15h, and is centrifuged, supernatant is removed, and the bacterial liquid with the concentration of 10 is obtained by using buffer solution to resuspend7-109CFU/mL。
Further, the concentration of the suspension was resuspended to 10 using 0.01M phosphate buffer7-109CFU/mL。
Further, the step (5) of washing the intestinal tissue is to wash the intestinal contents with a buffer.
Further, the intestinal tissue is washed in the step (5) by using a buffer solution at 2-6 ℃.
Further, the washing of the intestinal tissue in the step (5) uses a buffer solution at 4 ℃ to wash the intestinal contents.
Further, the step (5) of washing the intestinal tissue is to wash the intestinal contents with 0.01M phosphate buffer.
Further, the step (6) of segmenting the intestinal tissue comprises segmenting the whole segment of the intestinal tissue of the mouse into duodenum, jejunum, ileum, caecum and colon according to tissue characteristics, wherein the duodenum is 4 +/-0.5 cm behind the pylorus, the jejunum is arranged between the duodenum and the ileum, the ileum is 4 +/-0.5 cm in front of the caecum, and the colon is 6 +/-1 cm behind the caecum. Each section of intestine is divided into pieces of intestinal tissue of equal length using sterile scissors.
Further, the step (7) of inoculating the bacterial liquid comprises the step of flatly paving the intestinal tissue blocks in the pore plate, so that the intestinal mucosa layer faces the upper side.
Further, the bacterial liquid inoculated in the step (7) is inoculated in 10 holes7-109CFU/mL, the amount of the bacterial solution is 1-2 mL.
Further, the co-culture in the step (8) means that the intestinal tissue and the bacterial liquid are cultured in a constant temperature shaking incubator, the culture temperature is 35-38 ℃, and the rotation speed of the shaking incubator is 100-200 rpm.
Further, the co-culture in the step (8) refers to the culture of the intestinal tissue and the bacterial liquid in a constant temperature shaking incubator, and the culture time can be designed to be different culture times in 30min-2 h.
The invention provides a high-flux bacterial adhesion model based on mouse intestinal tissues, which is established by the construction method.
Furthermore, the model is used for successfully adhering bacteria on intestinal tissues and is mainly determined by calculating the total number of bacterial colonies adhered to the intestinal tissues, observing the adhesion condition of the bacteria by a scanning electron microscope, and detecting whether indexes such as the whole polysaccharide content, the protein content, the biofilm and the like are increased after the bacteria are adhered to the intestinal tissues.
Further, the successful adhesion of the bacteria on the intestinal tissue is mainly realized by calculating the total number of bacterial colonies adhered on the intestinal tissue, observing the successful adhesion of the bacteria on the intestinal tissue by a scanning electron microscope, and detecting the increase of the whole polysaccharide content, the protein content and the biofilm biomass after the bacteria are adhered on the intestinal tissue.
The invention also provides a high-flux bacterial adhesion model based on the intestinal tissue of the mouse, which is used for evaluating the bacterial adhesion capability and evaluating the activity of probiotic drinks, probiotic powder, health-care products and medicaments for promoting the bacterial adhesion.
Compared with the prior art, the invention has the following advantages:
(1) the model construction method is low in technical difficulty, and mouse intestinal tissues and probiotic bacteria liquid are taken for co-culture; the model construction material is easy to obtain, the intestinal tissue is taken from the intestinal tract of the mouse, and the bacterial liquid is freshly inoculated probiotic bacterial liquid; the culture conditions are simple, and the culture is carried out in a constant temperature shaking incubator. Intestinal tissues currently taken from the intestinal tract of mice are co-cultured with probiotics, and the adhesion condition of bacteria in real intestinal tissues is evaluated. The isolated intestinal tissue can still survive for 2 hours in vitro, the intestinal tissue has an intestinal epithelial cell nucleus intestinal mucosa layer, the model objectively reflects the adhesion condition of bacteria in a real intestinal environment, the experimental cost is low, the construction technology difficulty is low, and the model can be widely used for the evaluation of the adhesion capability of the bacteria and the activity evaluation of bacteria adhesion related food, health care products and medicines.
Drawings
FIG. 1 is a schematic diagram of a method for constructing a high-throughput bacterial adhesion model based on intestinal tissues of mice;
FIG. 2 is a scanning electron microscope photograph of bacteria adhered to colon after the bacterial adhesion models of examples 1-6 were constructed.
FIG. 3 is a histogram of the number of colonies of Lactobacillus plantarum attached to intestinal tissue after construction of the bacterial adhesion model of example 1.
FIG. 4 is a histogram of the number of colonies of Lactobacillus rhamnosus adhering to intestinal tissue after the bacterial adhesion model of example 4 was constructed.
FIG. 5 is a scanning electron microscope photograph of Lactobacillus plantarum adhered to intestinal tissue after construction of the bacterial adhesion model of example 1.
FIG. 6 is a scanning electron microscope photograph of Lactobacillus rhamnosus adherence to intestinal tissue after construction of the bacterial adherence model of example 4.
FIG. 7 is a histogram showing the measurement of polysaccharide content, protein content and biofilm biomass of Lactobacillus plantarum following construction of the bacterial adhesion model of example 1, to intestinal tissue.
FIG. 8 is a bar graph showing the measurement of polysaccharide content, protein content and biofilm biomass of Lactobacillus rhamnosus adhered to intestinal tissues after the bacterial adhesion model was constructed in example 4.
Detailed Description
The following description of the embodiments of the present invention is provided in connection with the accompanying drawings and examples, but the invention is not limited thereto. It is noted that the processes described below, if not specifically detailed, are all those that can be realized or understood by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
Example 1
Based on a mouse intestinal tissue high-flux bacterial adhesion model and a construction method and application thereof, fig. 1 is a schematic diagram of the construction method based on the mouse intestinal tissue high-flux bacterial adhesion model, and as shown in fig. 1, the construction method comprises the following steps:
(1) the study subjects were: selecting 2-3 month old C57BL/6 mice;
(2) preparing bacterial liquid: preparing a lactobacillus plantarum LP45 colony plate in advance, selecting a single colony to be inoculated in a fresh MRS culture medium with the inoculation amount of 2% (v/v), placing the single colony in a constant-temperature shaking incubator at 37 ℃ for 10h, centrifuging, removing supernatant, and using buffer solution to resuspend to obtain 10-concentration bacterial liquid7CFU/mL, and temporarily storing in a refrigerator at 4 ℃;
(3) dissecting the mice: anesthetizing a mouse by using pentobarbital, transferring the mouse into a beaker after the mouse is deeply anesthetized, soaking the mouse in alcohol for 5min, and dissecting the mouse in a super clean bench;
(4) taking intestinal tissues of the mice in a sterile environment;
(5) membrane, blood vessel, fat outside intestinal tissue: removing membranes, blood vessels and fat from the exterior of the intestinal tissue using sterile instruments;
(6) cleaning intestinal tissues: gently removing intestinal contents, and then slowly washing intestinal tissues by using a buffer solution at 4 ℃;
(7) intestinal tissue segmentation: according to the physiological structure, the colon is divided into 6 groups of 5mm long intestinal tissue blocks by using sterile scissors, wherein the duodenum is 4 +/-0.5 cm behind the pylorus, the jejunum is arranged between the duodenum and the ileum, the ileum is arranged at the front 4 +/-0.5 cm of the cecum, and the colon is arranged at the back 6 +/-1 cm of the cecum.
(8) Inoculating bacterial liquid: spreading each group of intestinal tissues in 24-well plate with intestinal mucosa layer facing upwards, inoculating 10 of intestinal mucosa into each well71mL of CFU/mL lactobacillus plantarum LP45 bacterial liquid; each well of the individual cultured intestine sections was inoculated with 1mL of 4 ℃ buffer.
(9) Placing the pore plate in a constant temperature shaking table incubator for cultivation at 37 deg.C with shaking table rotation speed of 100rpm, and culturing intestinal tissue and Lactobacillus plantarum LP45 bacteria liquid in the constant temperature shaking table incubator for 30min, 60min, and 120 min.
And (3) observing by using an electron microscope:
taking an intestinal segment co-cultured with lactobacillus plantarum LP45, placing the intestinal segment in a sterile 24-well plate, and fixing for 4 hours by using 2mL of 2.5% (v/v) glutaraldehyde; washed 3 times with 0.1M phosphate buffer (pH 7.3); washing the intestinal segment with distilled water at 4 deg.C for three times; after freeze-drying for 18h, observing under a scanning electron microscope.
Index detection:
(1) plate counting: taking an intestinal section co-cultured with lactobacillus plantarum LP45, adding 1mL of 0.1M phosphate buffer solution, shearing intestinal tissues into homogenate by using sterile scissors, and collecting with a sterile EP tube for later use; diluting the collected bacterial liquid to 10-1、10-2、10-3、10-4Coating a plate; count after anaerobic culture for 24 h.
(2) And (3) polysaccharide content determination: taking 200 mu L of intestinal tissue homogenate, and determining the content change of polysaccharide in the intestinal tissue model adhered with the probiotics by adopting a phenol-sulfuric acid method.
(3) Protein content determination: taking 200 mu L of intestinal tissue homogenate, and determining the change of protein content in the intestinal tissue model adhered with the probiotics by adopting a Coomassie brilliant blue G-250 method.
(4) Biofilm measurement: intestinal tissue homogenate 200. mu.L was taken, 200. mu.L of 10mg/L crystal violet was added, and cultured for 15min, and the sample was washed 3 times with 0.01M PBS, 1mL of 95% (v/v) ethanol was added, 200. mu.L was taken and transferred to a 96-well plate, and the absorbance at 570nm was measured.
Example 2
Based on a mouse intestinal tissue high-flux bacterial adhesion model and a construction method and application thereof, fig. 1 is a schematic diagram of the construction method based on the mouse intestinal tissue high-flux bacterial adhesion model, and as shown in fig. 1, the construction method comprises the following steps:
(1) study subjects: selecting 2-3 month old C57BL/6 mice;
(2) preparing bacterial liquid: preparing a lactobacillus plantarum LP45 colony plate in advance, selecting a single colony to be inoculated in a fresh MRS culture medium with the inoculation amount of 2% (v/v), placing the single colony in a constant-temperature shaking incubator at 35 ℃ for 10h, centrifuging, removing supernatant, and resuspending by using buffer solution to obtain 10-concentration bacterial liquid9CFU/mL, and temporarily storing in a refrigerator at 4 ℃;
(3) dissecting the mice: anaesthetizing a mouse by using pentobarbital, transferring the mouse into a beaker after the mouse is deeply anaesthetized, soaking the mouse in alcohol for 5min, and dissecting the mouse in a super clean bench;
(4) taking intestinal tissues of mice in a sterile environment;
(5) membrane, blood vessel, fat outside the intestinal tissue: removing membranes, blood vessels and fat from the exterior of the intestinal tissue using sterile instruments;
(6) cleaning intestinal tissues: gently removing intestinal contents, and then slowly washing intestinal tissues by using a buffer solution at 4 ℃;
(7) intestinal tissue segmentation: according to the physiological structure, the colon is divided into 6 groups of 5mm long intestinal tissue blocks by using sterile scissors, wherein the duodenum is 4 +/-0.5 cm behind the pylorus, the jejunum is arranged between the duodenum and the ileum, the ileum is arranged at the front 4 +/-0.5 cm of the cecum, and the colon is arranged at the back 6 +/-1 cm of the cecum.
(8) Inoculating bacterial liquid: spreading each group of intestinal tissues in 24-well plate with intestinal mucosa layer facing upwards, inoculating 10 of intestinal mucosa into each well91mL of CFU/mL lactobacillus plantarum LP45 bacterial liquid; each well of the individual cultured intestine sections was inoculated with 1mL of 4 ℃ buffer.
(9) Placing the pore plate in a constant temperature shaking table incubator for cultivation, wherein the cultivation temperature is 35 ℃, the shaking table rotation speed is 100rpm, and the cultivation time of the intestinal tissue and lactobacillus plantarum LP45 bacterial liquid in the constant temperature shaking table incubator is designed to be 30min, 60min and 120 min.
And (3) observing by an electron microscope:
taking an intestinal segment co-cultured with lactobacillus plantarum LP45 bacteria, placing the intestinal segment in a sterile 24-well plate, and fixing for 4 hours by using 2mL of 2.5% (v/v) glutaraldehyde; washed 3 times with 0.1M phosphate buffer (pH 7.3); washing the intestinal segment with distilled water at 4 deg.C for three times; after freeze-drying for 18h, observing under a scanning electron microscope.
Example 3
Based on a mouse intestinal tissue high-flux bacterial adhesion model and a construction method and application thereof, fig. 1 is a schematic diagram of the construction method based on the mouse intestinal tissue high-flux bacterial adhesion model, and as shown in fig. 1, the construction method comprises the following steps:
(1) study subjects: selecting 2-3 month old C57BL/6 mice;
(2) preparing bacterial liquid: preparing a lactobacillus plantarum LP45 colony plate in advance, picking a single colony for inoculationInoculating in fresh MRS culture medium with an inoculum size of 2% (v/v), culturing at 38 deg.C for 10 hr in constant temperature shaking incubator, centrifuging, removing supernatant, and resuspending with buffer to obtain 10% strain liquid9CFU/mL, and temporarily storing in a refrigerator at 4 ℃;
(3) dissecting the mice: anesthetizing a mouse by using pentobarbital, transferring the mouse into a beaker after the mouse is deeply anesthetized, soaking the mouse in alcohol for 5min, and dissecting the mouse in a super clean bench;
(4) taking intestinal tissues of the mice in a sterile environment;
(5) membrane, blood vessel, fat outside the intestinal tissue: removing membranes, blood vessels and fat from the exterior of the intestinal tissue using sterile instruments;
(6) cleaning intestinal tissues: gently removing intestinal contents, and then slowly washing intestinal tissues by using a buffer solution at 4 ℃;
(7) intestinal tissue segmentation: according to the physiological structure, the colon is divided into 6 groups of 5mm long intestinal tissue blocks by using sterile scissors, wherein the duodenum is 4 +/-0.5 cm behind the pylorus, the jejunum is arranged between the duodenum and the ileum, the ileum is arranged at the front 4 +/-0.5 cm of the cecum, and the colon is arranged at the back 6 +/-1 cm of the cecum.
(8) Inoculating bacterial liquid: spreading each group of intestinal tissues in 24-well plate with intestinal mucosa layer facing upwards, inoculating 10 of intestinal mucosa into each well92mL of CFU/mL lactobacillus plantarum LP45 bacterial liquid; each well of the individual cultured intestine sections was inoculated with 2mL of a buffer solution at 4 ℃.
(9) Placing the pore plate in a constant temperature shaking table incubator for cultivation at the cultivation temperature of 38 ℃ and the shaking table rotation speed of 100rpm, and culturing the intestinal tissue and the lactobacillus plantarum LP45 bacterial liquid in the constant temperature shaking table incubator for 30min, 60min and 120 min.
And (3) observing by using an electron microscope:
taking an intestinal section co-cultured with lactobacillus plantarum LP45 bacteria, placing the intestinal section in a sterile 24-well plate, and fixing for 4 hours by using 2mL of 2.5% (v/v) glutaraldehyde; washed 3 times with 0.1M phosphate buffer (pH 7.3); washing the intestinal segment with distilled water at 4 deg.C for three times; after freeze-drying for 18h, observing under a scanning electron microscope.
Example 4
Based on a mouse intestinal tissue high-flux bacterial adhesion model and a construction method and application thereof, fig. 1 is a schematic diagram of the construction method based on the mouse intestinal tissue high-flux bacterial adhesion model, and as shown in fig. 1, the construction method comprises the following steps:
(1) study subjects: selecting 2-3 month old C57BL/6 mice;
(2) preparing bacterial liquid: preparing a lactobacillus rhamnosus LRa05 colony plate in advance, selecting a single colony to be inoculated in a fresh MRS culture medium, wherein the inoculation amount is 2% (v/v), placing the single colony in a constant-temperature shaking incubator at 37 ℃ for 15h, centrifuging, removing a supernatant, and using a buffer solution to resuspend to obtain a bacterial liquid with the concentration of 107CFU/mL, and temporarily storing in a refrigerator at 4 ℃;
(3) dissecting the mice: anaesthetizing a mouse by using pentobarbital, transferring the mouse into a beaker after the mouse is deeply anaesthetized, soaking the mouse in alcohol for 5min, and dissecting the mouse in a super clean bench;
(4) taking intestinal tissues of the mice in a sterile environment;
(5) membrane, blood vessel, fat outside the intestinal tissue: removing membranes, blood vessels and fat from the exterior of the intestinal tissue using sterile instruments;
(6) cleaning intestinal tissues: gently removing intestinal contents, and then slowly washing intestinal tissues by using a buffer solution at 4 ℃;
(7) intestinal tissue segmentation: according to the physiological structure, the colon is divided into 6 groups of 5mm long intestinal tissue blocks by using sterile scissors, wherein the duodenum is 4 +/-0.5 cm behind the pylorus, the jejunum is arranged between the duodenum and the ileum, the ileum is arranged at the front 4 +/-0.5 cm of the cecum, and the colon is arranged at the back 6 +/-1 cm of the cecum.
(8) Inoculating bacterial liquid: spreading each group of intestinal tissues in 24-well plate with intestinal mucosa layer facing upwards, inoculating 10 of intestinal mucosa into each well71mL of CFU/mL of Lactobacillus rhamnosus LRa05 bacterial liquid; each well of the individual cultured intestine sections was inoculated with 1mL of 4 ℃ buffer.
(9) Placing the pore plate in a constant temperature shaking table incubator for culturing at 37 ℃ and 200rpm of shaking table, and culturing intestinal tissues and Lactobacillus rhamnosus LRa05 bacterial liquid in the constant temperature shaking table incubator for 30min, 60min and 120 min.
And (3) observing by an electron microscope:
taking an intestinal segment co-cultured with the lactobacillus rhamnosus LRa05, placing the intestinal segment in a sterile 24-well plate, and fixing for 4 hours by using 2mL of 2.5% (v/v) glutaraldehyde; washed 3 times with 0.1M phosphate buffer (pH 7.3); washing the intestinal segment with distilled water at 4 deg.C for three times; after freeze-drying for 18h, observing under a scanning electron microscope.
Index detection:
(1) plate counting: taking an intestinal section co-cultured with lactobacillus rhamnosus LRa05, adding 1mL of 0.1M phosphate buffer solution, shearing intestinal tissues into homogenate by using sterile scissors, and collecting with a sterile EP tube for later use; diluting the collected bacterial liquid to 10-1、10-2、10-3、10-4Coating a plate; count after anaerobic culture for 24 h.
(2) And (3) polysaccharide content determination: taking 200 mu L of intestinal tissue homogenate, and determining the content change of polysaccharide in the intestinal tissue model adhered with the probiotics by adopting a phenol-sulfuric acid method.
(3) Protein content determination: taking 200 mu L of intestinal tissue homogenate, and determining the change of protein content in the intestinal tissue model adhered with the probiotics by adopting a Coomassie brilliant blue G-250 method.
(4) Biofilm measurement: intestinal tissue homogenate 200. mu.L was taken, 200. mu.L of 10mg/L crystal violet was added, and cultured for 15min, and the sample was washed 3 times with 0.01M PBS, 1mL of 95% (v/v) ethanol was added, 200. mu.L was taken and transferred to a 96-well plate, and the absorbance at 570nm was measured.
Example 5
Based on a mouse intestinal tissue high-flux bacterial adhesion model and a construction method and application thereof, fig. 1 is a schematic diagram of the construction method based on the mouse intestinal tissue high-flux bacterial adhesion model, and as shown in fig. 1, the construction method comprises the following steps:
(1) study subjects: selecting 2-3 month old C57BL/6 mice;
(2) preparing bacterial liquid: preparing a lactobacillus rhamnosus LRa05 colony plate in advance, selecting a single colony to be inoculated in a fresh MRS culture medium, wherein the inoculation amount is 2% (v/v), placing the single colony in a constant temperature shaking table incubator at 35 ℃ for 15h, centrifuging, removing a supernatant, and using a buffer solution to resuspend to obtain a bacterial liquid with the concentration of 109CFU/mL, and temporarily storing in a refrigerator at 4 ℃;
(3) dissecting the mice: anesthetizing a mouse by using pentobarbital, transferring the mouse into a beaker after the mouse is deeply anesthetized, soaking the mouse in alcohol for 5min, and dissecting the mouse in a super clean bench;
(4) taking intestinal tissues of the mice in a sterile environment;
(5) membrane, blood vessel, fat outside the intestinal tissue: removing membranes, blood vessels and fat from the exterior of the intestinal tissue using sterile instruments;
(6) cleaning intestinal tissues: gently removing intestinal contents, and then slowly washing intestinal tissues by using a buffer solution at 4 ℃;
(7) intestinal tissue segmentation: according to the physiological structure, the colon is divided into 6 groups of 5mm long intestinal tissue blocks by using sterile scissors, wherein the duodenum is 4 +/-0.5 cm behind the pylorus, the jejunum is arranged between the duodenum and the ileum, the ileum is arranged at the front 4 +/-0.5 cm of the cecum, and the colon is arranged at the back 6 +/-1 cm of the cecum.
(8) Inoculating bacterial liquid: spreading each group of intestinal tissues in 24-well plate with intestinal mucosa layer facing upwards, inoculating 10 of intestinal mucosa into each well91mL of CFU/mL of Lactobacillus rhamnosus LRa05 bacterial liquid; each well of the individual cultured intestine sections was inoculated with 1mL of 4 ℃ buffer.
(9) Placing the pore plate in a constant temperature shaking table incubator for culturing at 35 deg.C with shaking table rotation speed of 200rpm, wherein the culture time of intestinal tissue and Lactobacillus rhamnosus LRa05 bacterial liquid in the constant temperature shaking table incubator is designed to be 30min, 60min, and 120 min.
And (3) observing by using an electron microscope:
taking an intestinal segment co-cultured with the lactobacillus rhamnosus LRa05, placing the intestinal segment in a sterile 24-well plate, and fixing for 4 hours by using 2mL of 2.5% (v/v) glutaraldehyde; washed 3 times with 0.1M phosphate buffer (pH 7.3); washing the intestinal segment with distilled water at 4 deg.C for three times; after freeze-drying for 18h, observing under a scanning electron microscope.
Example 6
Based on a mouse intestinal tissue high-flux bacterial adhesion model and a construction method and application thereof, fig. 1 is a schematic diagram of the construction method based on the mouse intestinal tissue high-flux bacterial adhesion model, and as shown in fig. 1, the construction method comprises the following steps:
(1) study subjects: selecting 2-3 month old C57BL/6 mice;
(2) preparing bacterial liquid:preparing a lactobacillus rhamnosus LRa05 colony plate in advance, selecting a single colony to be inoculated in a fresh MRS culture medium, wherein the inoculation amount is 2% (v/v), placing the single colony in a constant-temperature shaking incubator at 38 ℃ for 15h, centrifuging, removing a supernatant, and using a buffer solution to resuspend to obtain a bacterial liquid with the concentration of 109CFU/mL, and temporarily storing in a refrigerator at 4 ℃;
(3) dissecting the mice: anaesthetizing a mouse by using pentobarbital, transferring the mouse into a beaker after the mouse is deeply anaesthetized, soaking the mouse in alcohol for 5min, and dissecting the mouse in a super clean bench;
(4) taking intestinal tissues of the mice in a sterile environment;
(5) membrane, blood vessel, fat outside the intestinal tissue: removing membranes, blood vessels and fat from the exterior of the intestinal tissue using sterile instruments;
(6) cleaning intestinal tissues: gently removing intestinal contents, and then slowly washing intestinal tissues by using a buffer solution at 4 ℃;
(7) intestinal tissue segmentation: according to the physiological structure, the colon is divided into 6 groups of 5mm long intestinal tissue blocks by using sterile scissors, wherein the duodenum is 4 +/-0.5 cm behind the pylorus, the jejunum is arranged between the duodenum and the ileum, the ileum is arranged at the front 4 +/-0.5 cm of the cecum, and the colon is arranged at the back 6 +/-1 cm of the cecum.
(8) Inoculating bacterial liquid: spreading each group of intestinal tissues in 24-well plate with intestinal mucosa layer facing upwards, inoculating 10 of intestinal mucosa into each well92mL of CFU/mL of Lactobacillus rhamnosus LRa05 bacterial liquid; each well of the individual cultured intestine sections was inoculated with 2mL of a buffer solution at 4 ℃.
(9) Placing the pore plate in a constant temperature shaking table incubator for culturing at 38 ℃ and 200rpm, and culturing intestinal tissues and lactobacillus rhamnosus LRa05 bacterial liquid in the constant temperature shaking table incubator for 30min, 60min and 120 min.
And (3) observing by an electron microscope:
taking an intestinal segment co-cultured with the lactobacillus rhamnosus LRa05, placing the intestinal segment in a sterile 24-well plate, and fixing for 4 hours by using 2mL of 2.5% (v/v) glutaraldehyde; washed 3 times with 0.1M phosphate buffer (pH 7.3); washing the intestinal segment with distilled water at 4 deg.C for three times; after freeze-drying for 18h, observing under a scanning electron microscope.
And (4) analyzing results:
by taking an electron micrograph of adhesion of probiotics on colon, it was found that lactobacillus plantarum LP45 adhered better under the culture method of example 1 and lactobacillus rhamnosus LRa05 adhered better under the culture method of example 4. Therefore, the samples of examples 1 and 4 were subjected to plate counting and indicators of protein, polysaccharide, and biofilm.
The plate count results showed that the Lactobacillus plantarum LP45 in example 1 had adhesion in all of the duodenum, jejunum, ileum, caecum, and colon, with higher numbers of viable adhesion in the duodenum segment. The adhesion condition of the lactobacillus plantarum on the intestinal tissue can be visually observed through an electron microscope result, and the lactobacillus plantarum adheres to the intestinal tissue through forming a biological film. After culturing for 30min, the protein content of each intestinal section is obviously changed, and the content of other intestinal sections except the protein content of the colon section is increased is reduced; the content of other intestinal sections except the content of the polysaccharide in the duodenal sections is reduced; the content of other intestinal sections except the ileum section biofilm biomass is increased. After the cultivation is carried out for 60min, the polysaccharide content of each intestinal section is obviously changed, the polysaccharide content of the cecum and colon sections is increased compared with that of the intestinal section which is cultivated independently, and the polysaccharide content of the duodenum, jejunum and ileum is reduced compared with that of the intestinal section which is cultivated independently; the protein content of other intestinal sections is reduced except the protein content of the colon section is increased; the biomass of the colon section is less changed compared with the biofilm biomass of the blank intestinal tissue, and the biofilm biomass of other intestinal sections is reduced. After culturing for 120min, the content of other intestinal sections except the colon section is increased compared with the polysaccharide content of the blank intestinal tissue and is reduced; and the protein content of each intestinal section is reduced compared with the protein content of the blank intestinal tissue; compared with the blank intestinal tissue, the biomass of the biofilm in the ileum section is increased, the colon section has no obvious change, and the biomass of the biofilm in other intestinal sections is reduced compared with the blank intestinal tissue.
The plate count results show that lactobacillus rhamnosus LRa05 in example 4 has adhesion in duodenum, jejunum, ileum, caecum, and colon, wherein the number of adhered viable bacteria in colon segment is higher. The adhesion condition of the lactobacillus plantarum on the intestinal tissue can be visually observed through an electron microscope result, and the lactobacillus rhamnosus adheres to the intestinal tissue through forming a biological film. After culturing for 30min, the protein content of each intestinal segment is obviously changed, and the protein content of other intestinal segments except jejunum and colon segments is increased and reduced; the content of polysaccharide in other intestinal sections except for caecum and colon section is increased; the content of each intestinal segment except jejunum and colon segment biofilm biomass is reduced. After the intestinal sections are cultured for 60min, the polysaccharide content of each intestinal section is obviously changed, the polysaccharide content of the colon section is increased compared with that of the intestinal section which is cultured independently, and the polysaccharide content of other intestinal sections is reduced compared with that of the intestinal section which is cultured independently; the protein content of duodenum, jejunum and colon segment is increased; the biofilm biomass of duodenum, ileum and colon section is increased compared with blank intestine tissue, and the biofilm biomass of other intestine sections is reduced. After culturing for 120min, the content of other intestinal sections is reduced except that the content of polysaccharide in colon sections is increased compared with that in blank intestinal tissues; and the protein content of each intestinal section is reduced compared with the protein content of the blank intestinal tissue; compared with the blank intestinal tissue, the biomass of the biofilm in the ileum section is increased, the colon section has no obvious change, and the biomass of the biofilm in other intestinal sections is reduced compared with the blank intestinal tissue.
FIG. 2 is a scanning electron microscope photograph of the adhesion of Lactobacillus plantarum to the colon after the bacterial adhesion model was constructed according to examples 1-6. FIG. 2A is a scanning electron micrograph of Lactobacillus plantarum adhered colon under the conditions of example 1; FIG. 2B is a scanning electron micrograph of Lactobacillus plantarum adhered colon of example 2; FIG. 2C is a scanning electron micrograph of Lactobacillus plantarum adhered colon in the conditions of example 3; FIG. 2D is a scanning electron micrograph of a Lactobacillus plantarum adhered colon obtained under the conditions of example 4; in FIG. 2, E is a scanning electron microscope photograph of the adhered colon of Lactobacillus plantarum under the conditions of example 5, and F is a scanning electron microscope photograph of the adhered colon of Lactobacillus plantarum under the conditions of example 6, and it can be seen from A to F in FIG. 2 that Lactobacillus plantarum LP45 adheres better under the cultivation method of example 1 and Lactobacillus rhamnosus LRa05 adheres better under the cultivation method of example 4.
FIG. 3 is a histogram of the colony counts of Lactobacillus plantarum adhered to intestinal tissue after the bacterial adhesion model was constructed in example 1, as can be seen in FIG. 3: when the co-culture time is 30min and 60min, the number of adhered colonies of the lactobacillus plantarum in the duodenum segment is the highest compared with the jejunum, ileum, rectum and colon; the number of adhering colonies of lactobacillus plantarum in the colon segment was highest compared to the duodenum, jejunum, ileum and rectum at the co-cultivation time of 120 min.
FIG. 4 is a histogram of the number of colonies of Lactobacillus rhamnosus adhering to intestinal tissue after the bacterial adhesion model of example 4 was constructed, as can be seen from FIG. 4: the number of adhering colonies of lactobacillus rhamnosus in colon segment was highest when the co-culture time was 30min, 60min and 120min, compared to duodenum, jejunum, ileum and rectum.
FIG. 5 is a scanning electron microscope photograph of Lactobacillus plantarum adhered to intestinal tissue after construction of the bacterial adhesion model of example 1. FIG. 5A is a scanning electron micrograph of Lactobacillus plantarum adhered to the duodenum; FIG. 5B is a scanning electron micrograph of Lactobacillus plantarum adhered to the jejunum; FIG. 5C is a scanning electron micrograph of Lactobacillus plantarum adhered ileum; FIG. 5D is a scanning electron micrograph of Lactobacillus plantarum adhered caecum; in FIG. 5, E is the scanning electron microscope image of the adhesion colon of Lactobacillus plantarum, and the successful adhesion of Lactobacillus plantarum in the duodenum, jejunum, ileum, caecum, and colon segments can be observed from A to E in FIG. 5, respectively.
FIG. 6 is a scanning electron microscope photograph of Lactobacillus rhamnosus adherence to intestinal tissue after construction of the bacterial adherence model of example 4. FIG. 6A is a scanning electron micrograph of Lactobacillus rhamnosus adhering to the duodenum; FIG. 6B is a scanning electron micrograph of Lactobacillus rhamnosus adhering to the jejunum; FIG. 6C is a scanning electron micrograph of Lactobacillus rhamnosus adhering to the ileum; FIG. 6D is a scanning electron micrograph of Lactobacillus rhamnosus adhering to the cecum; in FIG. 6, E is a scanning electron micrograph showing the adhesion of Lactobacillus rhamnosus to the colon, and successful adhesion of Lactobacillus rhamnosus to the duodenum, jejunum, ileum, caecum and colon segment can be observed from A to E in FIG. 6, respectively.
FIG. 7 is a histogram showing the measurement of polysaccharide content, protein content and biofilm biomass of Lactobacillus plantarum following construction of the bacterial adhesion model of example 1, to intestinal tissue. FIG. 7A is a bar graph showing the change in polysaccharide content caused by the adhesion of Lactobacillus plantarum to each intestinal segment, as shown in FIG. 7A: adhesion of lactobacillus plantarum only increased the polysaccharide content in the colon segment compared to the intestinal segment cultured alone; FIG. 7B is a bar graph showing the change in protein content caused by the adhesion of Lactobacillus plantarum to each intestinal segment, as shown in FIG. 7B: adhesion of lactobacillus plantarum only increased the protein content in the duodenum and colon segments compared to the intestinal segments cultured alone; in FIG. 7, C is the change of biofilm biomass caused by the adhesion of Lactobacillus plantarum to each intestinal segment, and it can be seen from C in FIG. 7 that: adhesion of lactobacillus plantarum reduced biofilm biomass in the colon segment less than in the colon segment cultured alone.
FIG. 8 is a bar graph showing the measurement of polysaccharide content, protein content and biofilm biomass of Lactobacillus rhamnosus adhered to intestinal tissues after the bacterial adhesion model was constructed in example 4. In FIG. 8, A is a bar chart of the change of polysaccharide content caused by the adhesion of Lactobacillus rhamnosus to each intestinal segment, and it can be seen from A in FIG. 8 that: adhesion of lactobacillus rhamnosus only increased the polysaccharide content in the cecum and colon segments compared to the intestinal segments cultured alone; b in FIG. 8 is a bar graph showing the change in protein content caused by the adhesion of Lactobacillus rhamnosus to various intestinal segments, and B in FIG. 8 shows that: adhesion of lactobacillus rhamnosus only increased the protein content in the duodenum, jejunum and colon segments compared to the intestinal segments cultured alone; (ii) a C in figure 8 shows the change of biofilm biomass caused by the adhesion of lactobacillus rhamnosus to each intestinal section, and can be known from C in figure 8: adhesion of lactobacillus rhamnosus only increased biofilm biomass in the duodenum and colon segments compared to the intestinal segments cultured alone.
According to the high-flux bacteria adhesion model based on the intestinal tissues of the mice, the adhesion capability of the lactobacillus plantarum and the lactobacillus rhamnosus in different intestinal tracts is evaluated at different incubation times. The lactobacillus rhamnosus is found to have stronger adhesion capability in the colon, and the lactobacillus plantarum has stronger adhesion capability in the duodenum and the colon. The invention can quickly evaluate the adhesion capability of bacteria and the adhesion preference of each intestinal section by using a high-throughput bacteria adhesion model based on the intestinal tissues of mice. The method can be used for evaluating the activity of probiotic drinks, probiotic powder, health products and medicaments with bacteria adhesion, and can be used for quickly evaluating the adhesion characteristics of probiotic food and health products in intestinal tracts and screening compounds for promoting the adhesion of probiotics in the intestinal tracts by utilizing a mouse intestinal tissue-based high-flux bacteria adhesion model.
The above-mentioned embodiments are provided to further explain the objects, technical solutions and advantages of the present invention in detail, and it should be understood that the above-mentioned embodiments are only examples of the present invention and are not intended to limit the scope of the present invention. It should be understood that any modifications, equivalents, improvements and the like, which come within the spirit and principle of the invention, may occur to those skilled in the art and are intended to be included within the scope of the invention.
Claims (10)
1. A construction method of a high-flux bacterial adhesion model based on mouse intestinal tissues is characterized by comprising the following steps:
(1) preparing bacterial liquid;
(2) dissecting a mouse;
(3) taking intestinal tissues of the mice in a sterile environment;
(4) removing membranes, blood vessels and fat outside the intestinal tissue;
(5) cleaning intestinal tissue;
(6) intestinal tissue segmentation;
(7) inoculating bacterial liquid;
(8) and co-culturing the intestinal tissue and the bacterial liquid in a constant-temperature shaking incubator.
2. The method for constructing the mouse intestinal tissue-based high-throughput bacterial adhesion model according to claim 1, wherein the bacterial liquid obtained in the step (1) is a fresh seed liquid cultured for 10-15 hours, and is subjected to centrifugation, supernatant removal, and bacterial liquid concentration resuspension by using a buffer solution to be 107-109CFU/mL。
3. The method for constructing the mouse intestinal tissue-based high-throughput bacterial adhesion model according to claim 1, wherein the step (5) of washing the intestinal tissue is to wash the intestinal contents with a buffer.
4. The method for constructing a high-flux bacterial adhesion model based on mouse intestinal tissue according to claim 1, wherein the step (6) of segmenting the intestinal tissue comprises segmenting duodenum, jejunum, ileum, caecum and colon of the whole segment of the intestinal tissue of the mouse according to tissue characteristics, wherein the duodenum is 4 +/-0.5 cm behind the pylorus, the jejunum is located in the middle of the duodenum and the ileum, the ileum is located 4 +/-0.5 cm in front of the caecum, the colon is located 6 +/-1 cm behind the caecum, and each segment of the intestine is divided into intestinal tissue blocks with equal length by using sterile scissors.
5. The method for constructing the mouse intestinal tissue high-flux bacteria adhesion model according to claim 1, wherein the inoculating of the bacterial liquid in the step (7) comprises the step of paving intestinal tissue blocks in a pore plate, so that intestinal mucosa layers face upwards, and the amount of the inoculated bacterial liquid in each pore is 1-2 mL.
6. The method for constructing a high-flux bacterial adhesion model based on mouse intestinal tissue according to claim 1, wherein the co-culturing in step (8) is performed by culturing the intestinal tissue and bacterial liquid in a constant temperature shaking table incubator at 35-38 ℃ and at a shaking table rotation speed of 100-200 rpm.
7. The method for constructing the mouse intestinal tissue-based high-flux bacterial adhesion model according to claim 1, wherein the co-culture time in the step (8) is designed to be 30min-2 h.
8. The high-throughput bacterial adhesion model based on the intestinal tissue of the mouse, which is established by the construction method of any one of claims 1 to 7.
9. The high-throughput bacteria adhesion model based on mouse intestinal tissue according to claim 8, wherein the successful adhesion of bacteria on intestinal tissue is mainly performed by counting the total number of colonies adhered on intestinal tissue, observing the successful adhesion of bacteria on intestinal tissue by scanning electron microscope, and detecting the increase of total polysaccharide content, protein content and biofilm biomass after bacteria adhere to intestinal tissue.
10. The mouse intestinal tissue-based high-throughput bacterial adhesion model of claim 8, which is used for evaluating the bacterial adhesion capability and evaluating the activity of probiotic drinks, probiotic powder, health products and medicines for bacterial adhesion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210103431.5A CN114621910A (en) | 2022-01-27 | 2022-01-27 | High-flux bacteria adhesion model based on mouse intestinal tissue and construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210103431.5A CN114621910A (en) | 2022-01-27 | 2022-01-27 | High-flux bacteria adhesion model based on mouse intestinal tissue and construction method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114621910A true CN114621910A (en) | 2022-06-14 |
Family
ID=81898015
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210103431.5A Pending CN114621910A (en) | 2022-01-27 | 2022-01-27 | High-flux bacteria adhesion model based on mouse intestinal tissue and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114621910A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120196275A1 (en) * | 2011-01-27 | 2012-08-02 | Mezghanni Rosangela | Multicellular organotypic model of human intestinal mucosa |
| CN107236780A (en) * | 2017-05-17 | 2017-10-10 | 武汉市农业科学技术研究院畜牧兽医科学研究所 | Kit and method of a kind of detection bacterium to enteron aisle adhesion |
| RU2745654C1 (en) * | 2019-12-30 | 2021-03-30 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Вятский государственный университет" (ВятГУ) | Method of study of gastric and intestinal microbiota during suppression of colonization resistance of gastric mucosa of experimental animals |
| WO2021164591A1 (en) * | 2020-02-23 | 2021-08-26 | 浙江华康药业股份有限公司 | Method for evaluating sugar alcohol in-vitro intestinal microorganism |
-
2022
- 2022-01-27 CN CN202210103431.5A patent/CN114621910A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120196275A1 (en) * | 2011-01-27 | 2012-08-02 | Mezghanni Rosangela | Multicellular organotypic model of human intestinal mucosa |
| CN107236780A (en) * | 2017-05-17 | 2017-10-10 | 武汉市农业科学技术研究院畜牧兽医科学研究所 | Kit and method of a kind of detection bacterium to enteron aisle adhesion |
| RU2745654C1 (en) * | 2019-12-30 | 2021-03-30 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Вятский государственный университет" (ВятГУ) | Method of study of gastric and intestinal microbiota during suppression of colonization resistance of gastric mucosa of experimental animals |
| WO2021164591A1 (en) * | 2020-02-23 | 2021-08-26 | 浙江华康药业股份有限公司 | Method for evaluating sugar alcohol in-vitro intestinal microorganism |
Non-Patent Citations (2)
| Title |
|---|
| DUYGU ALP AND HAKAN KULEAŞAN: "Adhesion mechanisms of lactic acid bacteria: conventional and novel approaches for testing", WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, pages 1 - 9 * |
| 曾东 等: "乳酸菌和大肠杆菌对固定化犬肠道黏蛋白模型的黏附性", 中国兽医学报, vol. 31, no. 10, pages 1419 - 1423 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108220206B (en) | Bifidobacterium longum and application thereof | |
| Park et al. | Emulating host-microbiome ecosystem of human gastrointestinal tract in vitro | |
| CN108486000B (en) | Preparation method and application of bifidobacterium single-bacterium fermented milk | |
| CN114574390B (en) | Bifidobacterium longum subspecies infantis for relieving colonitis and application thereof | |
| CN109182184B (en) | Pediococcus acidilactici strain and application thereof | |
| CN110577912B (en) | Lactobacillus gasseri and application thereof in preparing fermented milk | |
| CN108728382A (en) | One plant of tool norcholesterol and lactobacillus plantarum and its application for promoting enteron aisle short chain fatty acids generation ability | |
| CN112899182B (en) | Lactobacillus crispatus capable of preventing and/or treating cervical squamous cell carcinoma | |
| CN112080445A (en) | Lactobacillus plantarum ZJ316 and application thereof in inhibiting helicobacter pylori | |
| CN110903997B (en) | Fusobacterium nucleatum obtained from colorectal cancer tumor tissue and application thereof | |
| CN105733978B (en) | One Enterococcus faecalis WEFA23 | |
| CN114381407A (en) | Lactobacillus rhamnosus R7970 and product and application thereof | |
| CN108485999B (en) | A kind of streptococcus novel species HTS25 and its application | |
| CN114621910A (en) | High-flux bacteria adhesion model based on mouse intestinal tissue and construction method and application thereof | |
| CN115895966A (en) | Bifidobacterium bifidum BL002 for assisting in relieving gout and application thereof | |
| CN112063566B (en) | Enterococcus faecium and application thereof | |
| CN105733977B (en) | Probiotic feed additive | |
| CN113621572A (en) | Application of lactobacillus rhamnosus in adhering colon cells | |
| CN112011489B (en) | Method for enriching and separating Francisella and/or its neighboring group species, culture medium and preparation method of culture medium | |
| CN117947171A (en) | Method for evaluating damage after infection of respiratory tract mucosa epithelium organoid bacteria | |
| CN117264841A (en) | Bacillus licheniformis AJQ separated from eel intestinal tract and application thereof | |
| CN118165868A (en) | Corynebacterium glutamicum for promoting intestinal barrier repair and GLP-1 expression, screening method and application | |
| AL-Jumaa et al. | Laboratory Diagnosis of Mycoplasma spp. from the Upper Respiratory Tract and Conjunctival Infections in Shelter Cats | |
| CN115838652A (en) | Lactobacillus reuteri LR21 and application thereof | |
| Akhila et al. | Characterization Of Probiotic Lactic Acid Bacteria From Honey And Assessment Of Their Effects On Consumption By Type-2-Diabetes Using Wistar Rat. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |