CN108660182B - Culture medium for separating and screening lactobacillus fermentum in human intestinal tract and application of culture medium - Google Patents
Culture medium for separating and screening lactobacillus fermentum in human intestinal tract and application of culture medium Download PDFInfo
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Abstract
The invention discloses a culture medium for separating and screening lactobacillus fermentum in human intestinal tracts and application thereof, and belongs to the technical field of microorganisms. On the basis of the traditional lactobacillus screening culture medium LAMVAB culture medium, the invention replaces carbon source and adds the concentration of 0.256 multiplied by 10‑3mg/mL streptomycin and 6.4X 10‑6The mg/mL gentamicin inhibits the growth of common lactobacillus and other lactic acid bacteria in human intestinal tracts and keeps the growth of the lactobacillus fermentum in the culture medium. The culture medium solves the problem of difficulty in screening the lactobacillus fermentum in the human intestinal tract, and improves the screening efficiency of the lactobacillus fermentum in the human intestinal tract.
Description
Technical Field
The invention relates to a culture medium for separating and screening lactobacillus fermentum in human intestinal tracts and application thereof, belonging to the technical field of microorganisms.
Background
Lactobacillus fermentum (Lactobacillus fermentum) belongs to the genus Lactobacillus in the family Lactobacillus, is heterolactobacillus fermentum, gram-positive, facultative anaerobic in metabolic type, and has an optimal growth temperature of 30-37 ℃. Lactobacillus fermentum is one of the resident flora of human intestinal tract, and mainly adheres to small intestinal epithelial cells. In 2011, lactobacillus fermentum is listed in the list of strains available for food, and is also recognized by the food and drug administration of the united states of america as GRAS (generally recognized as safe). The lactobacillus fermentum CECT5716 is listed in the infant edible strain List by the Ministry of health of China in 2016 (6 months).
Researches show that lactobacillus fermentum, as one of probiotics strains for food, can tolerate the environment of low acidity and high bile salt in intestinal tracts of human bodies and has strong adaptability in the environment of gastrointestinal tracts. The lactobacillus fermentum has good probiotic effects in the aspects of immunoregulation (colitis, irritable bowel syndrome and the like), pathogen antagonism (salmonella, staphylococcus aureus, candida, streptococcus and the like), oxidation resistance, cholesterol reduction and the like. The lactobacillus fermentum has great research value in the aspect of potential probiotic characteristics as a probiotic, so that the problem to be solved firstly is to quickly and efficiently screen excellent lactobacillus fermentum, and the screening culture medium is the most critical in the screening process.
At present, the lactobacillus fermentum is mainly selected from healthy human intestinal tracts and some fermented foods. The Chinese patent application with the application number of 201610827496.9 and the name of 'specific culture medium for lactobacillus fermentum and application thereof' discloses a specific culture medium for lactobacillus fermentum and application thereof, but the culture medium can only be applied to the specific culture of lactobacillus fermentum in fermented dairy products, and is not suitable for strain separation of complex bacterial phase samples. The human intestinal tract contains abundant and diverse microorganisms with the number of 1014More than 99% of the bacteria are bacteria, and 500-1000 different species exist. Lactobacillus including lactobacillus fermentum is an important probiotic in intestinal tracts, and the lactobacillus which is common in human intestinal tracts is not less than 20, but is often low in abundance and not easy to screen. The most effective medium for screening lactobacillus from intestinal contents is the LAMVAB (Lactobacillus Fermentarium MRS with Arabidopsis and Three antibiotics) medium, which inhibits the growth of other bacteria besides lactobacillus by lowering pH and adding vancomycin, but it is limited to screening lactobacillus and cannot be accurate to the bacterial level. Therefore, no specific culture medium for separating and screening lactobacillus fermentum in human intestinal tracts exists at present.
Disclosure of Invention
The invention aims to solve the technical problem of providing a culture medium for improving the efficiency of screening and separating lactobacillus fermentum in human intestinal tracts, which contains streptomycin, vancomycin, gentamicin, a carbon source and other substances and can inhibit the growth of non-lactobacillus and other common lactobacillus in the human intestinal tracts, thereby carrying out the separation and screening of the lactobacillus fermentum in the human intestinal tracts.
The invention also provides a method for separating and screening the lactobacillus fermentum in the intestinal tract of the human body, wherein the lactobacillus fermentum in the intestinal tract is enriched by utilizing the culture medium liquid pipe and then diluted and coated on the solid plate of the screening culture medium, so that the lactobacillus fermentum with very low abundance in the intestinal tract can be rapidly enriched.
In order to solve the above technical problems, a first object of the present invention is to provide a culture medium lfmata (lactobacillus Fermentaum MRS with Arabidopsis and Three antibiotics) for screening and isolating lactobacillus Fermentum in human intestinal tract, comprising the following components by weight: 8-15 g of peptone, 8-15 g of beef extract, 3-10 g of yeast powder, 801-3 g of tween, 2-3 g of dipotassium hydrogen phosphate, 2-5 g of sodium acetate, 1-3 g of diammonium citrate, 0.4-0.6 g of magnesium sulfate heptahydrate, 0.15-0.25 g of manganese sulfate monohydrate, 8-20 g of carbon source and 20 x 10g of vancomycin-3g, streptomycin 0.256g, gentamicin 6.4X 10-2g, 0.5g of L-cysteine and distilled water to reach the volume of 1000 mL.
In one embodiment of the invention, the culture medium further contains agar 10-20 g/L.
In one embodiment of the invention, the carbon source comprises arabinose.
In one embodiment of the invention, the sole carbon source of the medium is arabinose;
in one embodiment of the invention, the culture medium contains antibiotics in an amount of 1L by mass: vancomycin 20X 10-3g; streptomycin 0.256 g; gentamicin 6.4X 10-2g; all antibiotics were filter sterilized as solutions.
In one embodiment of the invention, the culture medium is sterilized at 115 ℃ for 20min except antibiotics, and the pH of the culture medium is 5.0 +/-0.1.
The second purpose of the invention is to provide the application of the culture medium in screening or culturing microorganisms.
A third object of the present invention is to provide a method for preparing the culture medium LFMATA, the method comprising the steps of:
(1) weighing 10g of peptone, 10g of beef extract, 5g of yeast powder, 2.6g of dipotassium phosphate, 2g of sodium acetate, 2g of diammonium hydrogen citrate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 20g of arabinose and 0.5g of L-cysteine, putting the materials into a container, adding distilled water to a constant volume of 969mL, adjusting the pH to 5.0 +/-0.1 by using 4M HCL, and sterilizing at 115 ℃ for 20 minutes;
(2) dissolving 0.1g of vancomycin in 50mL of water to prepare a solution A with the concentration of 2 mg/mL; dissolving 0.128g of gentamicin in 20mL of water to prepare a solution B with the concentration of 6.4 mg/mL; dissolving 0.512g streptomycin in 20mL water to prepare a solution C with the concentration of 25.6 mg/mL; sterilizing the solution A, B, C with filter membrane respectively;
(3) and (3) cooling the sterilized mixture obtained in the step (1) to 55 ℃, adding 10mL of the solution A, 10mL of the solution B and 10mL of the solution C obtained in the step (2), and uniformly mixing to obtain the LFMATA liquid culture medium.
In one embodiment of the invention, the method adds 20g of agar in the step (1), and the rest steps are unchanged, so as to prepare the LFMATA solid culture medium.
A fourth object of the present invention is to provide a method for isolating lactobacillus fermentum in a sample of human intestinal tract, comprising the steps of:
(1) enrichment culture: taking 0.1-0.5 g of intestinal contents, adding the intestinal contents into 5mL of LFMATA liquid culture medium under an aseptic condition, and carrying out enrichment culture for 18-24 h at 35-37 ℃ under an anaerobic condition;
(2) gradient dilution: oscillating and mixing uniformly by using a liquid tube oscillator in the step (1), putting 1mL of suspension into 9mL of physiological saline, and fully mixing uniformly to obtain 10-1Diluting the solution, repeating the above dilution steps to obtain 10-2、10-3、10-4、10-5、10-6Diluting the solution;
(3) coating culture: respectively suck 100uL of the above-mentioned 10-4、10-5、10-6Three gradient dilutions are placed on an LFMATA solid culture medium, uniformly coated by a coating rod, and cultured for 48 hours under the anaerobic condition at 37 ℃;
(4) primary purification culture: taking a dilution coating plate with the colony number within a range of 30-300, randomly selecting 10 single bacteria with different shapes and sizes from each sample, streaking the single bacteria on an MRS solid plate, and placing the single bacteria on an anaerobic condition at 37 ℃ for culturing for 48 hours.
(5) Secondary purification culture: and (4) inoculating a liquid MRS tube to a single colony on the streak plate in the step (4), and culturing for 20 hours at 37 ℃ under an anaerobic condition.
(6) And (3) strain preservation: mixing the two-stage purified culture bacterial suspension and sterilized 60% glycerol at a ratio of 1:1, packaging the mixture in a freezing tube, and freezing and storing in an ultra-low temperature refrigerator of-80 deg.C.
(7) And (3) strain identification: and (4) centrifuging the residual bacterial suspension obtained in the step (6) to obtain bacterial sludge, carrying out resuspension cleaning on the bacterial sludge by using sterile physiological saline, then carrying out 16S DNA PCR amplification, and carrying out detection and identification on an amplification sequence.
The invention also claims the application of the method in the aspects of separating and identifying the lactobacillus fermentum.
Has the advantages that: (1) the invention is improved on the basis of an LAMVAB screening culture medium, and is added with vancomycin, streptomycin and gentamicin, and the carbon source is limited to arabinose, so that the growth of non-lactobacillus and common lactobacillus except lactobacillus fermentum in human intestinal tracts is inhibited, and the efficiency of screening and separating lactobacillus fermentum from the human intestinal tracts is greatly improved. (2) The invention also details the preparation method and screening application steps of the culture medium for separating and screening the lactobacillus fermentum. Compared with the common bacteria screening process, the method provided by the invention has the advantages that the screening culture medium liquid pipe is used for enrichment culture of the lactobacillus fermentum, and then the common bacteria screening processes such as dilution coating, streak purification, liquid pipe connection and the like are carried out, so that the method is mainly used for screening the low-abundance lactobacillus fermentum in the intestinal tract of a human body.
Detailed Description
Example 1: research on composition of lactobacillus in human intestinal flora based on lactobacillus metagenome
The technical process of sequencing the human intestinal flora lactobacillus metagenome comprises the following steps:
(1) taking 10 intestinal tract content samples of the Nantong area from a freezing refrigerator at the temperature of-80 ℃, and taking out 0.2g of intestinal tract content samples for DNA extraction by using a gun head after thawing;
(2) separating and extracting genome DNA from the 10 intestinal contents in the step (1) by using a fecal DNA Kit (FastDNA Spin Kit For Feces, MP);
(3) lactobacillus groel gene amplification:
taking 1uL of the genome DNA in the step (2) as a PCR template.
(II) the PCR primers used are lactobacillus groel gene specific amplification primers: 308F-TGAAGAAYGTNRYNGCYGG and 806R-AANGTNCCVCGVATCTTGT, and the length of the amplified fragment is 499 bp.
(III) the PCR reaction system is as follows: 25uL of Premix Taq (TaKaRa Taq Version 2.0plus dye, TaKaRa), 1uL of 308F, 1uL of 806R, 2uL of DNA template, 21uL of double distilled water.
(IV) the PCR reaction conditions are as follows: 95 ℃ for 5 min; 35 cycles (95 ℃, 30 s; 55 ℃, 40 s; 72 ℃, 40 s); 72 ℃ for 5 min; 12 ℃ for 10 min.
(V) obtaining an intestinal content bacterium DNA lactobacillus groel gene amplification product through the PCR series process.
(4) Identification and recovery of PCR products:
and (I) taking 3uL of the PCR product obtained in the step (3), placing the spot on 1.0% agarose gel added with dye for electrophoresis, performing 110V electrophoresis for 30min, and then performing identification on an ultraviolet gel imager after the electrophoresis is finished.
(II) all positive PCR products are electrophoresed in macroporous recovery gel, the sample application amount is 45uL, the agarose concentration is 1.5%, and 110V electrophoresis is carried out for 30 min. Under an ultraviolet lamp, a DNA strip of about 499bp was cut out from a 100bp DNA Ladder Marker and placed in a 1.5mL EP tube.
(III) the DNA band was collected and purified using an agarose Gel recovery Kit (QIAquick Gel extraction Kit, QIAGEN).
(5) And (4) using a multifunctional microplate reader to perform concentration determination on the purified DNA in the step (4).
(6) Quantitative and equal-mass mixing and library construction.
(7) Sequencing on Miseq PE 300.
(8) And (5) off-line data processing and analysis.
Intestinal content samples of 10 human groups in southeast region (S1, S2, S3, S4, S5, S6, S7, S8, S9, S10) lactobacillus metagenome analysis results are as follows:
TABLE 1-1 analysis of the composition of Lactobacillus species in intestinal content of 10 populations (from southeast Asian region)
TABLE 1-2 analysis of Lactobacillus composition profiles in intestinal content samples of 10 populations (from Nantong area)
Note: l is an abbreviation of Lactobacillus.
As shown in tables 1-1 and 1-2, based on the results of lactobacillus metagenomic sequencing of 10 samples of human intestinal contents in the southeast region, we found that l.johnsonii, l.mucosae and l.salivariaus were most abundant in human intestinal tracts, while l.casei, l.crispatus, l.frumenti, l.reuteri and l.variaginalis were ubiquitous in human intestinal tracts, although low in content. Taking these ten population samples as an example, we selected these 21 lactobacilli as common lactobacilli in the intestinal tract of the population.
Example 2: application of intestinal content sample in strain screening on lactobacillus basal medium LAMVAB
The formula of the LAMVAB culture medium comprises the following components (calculated by 1L): 10g of peptone, 10g of beef extract, 5g of yeast powder, 2.6g of dipotassium phosphate, 2g of sodium acetate, 2g of diammonium citrate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 20g of arabinose, and 20 multiplied by 10 grams of vancomycin-3g, 0.5g of L-cysteine, 0.05g of bromocresol green, 801 g of Tween and 1000mL of water.
The preparation steps of the culture medium are as follows:
(1) weighing 10g of peptone, 10g of beef extract, 5g of yeast powder, 2.6g of dipotassium phosphate, 2g of sodium acetate, 2g of diammonium hydrogen citrate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate 2, 20g of glucose, 0.5g of L-cysteine and 20g of agar, adding distilled water to a constant volume of 969mL, adjusting the pH to 5.0 +/-0.1 by using 4M HCL, and sterilizing at 115 ℃ for 20 minutes;
(2) dissolving 0.1g of vancomycin in 50mL of water to prepare a solution with the concentration of 2 mg/mL;
(3) weighing 0.25g of bromocresol green, and dissolving in 100mL of 95% ethanol solution to prepare a bromocresol green solution with the concentration of 2.5 mg/mL;
(4) and (3) cooling the sterilized mixture obtained in the step (1) to 55 ℃, adding 10mL of the vancomycin solution obtained in the step (2) and 10mL of the bromocresol green solution obtained in the step (3), and uniformly mixing to obtain the solid culture medium.
The method for separating and screening the microorganisms in the human intestinal tract sample by using the culture medium comprises the following steps:
(1) dilution coating: inoculating the obtained intestinal contents in sterile physiological saline at an inoculation amount of 1%, and diluting to a concentration of 10-1-10-5From 10-3，10-4And 10-5Coating 100uL of the three gradient dilutions on the LAMVAB solid medium;
(2) primary purification culture: taking a diluted coating plate with the colony number within a range of 30-300, randomly selecting 10 single bacteria with different shapes and sizes from each sample, placing the single bacteria on an MRS solid plate, streaking, and culturing for 48 hours under an anaerobic condition at 37 ℃;
(3) secondary purification culture: taking a single colony on the streak plate in the step (2) to be connected with a liquid MRS tube, and culturing for 20h under the anaerobic condition at 37 ℃;
(4) and (3) strain preservation: uniformly mixing the secondary purified culture bacterial suspension and sterilized 60% glycerol at a ratio of 1:1 to obtain a final glycerol concentration of 30%, subpackaging the mixed solution in a freezing tube, and freezing and storing in an ultra-low temperature refrigerator at-80 ℃;
(5) and (3) strain identification: and (4) centrifuging the residual bacterial suspension obtained in the step (4) to obtain bacterial sludge, carrying out resuspension cleaning on the bacterial sludge by using sterile physiological saline, then using the bacterial sludge for 16SDNA PCR amplification, and carrying out detection and identification on an amplification sequence.
Taking the results of screening intestinal content samples (A1, A2, A3, A4 and A5) of 5 groups in Nantong area on a LAMVAB culture medium as an example, the experimental results are as follows (10 strains are selected in each sample):
TABLE 25 summary of the results of the bacterial screening of the population intestinal content samples on LAMVAB medium
As can be seen from Table 2, 5 intestinal samples from southern Tong area were co-screened on LAMVAB medium to give 50 strains, of which Lactobacillus fermentum was present in only 4 strains, the 4 strains being derived from 3 human samples. In addition to the lactobacilli which appear in example 1, other lactic acid bacteria were obtained, including Leuconostoc citreum, Wessella cibaria, Weissella parameteroides and Pediococcus pentosaceus, and their frequency of occurrence was high.
Example 3: carbon source selection and optimization of media
As described in examples 1-2, there are about 25 species of Lactobacillus commonly found in the human intestinal tract and Lactobacillus that can grow on the existing Lactobacillus-specific medium, LAMVAB. For these 25 species, we first explored their utilization of different carbon sources. The experimental results are as follows:
TABLE 3 utilization of different sugars by 25 common lactic acid bacteria in human intestinal tract
Note: "+" indicates available; "-" indicates unavailable; "/" indicates unknown.
According to the utilization of different sugars by various bacteria in human intestinal tract, arabinose was selected as a carbon source of a selection medium. In addition to Lactobacillus fermentum, 11 strains of bacteria can also utilize arabinose, Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus antri (Lactobacillus antri), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus mucosus (Lactobacillus mucosae), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus sakei (Lactobacillus sake), Leuconostoc citreum (leuconostrum), Pediococcus pentosaceus (Pediococcus), Lactobacillus fructici, Weissella mellifera (Weissella cibaria) and Weissella mesenteroides (Weissella paramesenteroides), respectively.
Example 4: antibiotic selection and optimization of media
This example carried out antibiotic susceptibility tests on 11 arabinose-utilizing strains (Lactobacillus fermentum, Lactobacillus antrum, Lactobacillus brevis, Lactobacillus mucilaginosus, Lactobacillus reuteri, Lactobacillus sake, Lactobacillus. frumenti, Leuconostoc citreum, Pediococcus pentosaceus, Weissella fusiformis and Weissella mesenterica) based on example 3, with specific reference to ISO 10932-. The experimental results are as follows.
TABLE 4 Minimum Inhibitory Concentrations (MIC) of different strains for different antibiotics
TABLE 5 Minimum Inhibitory Concentrations (MIC) of different strains for different antibiotics
| MIC(ug/mL) | Chloromycetin | Gentamicin | Vancomycin | Rifampicin | Ampicillin | Penicillin G |
| Lactobacillus fermentum | 2 | 128 | R | R | 0.125 | 0.125 |
| Lactobacillus antrum | 4 | 64 | R | / | / | / |
| Lactobacillus brevis | / | 128 | R | / | 0.125 | / |
| Lactobacillus mucilaginosus | 4 | 256 | R | / | / | / |
| Lactobacillus reuteri | 8 | 32 | R | / | / | / |
| Lactobacillus sake | / | / | R | / | / | / |
| Lactobacillus.frumenti | 4 | 128 | R | / | / | / |
| Leuconostoc citreum | 8 | 64 | R | / | / | / |
| Pediococcus pentosaceus | 4 | R | R | R | 2 | 1 |
| Weissella confusa | 4 | 32 | R | / | / | / |
| Weissella mesenteroides | 2 | 8 | R | / | / | / |
Note: s represents that the lowest concentration in the measurement range is still inhibited; r represents the highest concentration still endures in the measurement range; unknown representation
Wherein, the measurement concentration ranges of the antibiotics are as follows: clindamycin, 0.032ug/mL-16 ug/mL; ciprofloxacin, 0.25ug/mL-128 ug/mL; neomycin, 0.5ug/mL-256 ug/mL; streptomycin, 0.5ug/mL-256 ug/mL; tetracycline, 0.125ug/mL-64 ug/mL; erythromycin, 0.016ug/mL-8 ug/mL; chloramphenicol, 0.125ug/mL-64 ug/mL; gentamicin, 0.5ug/mL-256 ug/mL; vancomycin, 0.25ug/mL-128 ug/mL; rifampicin, 0.125ug/mL-64 ug/mL; ampicillin, 0.032ug/mL-16 ug/mL; penicillin G, 0.016ug/mL-8 ug/mL.
As can be seen from tables 4 and 5, the above-mentioned bacteria that can grow on the lactic acid bacteria medium can tolerate vancomycin at a high concentration, and thus vancomycin cannot be used as a screening condition thereof. The lactic acid bacteria showed significant differences in their resistance to streptomycin, while the Lactobacillus fermentum also tolerated the highest concentration of streptomycin, so that streptavidin was selected at a concentration of 256ug/mL to inhibit the growth of 7 species of Lactobacillus antratium, Lactobacillus brevis, Lactobacillus mucilaginosus, Lactobacillus sake, Lactobacillus frumenti, leuconostoc citreum and pediococcus pentosaceus. Although streptomycin inhibited the growth of these 7 lactic acid bacteria, it did not inhibit the growth of lactobacillus reuteri, weissella fusiformis and weissella mesenteroides, none of these 3 bacteria were as tolerant to gentamicin as seen from the gentamicin tolerance results as lactobacillus fermentum, so gentamicin was selected at a concentration of 64ug/mL to inhibit the growth of lactobacillus reuteri, weissella fusiformis and weissella mesenteroides. So far, a culture medium for screening lactobacillus fermentum in human intestinal tracts is formed by replacing a carbon source on the basis of a lactobacillus culture medium and adding streptomycin and gentamicin with specific concentrations.
Example 5: effect verification of LFMATA culture medium for separating and screening lactobacillus fermentum in human intestinal tract
The formula of the LFMATA solid culture medium comprises the following components (calculated by 1L): 10g of peptone, 10g of beef extract, 5g of yeast powder, 2.6g of dipotassium phosphate, 2g of sodium acetate, 2g of diammonium citrate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 20g of arabinose, and 20 multiplied by 10 grams of vancomycin-3g, streptomycin 0.256g, gentamicin 6.4X 10-2g, 0.5g of L-cysteine, 801 g of Tween and 1000mL of water.
The preparation steps of the culture medium are as follows:
(1) weighing 10g of peptone, 10g of beef extract, 5g of yeast powder, 2.6g of dipotassium phosphate, 2g of sodium acetate, 2g of diammonium hydrogen citrate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 20g of arabinose, 0.5g of L-cysteine and 20g of agar, putting the materials into a container, adding distilled water to fix the volume to 969mL, adjusting the pH to 5.0 +/-0.1 by using 4M HCL, and sterilizing at 115 ℃ for 20 minutes;
(2) dissolving 0.1g of vancomycin in 50mL of water to prepare a mother solution A with the concentration of 20 mg/mL; dissolving 0.128g of gentamicin in 20mL of water to prepare mother liquor B with the concentration of 6.4 mg/mL; 0.512g streptomycin is dissolved in 20mL water to prepare mother liquor C with the concentration of 25.6 mg/mL. Respectively filtering mother liquor A, B, C with a filter membrane for sterilization;
(3) and (3) cooling the sterilized mixture obtained in the step (1) to 55 ℃, adding 10mL of mother liquor A, 10mL of mother liquor B and 10mL of mother liquor C obtained in the step (2), and uniformly mixing to obtain the solid culture medium.
The application method of the culture medium in the separation of lactobacillus fermentum in the human intestinal tract sample comprises the following steps:
(1) enrichment culture: taking about 0.5g of intestinal contents, aseptically adding 5mL of culture medium liquid tube (agar is not added in the culture medium) for separating and screening lactobacillus fermentum in the human intestinal tract, and performing enrichment culture at 37 ℃ for 24h under anaerobic condition;
(2) gradient dilution: oscillating and mixing uniformly by using a liquid tube oscillator in the step (1), putting 1mL of suspension into 9mL of physiological saline, fully mixing uniformly to obtain 10-1 diluent, and repeating the dilution step to obtain 10-2、10-3、10-4、10-5、10-6Diluting the solution;
(3) coating culture: respectively suck 100uL of the above-mentioned 10-4、10-5、10-6Separating and screening the three gradient diluents in human intestinal tracts on a solid plate for a culture medium of lactobacillus fermentum, uniformly coating the solid plate with a coating rod, and culturing for 48 hours under an anaerobic condition at 37 ℃;
(4) primary purification culture: taking a dilution coating plate with the colony number within a range of 30-300, randomly selecting 10 single bacteria with different shapes and sizes from each sample, streaking the single bacteria on an MRS solid plate, and placing the single bacteria on an anaerobic condition at 37 ℃ for culturing for 48 hours.
(5) Secondary purification culture: and (4) inoculating a liquid MRS tube to a single colony on the streak plate in the step (4), and culturing for 20 hours at 37 ℃ under an anaerobic condition.
(6) And (3) strain preservation: mixing the two-stage purified culture bacterial suspension and sterilized 60% glycerol at a ratio of 1:1, packaging the mixture in a freezing tube, and freezing and storing in an ultra-low temperature refrigerator of-80 deg.C.
(7) And (3) strain identification: and (4) centrifuging the residual bacterial suspension obtained in the step (6) to obtain bacterial sludge, carrying out resuspension cleaning on the bacterial sludge by using sterile physiological saline, then using the bacterial sludge for 16SDNA PCR amplification, and carrying out detection and identification on an amplification sequence.
The results of screening 5 intestinal content samples (a1, a2, A3, a4, a5) on LFMATA medium as described in example 2 were as follows (10 strains were selected per sample):
TABLE 6 results of the screening of 5 intestinal contents described in example 2 on the medium according to the invention
Note: the strains in this table are all different strains that are present independently.
The results of the screening of the 5 intestinal content samples on the lamlab medium and the LFMATA medium were compared as follows (10 strains were selected for each sample):
table 7 summary of results of the 5 intestinal contents screening on two media as described in example 2
As is clear from tables 6 and 7, the 5 intestinal samples described in example 2 were co-screened on LFMATA medium to give 50 strains, of which the number of Lactobacillus fermentum reached 29, from 5 samples. Compared with the example 2, the occurrence frequency of the lactobacillus fermentum is improved from 4 to 29, and from 2 sample sources to 5 sample sources, and the screening efficiency is greatly improved. The culture medium has obvious advantages for screening lactobacillus fermentum with low abundance in human intestinal tracts.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (5)
1. A method for isolating lactobacillus fermentum in a sample of human intestinal tract, comprising the steps of:
(1) enrichment culture: taking 0.1-0.5 g of intestinal contents, adding the intestinal contents into a culture medium under an aseptic condition, and carrying out enrichment culture for 18-24 h under an anaerobic condition at the temperature of 35-37 ℃; the culture medium is as follows: each L comprises the following components by weight: 8-15 g of peptone, 8-15 g of beef extract, 3-10 g of yeast powder, 801-3 g of tween, 2-3 g of dipotassium hydrogen phosphate, 2-5 g of sodium acetate, 1-3 g of diammonium citrate, 0.4-0.6 g of magnesium sulfate heptahydrate, 0.15-0.25 g of manganese sulfate monohydrate, 8-20 g of arabinose, 15-25 multiplied by 10 of vancomycin-3 g, streptomycin 0.2-0.3 g, gentamicin 6-8 × 10-2 g, 0.4-0.8 g of L-cysteine;
(2) gradient dilution: oscillating and mixing uniformly by using a liquid tube oscillator in the step (1), putting 1mL of suspension into 9mL of physiological saline, and fully mixing uniformly to obtain 10-1Diluting the solution, repeating the above dilution steps to obtain 10-2、10-3、10-4、10-5、10-6Diluting the solution;
(3) coating culture: sucking 100 μ L of the above 10-4、10-5、10-6The three gradient dilutions are placed on a culture medium added with 10-20 g of agar, uniformly coated by a coating rod, and cultured for 48 hours under the anaerobic condition at 37 ℃;
(4) primary purification culture: taking a diluted coating plate with the colony number within a range of 30-300, randomly selecting 10 single bacteria with different shapes and sizes from each sample, placing the single bacteria on an MRS solid plate, streaking, and culturing for 48 hours under an anaerobic condition at 37 ℃;
(5) secondary purification culture: taking a single colony on the streak plate in the step (4) to be connected with a liquid MRS tube, and culturing for 20h under the anaerobic condition at 37 ℃;
(6) and (3) strain preservation: uniformly mixing the secondary purified culture bacterial suspension and sterilized 60% glycerol at a ratio of 1:1 to obtain a final glycerol concentration of 30%, subpackaging the mixed solution in a freezing tube, and freezing and storing in an ultra-low temperature refrigerator at-80 ℃;
(7) and (3) strain identification: and (4) centrifuging the residual bacterial suspension obtained in the step (6) to obtain bacterial sludge, carrying out resuspension cleaning on the bacterial sludge by using sterile physiological saline, then carrying out 16S DNA PCR amplification, and carrying out detection and identification on an amplification sequence.
2. The method according to claim 1, wherein the culture medium contains the following antibiotics in an amount of 1L: vancomycin 20X 10-3g; streptomycin 0.256 g; gentamicin 6.4X 10-2g。
3. The method of claim 2, wherein the method of preparing the culture medium comprises the steps of:
(1) weighing 10g of peptone, 10g of beef extract, 5g of yeast powder, 2.6g of dipotassium phosphate, 2g of sodium acetate, 2g of diammonium hydrogen citrate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 20g of arabinose, 0.5g of L-cysteine, adding distilled water to a constant volume of 969mL, adjusting the pH to 5.0 +/-0.1 by using HCL, and sterilizing at 110-115 ℃ for 15-20 minutes;
(2) preparing a vancomycin solution with the concentration of 2 mg/mL; preparing a gentamicin solution with the concentration of 6.4 mg/mL; preparing a streptomycin solution with the concentration of 25.6 mg/mL; sterilizing the solutions by filtering membranes respectively;
(3) and (3) cooling the sterilized mixture obtained in the step (1) to 50-55 ℃, adding 10mL of the solution obtained in the step (2) respectively, and uniformly mixing.
4. The method of claim 3, wherein the method of preparing the culture medium comprises the steps of: (1) weighing 10g of peptone, 10g of beef extract, 5g of yeast powder, 2.6g of dipotassium phosphate, 2g of sodium acetate, 2g of diammonium hydrogen citrate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 20g of arabinose, 0.5g of L-cysteine and 20g of agar, adding distilled water to a constant volume of 969mL, adjusting the pH to 5.0 +/-0.1 by using HCL, and sterilizing at 110-115 ℃ for 15-20 minutes;
(2) preparing a vancomycin solution with the concentration of 2 mg/mL; preparing a gentamicin solution with the concentration of 6.4 mg/mL; preparing a streptomycin solution with the concentration of 25.6 mg/mL; sterilizing the solutions by filtering membranes respectively;
(3) and (3) cooling the sterilized mixture obtained in the step (1) to 50-55 ℃, adding 10mL of the solution obtained in the step (2) respectively, and uniformly mixing.
5. Use of the method according to any one of claims 1 to 4 for the isolation and identification of Lactobacillus fermentum.
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