阅读说明：本技术 基于Caco-2细胞高效诱变筛选肠道定植益生菌的育种方法 (Breeding method for screening intestinal tract colonization probiotics based on Caco-2 cell high-efficiency mutagenesis ) 是由 王加众 王春平 秦贵平 王海洲 于 2021-08-27 设计创作，主要内容包括：本发明公开了一种基于Caco-2细胞高效诱变筛选肠道定植益生菌的育种方法,属于益生菌培育技术领域,包括以下步骤：步骤1、制备益生菌株；步骤2、诱变筛选；步骤3、基于体外模拟肠道环境耐受性筛选；步骤4、基于Caco-2细胞模型黏附性筛选。与现有技术相比较具有体外快速高效诱变筛选能够长时间定植于动物肠道的益生菌的特点。(The invention discloses a breeding method for screening probiotics for intestinal tract colonization based on Caco-2 cell high-efficiency mutagenesis, which belongs to the technical field of probiotic cultivation and comprises the following steps: step 1, preparing probiotic strains; step 2, mutagenesis screening; step 3, screening based on in vitro simulated intestinal environment tolerance; and 4, performing adhesion screening based on the Caco-2 cell model. Compared with the prior art, the method has the characteristics of in vitro rapid and efficient mutagenesis and screening of the probiotics capable of being planted in the intestinal tracts of animals for a long time.)

1. A breeding method for screening probiotics planted in intestinal tracts based on Caco-2 cell high-efficiency mutagenesis is characterized by comprising the following steps:

step 1, preparing probiotic strains

1-1, streaking and inoculating probiotic strains to a microbial culture medium, and culturing at constant temperature of 30-41 ℃ until single colonies appear;

1-2, selecting the single colony in the step 1-1, inoculating the single colony in a microorganism liquid culture medium, performing shake culture at the temperature of 30-41 ℃ and the speed of 180-220 r/min for 8-12 hours to obtain a bacterial liquid;

1-3, transferring the bacterial liquid obtained in the step 1-2 into a sterilizing centrifuge tube, centrifuging for 5-10 min at 3000-5000 r/min, and removing supernatant to obtain bacterial precipitate;

1-4, placing the bacterial precipitates obtained in the step 1-3 into a centrifuge tube, adding phosphate buffer saline solution for resuspension, and adjusting the concentration of the bacterial liquid to 1-2 multiplied by 10⁷Obtaining bacterial suspension per mL;

step 2, mutagenesis screening

2-1, placing the bacterial suspension obtained in the step 1-4 into a centrifugal tube, adding 0.2 part of mutagen (parts are mass units, the same below), keeping out of the sun, and standing for 30-40 minutes at normal temperature to obtain a mutant bacterial liquid;

2-2, taking the mutagenic bacterium liquid obtained in the step 2-1, keeping out of the sun, centrifuging at 3000-5000 r/min for 5-10 min, and removing supernatant to obtain mutagenic bacterium precipitate;

2-3, taking the mutagenic bacteria precipitate obtained in the step 2-2, keeping out of the sun, adding phosphate buffer solution for heavy suspension, centrifuging for 5-10 min at 3000-5000 r/min, and removing supernatant to obtain the mutagenic bacteria precipitate;

step 3, screening based on in-vitro simulated intestinal environment tolerance

3-1, preparing a simulated gastric acid and bile salt screening solution;

3-2, inoculating the mutagenized bacterial precipitate obtained in the step 2-3 into the simulated gastric acid and cholate screening solution in the step 3-1, and culturing for 18-24 hours at 37-41 ℃ by shaking to obtain a gastric acid and cholate screening bacterial solution;

3-3, centrifuging the screened gastric acid and cholate bacterial liquid obtained in the step 3-2 at 37-41 ℃ for 5-10 min at 3000-5000 r/min, removing supernatant, and adding phosphate buffer solution for heavy suspension to obtain screened gastric acid and cholate bacterial liquid;

step 4, adhesion screening based on Caco-2 cell model

4-1, Caco-2 cell activation and passage;

4-2, counting by using a blood counting chamber, and adjusting the concentration of Caco-2 cells to 1-2 × 10 by using a cell culture medium⁵Inoculating the cells/mL in a cell culture dish, and culturing at 37-41 ℃ in 5% CO₂Culturing for 15-20 days, changing the cell culture solution 1-2 times per week until the typical intestinal cell differentiation characteristics can be seen by microscopic examination;

4-3, adding the screened gastric acid bile salt bacterial liquid obtained in the step 3-4 into the cell culture dish in the step 4-2, mixing the bacterial liquid and the cell culture dish gently and uniformly, and performing reaction at the temperature of 37-41 ℃ and 5% CO₂Culturing for at least longer than the time for screening the adhesion Caco-2 cells, and gently cleaning the cells by using a cell culture medium every 5 hours;

4-4, removing the culture medium after the culture is finished, and washing the cells by phosphate buffer solution; stripping Caco-2 cells in the step 4-3 by using a cell scraper, scraping the obtained combination of the probiotics and the Caco-2 cells, inoculating the combination into a microorganism liquid culture medium, culturing at 30-41 ℃ at 180-220 r/min for 8-12 h by using a shaking table to obtain a cell screened bacterial liquid;

4-5, dipping the screened bacterial liquid obtained in the step 4-4 by using an inoculating ring, streaking and inoculating the bacterial liquid to a microorganism solid culture medium, and culturing at constant temperature of 30-41 ℃ until a single colony appears;

4-6, selecting the single colony in the step 4-5 for culture, repeating the step 1-the step 4, and finally storing for later use after molecular biological identification.

2. A breeding method for screening probiotics for intestinal colonization based on Caco-2 cell high-efficiency mutagenesis as recited in claim 1, wherein the mutagen is 1-methyl-3-nitro-1-nitrosoguanidine.

3. A breeding method for screening probiotics for intestinal colonization based on Caco-2 cell high-efficiency mutagenesis as claimed in claim 1, wherein in the step 3-1, the preparation of the gastric acid and bile salt simulating screening solution is realized by: and (3) taking concentrated hydrochloric acid, adding phosphate buffer salt solution to adjust the pH value to 2.0, adding 1 part of pepsin and 0.3 part of oxgall salt into each 100 parts of liquid, mixing uniformly, and filtering by using a filter to obtain the simulated gastric acid and gall salt screening liquid.

4. A breeding method for screening intestinal colonizing probiotics based on Caco-2 cell high efficiency mutagenesis as claimed in claim 1, wherein the probiotic species comprise fungi and bacteria.

5. A breeding method for screening intestinal colonizing probiotics based on Caco-2 cell high efficiency mutagenesis as claimed in claim 4, wherein the fungi comprises Saccharomyces cerevisiae, Saccharomyces boulardii and yarrowia lipolytica.

6. A breeding method for screening probiotics for intestinal colonization based on Caco-2 cell high-efficiency mutagenesis as claimed in claim 4, wherein the bacteria comprise lactobacillus and bifidobacterium.

Technical Field

The invention relates to the technical field of probiotic cultivation, and particularly belongs to a breeding method for screening probiotics for intestinal tract colonization based on Caco-2 cell high-efficiency mutagenesis.

Background

Mutation breeding is a breeding method in which, in order to increase the random mutation frequency of genes and expand the variation range, the genetic characteristics of organisms are induced to vary by physical and chemical factors under artificial conditions, and then individuals meeting certain requirements of people are selected from variation groups, thereby further cultivating new animal and plant or microorganism varieties.

Mutation breeding is largely divided into random mutagenesis, which is a traditional means of mutation breeding, and directed mutagenesis, in which a cell or organism is exposed to a mutagen to select for mutants having desired characteristics. Mutagens, which essentially change the genetic material of an organism, may also be carcinogenic because many mutations may cause cancer, and commonly used mutagens include physical, chemical and biological mutagens. At present, the mutagenesis mode of probiotics is mainly random mutagenesis, the mutagenesis efficiency is low, the workload is large, and ideal mutant strains cannot be obtained.

The method comprises the steps of firstly adding a mutagen to randomly mutate probiotics, then inoculating the probiotics in a stomach-irrigation mode, separating suspected mutant probiotics from animal intestines or excrement at different time periods, carrying out PCR identification, and repeating the mutagenesis method until stably inherited probiotics capable of being colonized in animal intestines for a long time are screened. The method for screening the animal intestinal tract colonizing probiotics by mutagenesis has the defects of complex operation, large workload and low mutagenesis efficiency.

Disclosure of Invention

The invention aims to provide a breeding method for screening probiotics planted in intestinal tracts based on Caco-2 cell high-efficiency mutagenesis, so as to achieve the aim of screening the probiotics capable of being planted in the intestinal tracts of animals for a long time in vitro and quickly and efficiently.

The breeding method for screening the probiotics for intestinal tract colonization based on Caco-2 cell high-efficiency mutagenesis is characterized by comprising the following steps:

step 1, preparing probiotic strains

1-1, streaking and inoculating probiotic strains to a microbial culture medium, and culturing at constant temperature of 30-41 ℃ until single colonies appear;

1-2, selecting the single colony in the step 1-1, inoculating the single colony in a microorganism liquid culture medium, performing shake culture at the temperature of 30-41 ℃ and the speed of 180-220 r/min for 8-12 hours to obtain a bacterial liquid;

1-3, transferring the bacterial liquid obtained in the step 1-2 into a sterilizing centrifuge tube, centrifuging for 5-10 min at 3000-5000 r/min, and removing supernatant to obtain bacterial precipitate;

1-4, placing the bacterial precipitates obtained in the step 1-3 into a centrifuge tube, adding phosphate buffer saline solution for resuspension, and adjusting the concentration of the bacterial liquid to 1-2 multiplied by 10⁷Obtaining bacterial suspension per mL;

step 2, mutagenesis screening

2-1, placing the bacterial suspension obtained in the step 1-4 into a centrifugal tube, adding 0.2 part of mutagen (parts are mass units, the same below), keeping out of the sun, and standing for 30-40 minutes at normal temperature to obtain a mutant bacterial liquid;

2-2, taking the mutagenic bacterium liquid obtained in the step 2-1, keeping out of the sun, centrifuging at 3000-5000 r/min for 5-10 min, and removing supernatant to obtain mutagenic bacterium precipitate;

2-3, taking the mutagenic bacteria precipitate obtained in the step 2-2, keeping out of the sun, adding phosphate buffer solution for heavy suspension, centrifuging for 5-10 min at 3000-5000 r/min, and removing supernatant to obtain the mutagenic bacteria precipitate;

step 3, screening based on in-vitro simulated intestinal environment tolerance

3-1, preparing a simulated gastric acid and bile salt screening solution;

3-2, inoculating the mutagenized bacterial precipitate obtained in the step 2-3 into the simulated gastric acid and cholate screening solution in the step 3-1, and culturing for 18-24 hours at 37-41 ℃ by shaking to obtain a gastric acid and cholate screening bacterial solution;

3-3, centrifuging the screened gastric acid and cholate bacterial liquid obtained in the step 3-2 at 37-41 ℃ for 5-10 min at 3000-5000 r/min, removing supernatant, and adding phosphate buffer solution for heavy suspension to obtain screened gastric acid and cholate bacterial liquid;

step 4, adhesion screening based on Caco-2 cell model

4-1, Caco-2 cell activation and passage;

4-2, counting by using a blood counting chamber, and adjusting the concentration of Caco-2 cells to 1-2 × 10 by using a cell culture medium⁵Inoculating the cells/mL in a cell culture dish, and culturing at 37-41 ℃ in 5% CO₂Culturing for 15-20 days, changing the cell culture solution 1-2 times per week until the typical intestinal cell differentiation characteristics can be seen by microscopic examination;

4-3, adding the screened gastric acid bile salt bacterial liquid obtained in the step 3-4 into the cell culture dish in the step 4-2, mixing the bacterial liquid and the cell culture dish gently and uniformly, and performing reaction at the temperature of 37-41 ℃ and 5% CO₂Culturing for at least longer than the time of screening for adherent Caco-2 cellsThe cells are washed by cell culture medium gently every 5 hours;

4-4, removing the culture medium after the culture is finished, and washing the cells by phosphate buffer solution; stripping Caco-2 cells in the step 4-3 by using a cell scraper, scraping the obtained combination of the probiotics and the Caco-2 cells, inoculating the combination into a microorganism liquid culture medium, culturing at 30-41 ℃ at 180-220 r/min for 8-12 h by using a shaking table to obtain a cell screened bacterial liquid;

4-5, dipping the screened bacterial liquid obtained in the step 4-4 by using an inoculating ring, streaking and inoculating the bacterial liquid to a microorganism solid culture medium, and culturing at constant temperature of 30-41 ℃ until a single colony appears;

4-6, selecting the single colony in the step 4-5 for culture, repeating the step 1-the step 4, and finally storing for later use after molecular biological identification.

Further, the mutagen is 1-methyl-3-nitro-1-nitrosoguanidine.

Further, in the step 3-1, the preparation of the simulated gastric acid and bile salt screening solution is realized by: and (3) taking concentrated hydrochloric acid, adding phosphate buffer salt solution to adjust the pH value to 2.0, adding 1 part of pepsin and 0.3 part of oxgall salt into each 100 parts of liquid, mixing uniformly, and filtering by using a filter to obtain the simulated gastric acid and gall salt screening liquid.

Further, the probiotic species include fungi and bacteria.

Further, the fungi include Saccharomyces cerevisiae, Saccharomyces boulardii, and yarrowia lipolytica.

Further, the bacteria include lactobacillus and bifidobacterium.

The premise that the intestinal probiotics can live in the intestinal tracts of animals and stay in the intestinal tracts of the animals for a long time without being discharged out of the bodies along with the gastrointestinal peristalsis is that the longer the intestinal probiotics stay in the intestinal tracts, the better the probiotic effect is. Based on the conclusion, the probiotic effect of the probiotics can be improved by mutagenizing and screening the probiotics which are planted in the intestinal tracts of the animals for a long time. In the prior art, a living body, namely an animal intestinal canal, is generally adopted to screen strains with strong adhesive capacity, and the strains are inoculated by microorganisms, slaughtered and separated and purified by microorganisms, so that the method has the advantages of long experimental period, low efficiency and fussy operation.

The method adopts Caco-2 cells for in vitro screening, the Caco-2 cells are human colon cancer cells, can be differentiated to form a structure similar to intestinal epithelium under the condition of cell culture, form a continuous monolayer, and have the cell polarity similar to the intestinal epithelium and tight connection. The variant strain of the gastric acid and bile salt resistance is screened out by simulating the gastric acid and bile salt in vitro, Caco-2 cell model adhesion screening is established, and finally the probiotics which can be planted in the intestinal tracts of animals for a long time is obtained by in vitro screening.

The microorganism has the characteristic of gene self-repair, and a part of mutant strains obtained by mutagenesis screening can restore the original characters after being subjected to gene repair for several times of passage, and the mutant strains which can always maintain the target characters after being subjected to continuous passage for several times have genetic stability.

The breeding method for screening the probiotics for intestinal tract colonization based on Caco-2 cell high-efficiency mutagenesis has the following positive effects:

1. according to the invention, animals are replaced by Caco-2 cell differentiated intestinal epithelial-like structures for screening, so that the mutagenesis screening period is effectively saved, and the mutagenesis efficiency is improved;

2. according to the invention, Caco-2 cells are adopted to replace animals for mutation screening breeding, so that the test period is greatly shortened;

3. the invention adopts Caco-2 cells without slaughtering animals to take target strains from intestinal tracts;

4. compared with animal-level mutagenesis screening, the cell-level mutagenesis screening breeding is easy to operate and high in accuracy.

Drawings

The accompanying drawings disclose, in part, specific embodiments of the present invention, wherein,

FIG. 1 is a Caco-2 differentiated intestinal epithelial structure in example 1 of the present invention;

FIG. 2 is a comparison chart of gene sequence sequencing in example 1 of the present invention;

FIG. 3 is a schematic diagram showing the genetic stability of the strain in example 1 of the present invention.

Detailed Description

The invention is further illustrated by the following examples in conjunction with the accompanying drawings.

Example (b): taking Saccharomyces boulardii as an example

1. Preparation of Saccharomyces boulardii Strain

And (3) streaking and inoculating the Saccharomyces boulardii strain to a YPD solid culture medium, culturing in a constant-temperature incubator at 37 ℃ until a single colony appears, selecting the single colony on a flat plate, inoculating the single colony in a test tube filled with 3 mL of YPD liquid culture medium, culturing at 37 ℃ for 220 r/min by a shaking table for 12 h to obtain a bacterial liquid. Transferring 1 mL of the cultured bacterial liquid into a 1.5 mL sterile centrifuge tube, centrifuging at 5000 r/min for 5 min, discarding the supernatant, adding phosphate buffer saline solution for resuspension, and adjusting the concentration of the bacterial liquid to 2 × 10⁷one/mL.

2. Mutagenesis screening

Taking the concentration of the step 1 as 2 multiplied by 10⁷Putting 4 mL of bacterial suspension/mL into a 10 mL centrifuge tube, adding 200 mg of 1-methyl-3-nitro-1-nitrosoguanidine, keeping out of the sun, acting for 40 minutes at normal temperature, centrifuging at 4 ℃ and 5000 r/min for 5 min, removing supernatant, adding 5 mL of phosphate buffer salt solution to clean the tube wall under the condition of keeping out of the sun, resuspending bacterial solution, centrifuging at 5000 r/min for 5 min, and removing supernatant to obtain the mutagenized Saccharomyces boulardii precipitate.

3. In-vitro intestinal environment tolerance simulation based screening

1 mol/mL concentrated hydrochloric acid is taken, phosphate buffer salt solution is added to adjust the pH value to 2.0, 1 g of pepsin and 0.3g of ox gall salt are respectively added into each 100 mL of liquid, the mixture is mixed evenly, and a filter with the diameter of 0.2 mu m is used for filtering, so that the simulated gastric acid and gall salt screening liquid is obtained. Inoculating a proper amount of mutagenized Saccharomyces boulardii sediment into a simulated gastric acid bile salt screening solution, and performing shake culture at 37 ℃ for 24h to obtain the gastric acid bile salt screening solution. Centrifuging at 37 deg.C for 5 min at 5000 r/min, removing supernatant, completely removing hydrochloric acid and bile salt, preventing Caco-2 cells from invasion, adding phosphate buffer solution, and resuspending to obtain Saccharomyces boulardii liquid after screening gastric acid and bile salt.

4. Adhesion screening based on Caco-2 cell model

Caco-2 cells were activated and passaged according to a conventional method, counted using a hemocytometer, and adjusted to a cell concentration of 1X 10 with DMEM medium⁵seed/mL, in cell culture dishes at 37 ℃ with 5% CO₂The culture is carried out for 20 days,the cell culture solution is changed for 1-2 times every week until the typical intestinal cell differentiation characteristics can be seen by microscopic examination. As shown in FIG. 1, it can be seen that the Caco-2 cells have a fine villus-like structure with regular small intestine on the top, and a tight connection structure is formed on the top between the adjacent cells, and the tight connection is located on the membranes on both sides.

Adding the screened gastric acid bile salt cholate saccharomyces boulardii liquid obtained in the step 3 into the culture dish, softly and uniformly mixing, and performing reaction at 37 ℃ and 5% CO₂The cells were cultured for 20h, and the cells were washed gently with DMEM medium every 5 h. After the culture is finished, removing the culture solution, adding 2mL of phosphate buffer salt solution to clean cells, peeling Caco-2 cells attached to a culture dish by using a cell spatula, adhering the probiotics to the Caco-2 cells, inoculating the scraped combination of the probiotics and the Caco-2 cells into a test tube filled with 3 mL of YPD liquid culture medium, and culturing on a shaking table at 30 ℃ and 220 r/min for 12 hours to realize amplification of the Saccharomyces boulardii, wherein the Caco-2 cells do not grow in the YPD liquid culture medium and die quickly, so that the screened Saccharomyces boulardii liquid is obtained. And (3) dipping the bacterial liquid obtained after the cell screening by using an inoculating ring, streaking and inoculating the bacterial liquid to an YPD solid culture medium, and culturing at 37 ℃ for 24h to obtain a single colony. And (3) selecting a single colony to perform PCR reaction and performing sequencing analysis by using a fungus universal primer, wherein as shown in figure 2, the consistency of 11 strains of bacteria and the standard strain SBSC is more than 98 percent, which indicates that the 11 strains of bacteria are screened and are all saccharomyces boulardii. The 11 strains identified by molecular biology are subjected to the steps 1 to 4 to obtain the character capable of stably inheriting the target, after 15 times of repetition, the strain with the number SD-9 can still adhere to Caco-2 cells for 20 hours, so that the adhesion property of the strain can be stably inherited, and the Saccharomyces boulardii SD-9 is the finally obtained target strain, as shown in figure 3.

It should be noted that while the invention has been described in detail with respect to specific embodiments thereof, it will be apparent to those skilled in the art that various obvious changes can be made therein without departing from the spirit and scope of the invention.