CN116287095A - Directional screening method for human intestinal Lacticaseibacillus paracasei strain - Google Patents
Directional screening method for human intestinal Lacticaseibacillus paracasei strain Download PDFInfo
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- CN116287095A CN116287095A CN202310345500.8A CN202310345500A CN116287095A CN 116287095 A CN116287095 A CN 116287095A CN 202310345500 A CN202310345500 A CN 202310345500A CN 116287095 A CN116287095 A CN 116287095A
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Abstract
The invention relates to a directional screening method of a human intestinal Lacticaseibacillus paracasei strain, which comprises the following steps: step 1, preparing directional solid and liquid culture mediums; step 2, weighing the feces, and culturing by using the culture medium in the step 1; and step 3, screening the bacterial liquid obtained in the step 2 after enrichment by using a Lacticaseibacillus paracasei bacterial strain to obtain the Lacticaseibacillus paracasei bacterial strain. The invention designs and develops the directional screening method by aiming at the growth characteristics of intestinal flora, the physiological and biochemical properties of Lacticaseibacillus paracasei and the like, so that the screening efficiency and quality of the strain are higher.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a directional screening method of a human intestinal probiotics Lacticaseibaci llus paracasei strain.
Background
The normal flora of human body has more than 1000 species, the weight of normal flora is about 1.5kg, the number of normal flora is 100 trillion, is 10 times of human body cells, is widely distributed on the skin, gastrointestinal tract, oral cavity, respiratory tract, genitourinary tract and other parts of human body, participates in the whole processes of growth, development, digestion, absorption, nutrition, immunity, biological antagonism and the like of human body, and has profound influence on the function of a regulatory host. The probiotics can be planted in the intestinal canal to fight against harmful bacteria, maintain the balance of intestinal flora, and regulate the immune function of the organism by enhancing the barrier function of intestinal mucosa, inhibiting the growth and adhesion of pathogenic bacteria, regulating and controlling the activity of immune cells, promoting the production of immune factors and the like. And intestinal probiotics are some active microorganisms beneficial to our human body. As the name suggests, probiotics are microorganisms that can exert beneficial effects, are active, and are beneficial. It can truly play a certain role in intestinal health in the field and can maintain the ecological balance of intestinal tracts. Some common probiotics are lactobacillus, yeast, bifidobacteria, clostridium butyricum, etc. The probiotics sold on the market are also various and may be as many as tens of. It co-grows with some other bacteria of the intestine but it plays a major role, for example probiotics are in a certain proportion for escherichia coli, and may inhibit the growth of escherichia coli, thus forming a balanced healthy development for the intestine.
Cheese bacillus paracasei (Lacticaseibacillus paracasei) is one of the intestinal probiotics and plays an important role in intestinal health. Lacticaseibacillus paracasei is a facultative anaerobic, gram-positive, heterofermentative lactic acid bacterium, widely found in the human mouth, intestinal tract, fermented dairy products and plant raw material fermented products (kimchi and feed). The lactobacillus casei has the characteristic of lactobacillus casei, can decompose protein to generate small molecular substances such as small molecular peptide and the like through the action of extracellular enzyme, and plays an important role in reducing blood pressure, resisting tumor, resisting oxidization, reducing cholesterol and the like. Meanwhile, the cheese bacillus paracasei has no toxic or side effect and drug resistance, and has the functions of regulating intestinal balance, enhancing non-specific immunity, promoting absorption and the like. For example, patent publication No. (CN 110959865A) discloses a novel use of Lactobacillus paracasei K56 for regulating the balance of gastrointestinal flora, and mentions that Lactobacillus paracasei K56 has the ability to significantly promote the growth of bifidobacteria and lactic acid bacteria in the intestinal tract, is capable of inhibiting Vibrio desulphus and/or Enterobacter in the intestinal tract, and is capable of inhibiting helicobacter pylori and/or Escherichia Shigella. Patent publication No. (CN 114504599A) discloses a new application of Lactobacillus paracasei ET-22 in resisting aging and improving innate immunity, and mentions that the Lactobacillus paracasei ET-22 has the effects of resisting aging, improving innate immunity of organisms, improving the resistance of the organisms to staphylococcus aureus infection and the like.
However, the Lacticaseibacillus paracasei strain isolated to human intestinal tracts is rarely reported, and few methods for screening human intestinal probiotics are available, particularly, a method for directionally screening human intestinal tracts Lacticaseibacillus paracasei is not reported.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention makes up the problems that the resources of the human intestinal probiotics Lacticaseibacillus paracasei strain are less and the accurate directional screening cannot be achieved in the common plate screening process, and the defects of the human intestinal probiotics Lacticaseibacillus paracasei are difficult to obtain.
The invention further aims to provide a directional screening method of the human intestinal Lacticaseibacillus paracasei strain, which can realize directional and accurate screening of the strain of the human intestinal probiotics Lacticaseibacillus paracasei strain and provide abundant backup strains for development of probiotics.
In order to achieve the purpose, the invention adopts the following technical scheme:
a directional screening method of a human intestinal Lacticaseibacillus paracasei strain, comprising the following steps:
step 1, preparing directional solid and liquid culture mediums;
wherein, the formula of the culture medium is expressed according to the concentration and mainly comprises the following components:
solution 1:
1) Peptone 10.00-15.50g/L
2) Glucose 3.50-6.50g/L
3) 8.50-10.70g/L yeast powder
4) Sodium chloride 0.04-0.90g/L
5) Cysteine hydrochloride 0.30-0.80g/L
6) 0.004-0.009g/L of calcium chloride
7) Magnesium sulfate 0.005-0.100g/L
8) Dipotassium hydrogen phosphate 0.02-0.08g/L
9) Potassium dihydrogen phosphate 0.04-0.09g/L
10 Sodium bicarbonate 0.25-0.75g/L
11 Tween 80.80-1.80 ml/L
12 0.06-0.15mg/L of resazurin
Solution 2:
vitamin K1 solution 0.35-1.25g/L
Solution 3:
0.02-0.08g/L of chlorhematin solution
Solution 4:
antibiotic solution
Vancomycin 2.0-7.0mg/ml
Gentamicin 5.0-11.0mg/ml
Kanamycin 6.0-14.0mg/ml
pH (25 ℃ C. Measurement) of 5.10-6.60
Solid culture medium agar 17.00-25.00g/L
The culture mediums are respectively configured for standby;
in particular, the method comprises the steps of,
1.1, accurately weighing each component in the solution 1 according to the concentration, putting the components into a 1.5L glass beaker, adding 200ml of ultrapure water for dissolution, and finally, using a 1000ml volumetric flask to fix the volume to 1000ml to obtain the solution 1;
1.2, preparing 100ml of solution 2 for later use; firstly, weighing about 11 g of vitamin K by using an analytical balance, adding absolute ethyl alcohol to enable the concentration to reach 0.35-1.25g/L, then filtering and sterilizing by using a 0.22um filter membrane to obtain a solution 2, and placing the solution 2 in a refrigerator at 4 ℃ for later use;
1.3, preparing 100ml of solution 3 for later use; firstly, weighing about 0.5g of hemin by using an analytical balance, dissolving in 1ml of 1mol/L sodium hydroxide solution, adding distilled water to 100ml to enable the concentration to reach 0.02-0.08g/L, filtering and sterilizing by using a 0.22um filter membrane to obtain a solution 3, and placing in a refrigerator at the temperature of 4 ℃ for later use;
1.4, preparing 10ml of solution 4 for later use; accurately weighing each component in the solution 4 by using an analytical balance, dissolving in 10ml of distilled water, then filtering and sterilizing by using a 0.22um filter membrane to obtain the solution 4, and placing in a refrigerator at 4 ℃ for later use;
1.5, adjusting the pH value of the solution 1 prepared in the step 1.1 to 5.10-6.60 (measured at 25 ℃) by using hydrochloric acid and sodium hydroxide solution;
further, if the liquid medium is prepared without adding agar, if the solid medium is prepared, 17.00-25.00g/L of agar is added.
1.6, autoclaving the solution obtained in step 1.5 at 121 ℃ for 20 minutes, and adding about 2.5ml of the solution, about 3.5 ml of the solution and about 0.2ml of the solution 4 when the temperature is reduced to 50-55 ℃ to obtain the directional screening culture medium.
Step 2, weighing the feces, and culturing by using the culture medium in the step 1;
specifically, 0.1g of fresh healthy children's feces is dissolved in 1ml of sterile physiological saline; then 100ul of fecal solution is inoculated into 10ml of prepared liquid directional screening culture medium by using a pipette, and cultured in an anaerobic incubator at 37 ℃ for 72-96 hours (the gases in the anaerobic incubator are divided into 2 types- (high purity nitrogen 99.999 percent, mixed gas-nitrogen: hydrogen: carbon dioxide=85%: 5%: 10%).
Step 3, screening Lacticaseibacillus paracasei strains, namely flat plate screening and high-throughput screening of intestinal strains, of the enriched healthy children fecal bacterial liquid in 2 modes; thus obtaining Lacticaseibacillus paracasei strain.
The method further comprises the following steps:
step 4, identifying strains: extracting the bacterial liquid DNA taken out in the step 3, amplifying a 16S gene sequence by PCR, detecting an amplified strip by electrophoresis, carrying out sample feeding and sequencing, checking the quality of a sequenced peak diagram by utilizing software, comparing the gene sequences by NCBI websites, and summarizing and sorting the comparison results;
and 5, strain preservation: screening the strain numbers to be preserved according to the strain identification results obtained by comparing the step 4, and preserving the bacterial solutions cultured in the test tube or the 96-well culture plate in the step 3 according to the corresponding numbers; meanwhile, in order to ensure no environmental pollution of the bacterial liquid, 1ul of disposable inoculating loop is used for dipping part of the preserved bacterial liquid to carry out plate 3 division line for verification.
The specific method for plate screening and high-throughput screening of intestinal strains is as follows:
plate screening method:
gradient dilution of the enriched healthy children fecal bacteria liquid is coated into a directional screening Lacticaseibacillus paracasei culture medium, and the culture medium is operated in an anaerobic workstation, the gas of an anaerobic tank is divided into 2 species (-99.999% of high-purity nitrogen, and the gas of mixed gas-nitrogen: hydrogen: carbon dioxide=85%: 5%: 10%), after 2-4d of culture, single colony is picked up, 3 rounds of streak purification is carried out on a corresponding directional screening Lacticaseibacillus paracasei agar plate, the purified single colony is inoculated into a directional screening Lacticaseibacillus paracaseis liquid culture medium, and the culture medium is subjected to shaking anaerobic culture for 24-48h at 37 ℃ and 200 rpm;
intestinal strain high throughput screening method:
the total bacterial amount and the viable bacteria rate in the enriched healthy children faeces sample diluent are measured by utilizing a flow cytometry viable bacteria detection technology, the total viable bacteria amount is calculated, limiting dilution is carried out according to the total viable bacteria amount, the diluted bacterial culture solution is transferred into a sterilized 96-hole deep hole plate, sealing is carried out by using a sealing film, the bacteria culture solution is placed in an anaerobic box at 37 ℃ for culture (the gas of the anaerobic box is divided into 2 species- (99.999% of high-purity nitrogen gas, the mixed gas-nitrogen gas: hydrogen gas: carbon dioxide=85%: 5%: 10%), the plate with proper growth amount is selected for bacterial strain selection after 2-4 days, the bacteria culture solution is transferred into the sterilized 96-hole deep hole plate for culture, 50-100ul bacterial solution is transferred into each hole for bacterial DNA extraction, and a proper amount of liquid culture medium is added into the 96-hole deep hole plate for continuous culture of the residual bacterial solution.
Compared with the prior art, the invention has the beneficial effects that:
1. the screening method can accurately screen and obtain the human intestinal tract strain Lacticaseibacillus paracasei, and 97.0% or more of all screened strains are Lacticaseibacillus paracasei strains.
2. The screening method has long culture time and simple operation in the strain enrichment and screening process, can be simultaneously carried out in multiple samples and multiple batches, and improves the screening efficiency of the intestinal probiotics Lacticaseibacillus paracasei.
3. The screening method is not suitable for pollution due to the addition of the compound antibiotics in the preparation process of the culture medium, and the quality guarantee period of the liquid and solid culture medium is longer (1 month).
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the statistics of the isolated strain in the plate screening Lacticaseibacillus paracasei method according to example 1 of the present invention.
FIG. 2 shows the strain statistics obtained by screening in the high throughput screening of intestinal strains according to example 1 of the present invention.
Fig. 3 is the strain statistics obtained by screening in classical MRS medium in comparative example.
FIG. 4 is a graph showing statistics of percentage of Lacticaseibacillus paracasei obtained by screening by different screening methods in the examples of the present invention to all strains.
FIG. 5 shows the result of Lacticaseibacillus paracasei in NCBI.
Detailed Description
Embodiments of the present invention will now be described in detail with reference to the following examples, which are only illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used are not manufacturer specific and are commercially available as conventional products.
The invention discloses a directional screening method of a human intestinal probiotics Lacticaseibacillus paracasei strain, which comprises the following steps:
1. preparing a solid and liquid culture medium for directional screening Lacticaseibacillus paracasei, and specifically preparing a flow according to a Lacticaseibacillus paracasei culture medium for directional screening, wherein the flow is as follows;
1.1, accurately weighing each component in the solution 1 according to the concentration, putting the components into a 1.5L glass beaker, adding 200ml of ultrapure water for dissolution, and finally, using a 1000ml volumetric flask to fix the volume to 1000ml to obtain the solution 1.
1.2, preparing 100ml of solution 2 for later use; firstly, weighing about 11 g of vitamin K by using an analytical balance, adding absolute ethyl alcohol to enable the concentration to reach 0.35-1.25g/L, then filtering and sterilizing by using a 0.22um filter membrane to obtain a solution 2, and placing the solution in a refrigerator at 4 ℃ for later use.
1.3, preparing 100ml of solution 3 for later use; firstly, weighing about 0.5g of hemin by using an analytical balance, dissolving in 1ml of 1mol/L sodium hydroxide solution, adding distilled water to 100ml, enabling the concentration to reach 0.02-0.08g/L, then filtering and sterilizing by using a 0.22um filter membrane to obtain a solution 3, and placing in a refrigerator at the temperature of 4 ℃ for standby.
1.4, preparing 10ml of solution 4 for later use; each component in the solution 4 is accurately weighed by an analytical balance, dissolved in 10ml of distilled water, filtered and sterilized by a 0.22um filter membrane to obtain the solution 4, and placed in a refrigerator at 4 ℃ for standby.
1.5, adjusting the pH value of the solution 1 prepared in the step 1.1 to 5.10-6.60 (measured at 25 ℃) by using hydrochloric acid and sodium hydroxide solution; if the liquid culture medium is prepared without adding agar, if the solid culture medium is prepared, 17.00-25.00g/L of agar is added.
1.6, autoclaving the solution obtained in step 1.5 at 121 ℃ for 20 minutes, and adding about 2.5ml of the solution, about 3.5 ml of the solution and about 0.2ml of the solution 4 when the temperature is reduced to 50-55 ℃ to obtain the directional screening culture medium.
Wherein, the formula of the culture medium is expressed according to the concentration and mainly comprises the following components:
solution 1:
1) Peptone 10.00-15.50g/L
2) Glucose 3.50-6.50g/L
3) 8.50-10.70g/L yeast powder
4) Sodium chloride 0.04-0.90g/L
5) Cysteine hydrochloride 0.30-0.80g/L
6) 0.004-0.009g/L of calcium chloride
7) Magnesium sulfate 0.005-0.100g/L
8) Dipotassium hydrogen phosphate 0.02-0.08g/L
9) Potassium dihydrogen phosphate 0.04-0.09g/L
10 Sodium bicarbonate 0.25-0.75g/L
11 Tween 80.80-1.80 ml/L
12 0.06-0.15mg/L of resazurin
Solution 2:
vitamin K1 solution 0.35-1.25g/L
Solution 3:
0.02-0.08g/L of chlorhematin solution
Solution 4:
antibiotic solution
Vancomycin 2.0-7.0mg/ml
Gentamicin 5.0-11.0mg/ml
Kanamycin 6.0-14.0mg/ml
pH (25 ℃ C. Measurement) of 5.10-6.60
Solid culture medium agar 17.00-25.00g/L
Classical intestinal strain screening MRS solid and liquid media MRS media formulation—mrs media formulation: 10g/L of casein enzyme digest; 10g/L of beef extract powder; 4g/L of tri-ammonium citrate; 5g/L sodium acetate; 0.2g/L of magnesium sulfate heptahydrate; manganese sulfate tetrahydrate 0.05g/L; 2g/L of dipotassium hydrogen phosphate; glucose 20g/L; tween-80.08 g/L; final pH 5.7+ -0.2, sterilizing at 115deg.C for 25 min)
Classical MRS medium is the most effective medium for screening Lacticaseibacillus paracasei probiotics.
2. Lacticaseibacillus paracasei classical MRS screening of probiotics- -fresh faeces from the same healthy child were sampled at 1g:1000 μl of physiological saline is added, and mixed by shaking. The method comprises the steps of diluting with sterile normal saline 10 times, inoculating into a serum culture bottle for enrichment culture of human intestinal flora, absorbing enrichment culture solution in 3 and 6 days, respectively coating the culture solution into MRS agar plates by using sterile normal saline gradient dilution, culturing for 24-72 hours in an anaerobic incubator at 37 ℃, picking single colony, carrying out 3 rounds of streak purification on the corresponding agar plates, inoculating the purified single colony into MRS liquid culture medium, and culturing for 24-48 hours in a shaking table at 200rpm at 37 ℃.
3. Directional screening of human intestinal probiotics Lacticaseibacillus paracasei:
plate screening: the culture solution enriched by the directional screening liquid culture medium of Lacticaseibacillus paracasei is diluted and coated on a directional screening Lacticaseibacillus paracasei solid culture medium by using sterile physiological saline in a gradient way, after 7-14d of culture in an anaerobic incubator at 37 ℃, single colonies are picked up and subjected to 3 rounds of streak purification on a corresponding directional screening Lacticaseibacillus paracasei agar plate, and the purified single colonies are inoculated into a directional screening Lacticaseibacillus paracasei liquid culture medium and subjected to shaking anaerobic culture for 48-72h at 200rpm at 37 ℃. Extracting genome DNA from bacterial liquid cultured in a shaking table by using a bacterial genome DNA rapid extraction Kit (T5 Direct PCR Kit), and taking the extracted genome DNA as a template.
High throughput screening of intestinal strains: the total bacterial amount and the bacterial rate in the dilution liquid of the healthy children faeces sample enriched by the directionally screened liquid culture medium of Lacticaseibacillus paracasei are measured by utilizing a flow cytometry living bacterial detection technology, the total living bacterial amount is calculated, limiting dilution is carried out according to the total living bacterial amount, the diluted bacterial culture solution is transferred into a sterilized 96-hole deep-hole plate, sealing is carried out by using a sealing film, the bacteria culture solution is placed in an anaerobic box for culture at 37 ℃ (the gas of the anaerobic box is divided into 2 species- (high-purity nitrogen 99.999 percent), the mixed gas of nitrogen and hydrogen is 5 percent of 10 percent of 5 percent) and after 2-4 days, the plate with proper growth amount is selected for bacterial strain selection, the bacteria culture solution is transferred into the sterilized 96-hole deep-hole plate for culturing, and 50-100ul bacterial solution is transferred into the 96-hole deep-hole plate for bacterial DNA extraction, and a proper amount of liquid culture medium is added for continuous culture of the rest bacterial solution.
4. Extracting genome DNA from bacterial liquid cultured in a shaking table by using a bacterial genome DNA rapid extraction Kit (T5 Direct PCR Kit), and taking the extracted genome DNA as a template.
The PCR reaction system was prepared to amplify the 16S DNA sequence of the strain, 2X Taq Plus Master Mix (Optimum, prinsepia, china) 10.6. Mu.l, the upstream primer 27F (5'AGAGTTTGATCCTGGCTCAG 3') 0.2. Mu.l, the downstream primer 1492R (5'TACGGCTACCTTGTTACGACTT 3') 0.2. Mu.l, the strain DNA template 2. Mu.l, and water to make up 30. Mu.l. The mixture was put into a PCR apparatus to conduct a reaction, and was subjected to a pre-denaturation at 95℃for 3min and 28 cycles (denaturation at 95℃for 15s, renaturation at 60℃for 15s and extension at 72℃for 30 s), followed by a thorough extension at 72℃for 5min.
2 μl of the PCR reaction product was taken for electrophoresis, and whether the band was in accordance with the expected size was detected. The PCR products were purified and then sequenced for one generation, and the sequencing results were placed in the 16S ribosomal RNA sequences (Bacteria and Archaea) database of the National Center for Biotechnology Information (NCBI) website for sequence alignment.
5. And (5) preserving the new strains and the potential probiotics obtained by comparison.
Intestinal strain isolation culture is carried out on 2 child faeces samples by utilizing an intestinal strain Lacticaseibacillus paracasei directional screening culture medium (plate screening and intestinal strain high-throughput screening) and classical MRS, and 186 strains are obtained by co-isolation culture. Wherein Lacticaseibacillus paracasei directional screening method (plate screening) is used for separating and obtaining 5 60 intestinal strains as shown in figure 1; lacticaseibacillus paracasei directional screening method (high throughput screening of intestinal strains) to obtain 5 kinds of 60 intestinal strains (figure 1); the classical GAM medium screening method is used for separating and obtaining 10 67 intestinal strains as shown in figure 3, which contain intestinal strains reported by related literature data, such as: enterococcus gallinarum, enterococcus lactis, bifidobacterium pseudocatenulatum, bifidobacterium faecale, lacticaseibacillus paracasei, streptococcus salivarius, brachybacterium rhamnosum, lacticaseibacillus paracasei subsp.
Carrying out data analysis on strains obtained by screening 2 fecal samples by 2 different screening methods, wherein 60 strains 53-Lacticaseibacillus paracasei, 2-Bifidobacterium pseudocatenulatum, 2-Enterococcus faecalis, 2-Bifidobacterium longum and 1-Parabacteroides distasonis obtained by an intestinal strain Lacticaseibacillus paracasei directional screening method (plate screening) achieve the effect of strain directional screening, and the percentage content of Lacticaseibacillus paracasei strains is 88.33%; the 79 strain 77 obtained by the directional screening method (high-throughput screening of intestinal strains) of the intestinal strain Lacticaseibacillus paracasei is Lacticaseibacillus paracasei, the 2 strain is Lacticaseibacillus paracasei subsp.tolarans, the directional screening effect of the strain is achieved, and the percentage content of the Lacticaseibacillus paracasei strain is 97.47%; the Lacticaseibacillus paracasei obtained from classical GAM screening medium represents 8.96% of all screened strains, and the ratios of the targeted strain Lacticaseibacillus paracasei obtained by the directional screening method of the present invention are 88.33%, 97.47% and 8.96%, respectively, as shown in fig. 4.
The results of sequence alignment of strain Lacticaseibacillus paracasei obtained by directed screening of intestinal strain Lacticaseibacillus paracasei in database 16S ribosomal RNA sequences (Bacteria and Archaea) of the National Center for Biotechnology Information (NCBI) website are shown in FIG. 5. The strain is shown to be a strain (Lacticaseibacillus paracasei), the 16S rDNA sequence of the strain is shown as SEQ ID No.1, and the colony is milky white, opaque, round convex, smooth in surface and neat in edge.
SEQ ID No.1：
GCAAGTCGACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAAC
ATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTA
AGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAG
AACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATG
GACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGA
TGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGAC
ACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGG
ACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGT
CGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTC
GGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAG
CCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAA
AGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAAC
CGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAG
TGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACC
AGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAG
CATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACG
ATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACG
CATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAG
GAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAA
GCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAG
ATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGT
CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC
TTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGT
GACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTAT
GACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGA
CCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGG
CTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGC
ACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC
ATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCTAA。
In conclusion, the method for directionally screening the human intestinal probiotics Lacticaseibacillus paracasei can accurately screen and obtain the probiotics Lacticaseibacillus paracasei which can inhibit vibrio and/or enterobacter in intestinal tracts, inhibit helicobacter pylori and/or escherichia-shigella and have the potential of reducing blood pressure, resisting tumors, resisting oxidization and reducing cholesterol. One bright spot in this screening method is the easier availability of human intestinal probiotics Lacticaseibacillus paracasei. The number of the probiotics strains in the strain resource library of Feng Furen-source intestinal probiotics achieves the aim of accurately screening the human-source intestinal probiotics Lacticaseibacillus paracasei, saves a large amount of manpower and material resources, and improves the screening efficiency. Provides rich probiotic resources for the food and pharmaceutical preparation of the probiotics Lacticaseibacillus paracasei.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (6)
1. A directional screening method of a human intestinal tract Lacticaseibacillus paracasei strain, which is characterized by comprising the following steps:
step 1, preparing directional solid and liquid culture mediums;
wherein, the formula of the culture medium is expressed according to the concentration and mainly comprises the following components:
solution 1:
solution 2:
vitamin K1 solution 0.35-1.25g/L
Solution 3:
0.02-0.08g/L of chlorhematin solution
Solution 4:
the culture mediums are respectively configured for standby;
step 2, weighing the feces, and culturing by using the culture medium in the step 1;
and step 3, screening the bacterial liquid obtained in the step 2 after enrichment by using a Lacticaseibacillus paracasei bacterial strain to obtain the Lacticaseibacillus paracasei bacterial strain.
2. The method for directional screening of a human intestinal Lacticaseibacillus paracasei strain according to claim 1, wherein in step 1, the culture medium is prepared by the steps of;
1.1, accurately weighing each component in the solution 1 according to the concentration, putting the components into a 1.5L glass beaker, adding 200ml of ultrapure water for dissolution, and finally, using a 1000ml volumetric flask to fix the volume to 1000ml to obtain the solution 1;
1.2, preparing 100ml of solution 2 for later use; firstly, weighing about 11 g of vitamin K by using an analytical balance, adding absolute ethyl alcohol to enable the concentration to reach 0.35-1.25g/L, then filtering and sterilizing by using a 0.22um filter membrane to obtain a solution 2, and placing the solution 2 in a refrigerator at 4 ℃ for later use;
1.3, preparing 100ml of solution 3 for later use; firstly, weighing about 0.5g of hemin by using an analytical balance, dissolving in 1ml of 1mol/L sodium hydroxide solution, adding distilled water to 100ml to enable the concentration to reach 0.02-0.08g/L, filtering and sterilizing by using a 0.22um filter membrane to obtain a solution 3, and placing in a refrigerator at the temperature of 4 ℃ for later use;
1.4, preparing 10ml of solution 4 for later use; accurately weighing each component in the solution 4 by using an analytical balance, dissolving in 10ml of distilled water, then filtering and sterilizing by using a 0.22um filter membrane to obtain the solution 4, and placing in a refrigerator at 4 ℃ for later use;
1.5, adjusting the pH value of the solution 1 prepared in the step 1.1 to 5.10-6.60 (measured at 25 ℃) by using hydrochloric acid and sodium hydroxide solution;
1.6, autoclaving the solution obtained in step 1.5 at 121 ℃ for 20 minutes, and adding about 2.5ml of the solution, about 3.5 ml of the solution and about 0.2ml of the solution 4 when the temperature is reduced to 50-55 ℃ to obtain the directional screening culture medium.
3. The method for directional screening of human intestinal Lacticaseibacillus paracasei strain according to claim 1, wherein in step 1.5, agar is not required to be added if a liquid medium is prepared, and 17.00-25.00g/L agar is required to be added if a solid medium is prepared.
4. The directional screening method of human intestinal Lacticaseibacillus paracasei strain according to claim 1, wherein in step 2, 0.1g of fresh healthy children's feces is dissolved in 1ml of sterile physiological saline; then 100ul of fecal solution is inoculated into 10ml of prepared liquid directional screening culture medium by using a pipette, and cultured in an anaerobic incubator at 37 ℃ for 72-96 hours.
5. The directional screening method of human intestinal Lacticaseibacillus paracasei strain according to claim 1, wherein in step 3, the screening comprises plate screening and high throughput screening of intestinal strain, and the specific method of plate screening and high throughput screening of intestinal strain is as follows:
plate screening method:
gradient dilution of the enriched healthy children fecal bacteria liquid is coated into a directional screening Lacticaseibacillus paracasei culture medium, the operation is carried out in an anaerobic workstation, after 2-4d of culture, single colony is selected to carry out 3 rounds of streak purification on a corresponding directional screening Lacticaseibacillus paracasei agar plate, the purified single colony is inoculated into a directional screening Lacticaseibacillus paracaseis liquid culture medium, and the culture is carried out for 24-48h by shaking table anaerobic culture at 37 ℃ and 200 rpm;
intestinal strain high throughput screening method:
the total bacterial amount and the viable bacteria rate in the enriched healthy children faeces sample diluent are measured by utilizing a flow cytometry viable bacteria detection technology, the total viable bacteria amount is calculated, limiting dilution is carried out according to the total viable bacteria amount, the diluted bacterial culture solution is transferred into a sterilized 96-hole deep-hole plate, sealing is carried out by using a sealing film, the sealing film is placed in an anaerobic box at 37 ℃ for culturing for 2-4 days, a plate with proper growth quantity is selected for strain selection, the bacterial strain selection is transferred into a sterile 96-hole deep-hole plate for culturing, 50-100ul bacterial solution is transferred into each hole for bacterial strain DNA extraction, and a proper amount of liquid culture medium is added into the 96-hole deep-hole plate for continuous culturing of the residual bacterial solution.
6. The directional screening method of a human intestinal Lacticaseibacillus paracasei strain according to claim 1, further comprising the steps of:
step 4, identifying strains: extracting the bacterial liquid DNA taken out in the step 3, amplifying a 16S gene sequence by PCR, detecting an amplified strip by electrophoresis, carrying out sample feeding and sequencing, checking the quality of a sequenced peak diagram by utilizing software, comparing the gene sequences by NCBI websites, and summarizing and sorting the comparison results;
and 5, strain preservation: screening the strain numbers to be preserved according to the strain identification results obtained by comparing the step 4, and preserving the bacterial solutions cultured in the test tube or the 96-well culture plate in the step 3 according to the corresponding numbers;
meanwhile, in order to ensure no environmental pollution of the bacterial liquid, 1ul of disposable inoculating loop is used for dipping part of the preserved bacterial liquid to carry out plate 3 division line for verification.
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