A method for the directed screening of human gut lacticaseibacillus paracasei strains

Abstract

The application discloses a directional screening method of a human intestinal Lacticaseibacillus paracasei strain, and comprises the following steps: step 1, preparation of directional solid and liquid culture media; step 2, weighing of feces and culture by using the culture media in step 1; and step 3, screening of the Lacticaseibacillus paracasei strain from the bacterial liquid obtained after enrichment in step 2, so as to obtain the Lacticaseibacillus paracasei strain. The directional screening method is developed according to the growth characteristics of intestinal flora and the physiological and biochemical properties of the Lacticaseibacillus paracasei, so that the efficiency and quality of the strain screening are higher.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a method for targeted screening of human intestinal probiotic strains of Lacticaseibacillus paracasei. Background Technology

[0002] The human body has over 1,000 species of normal flora, weighing approximately 1.5 kg and numbering in the hundreds of trillions—ten times the number of human cells. Widely distributed in the skin, gastrointestinal tract, oral cavity, respiratory tract, and urinary and reproductive tracts, it participates in the entire process of human growth, development, digestion, absorption, nutrition, immunity, and biological antagonism, profoundly influencing host function. Probiotics can colonize the gut, fighting harmful bacteria and maintaining gut microbiota balance. They can regulate the body's immune function by enhancing the intestinal mucosal barrier function, inhibiting the growth and adhesion of pathogenic bacteria, regulating immune cell activity, and promoting the production of immune factors. Gut probiotics are live microorganisms beneficial to the human body. As the name suggests, probiotics are live, beneficial microorganisms that can exert beneficial effects. They can genuinely contribute to gut health and maintain the ecological balance of the gut. Some common probiotics include lactobacillus, yeast, bifidobacteria, and clostridium butyricum. There are various types of probiotics available on the market, sometimes as many as dozens. They grow alongside other bacteria in the gut, but play a key role. For example, probiotics, when grown in a specific ratio to E. coli, may inhibit the growth of E. coli, thus promoting a balanced and healthy gut development.

[0003] Lactobacillus paracasei is a type of probiotic that plays an important role in gut health. It is a facultative anaerobic, Gram-positive, heterotrophic fermenting lactic acid bacterium widely found in the human mouth, intestines, fermented dairy products, and fermented plant materials (kimchi and animal feed). It possesses characteristics of the Lactobacillus genus, breaking down proteins into small molecules such as peptides through the action of extracellular enzymes. These peptides play a significant role in lowering blood pressure, anti-tumor activity, anti-oxidation, and cholesterol reduction. Furthermore, Lactobacillus paracasei has no toxic side effects, does not develop drug resistance, and has functions such as regulating intestinal balance, enhancing non-specific immunity, and promoting absorption. For example, patent publication number (CN110959865A) discloses a new application of *Lactobacillus paracasei* K56, which can regulate the balance of gastrointestinal flora. It mentions that *Lactobacillus paracasei* K56 has a significant ability to promote the growth of Bifidobacteria and Lactobacillus in the intestine, and can inhibit *Desulfovibrio* and / or *Enterobacter* spp. in the intestine, as well as inhibit *Helicobacter pylori* and / or *Escherichia coli*-Shigella spp. Patent publication number (CN114504599A) discloses a new application of *Lactobacillus paracasei* ET-22 in anti-aging and enhancing innate immunity, mentioning that *Lactobacillus paracasei* ET-22 has effects such as anti-aging, enhancing innate immunity, and improving the body's resistance to *Staphylococcus aureus* infection.

[0004] However, there are few reports of Lacticaseibacillus paracasei strains isolated into the human gut, and there are few methods for screening human gut probiotics, especially methods for targeted screening of human gut Lacticaseibacillus paracasei. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, and to overcome the limitations of limited human-derived intestinal probiotic strains of *Lacticaseibacillus paracasei* and the inability to achieve precise targeted screening during conventional plate screening, this invention provides a targeted screening method for human-derived intestinal probiotic *Lacticaseibacillus paracasei* strains. This method, developed by analyzing the growth characteristics of intestinal flora and the physiological and biochemical properties of *Lacticaseibacillus paracasei*, improves the efficiency and quality of strain screening, achieving the goal of precisely screening human-derived intestinal probiotics.

[0006] Another objective of this invention is to provide a method for targeted screening of human intestinal Lacticaseibacillus paracasei strains. This method enables targeted and precise screening of human intestinal probiotic Lacticaseibacillus paracasei strains, providing a rich pool of reserve strains for probiotic development.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] A method for targeted screening of human intestinal Lacticaseibacillus paracasei strains includes the following steps:

[0009] Step 1: Preparation of directional solid and liquid culture media;

[0010] The culture medium formulation used is expressed in terms of concentration and mainly includes the following components:

[0011] Solution 1:

[0012] 1) Peptone 10.00-15.50g / L

[0013] 2) Glucose 3.50-6.50 g / L

[0014] 3) Yeast powder 8.50-10.70g / L

[0015] 4) Sodium chloride 0.04-0.90 g / L

[0016] 5) Cysteine ​​hydrochloride 0.30-0.80 g / L

[0017] 6) Calcium chloride 0.004-0.009 g / L

[0018] 7) Magnesium sulfate 0.005-0.100 g / L

[0019] 8) Dipotassium hydrogen phosphate 0.02-0.08 g / L

[0020] 9) Potassium dihydrogen phosphate 0.04-0.09 g / L

[0021] 10) Sodium bicarbonate 0.25-0.75 g / L

[0022] 11) Tween 80 0.80-1.80 ml / L

[0023] 12) Rhazora 0.06-0.15 mg / L

[0024] Solution 2:

[0025] Vitamin K1 solution 0.35-1.25 g / L

[0026] Solution 3:

[0027] Heme chloride solution 0.02-0.08 g / L

[0028] Solution 4:

[0029] antibiotic solution

[0030] Vancomycin 2.0-7.0 mg / ml

[0031] Gentamicin 5.0-11.0 mg / ml

[0032] Kanamycin 6.0-14.0 mg / ml

[0033] pH value (measured at 25℃): 5.10-6.60

[0034] Solid culture medium (agar): 17.00-25.00 g / L

[0035] Prepare the above culture media separately and set aside for later use;

[0036] Specifically,

[0037] 1.1. Weigh each component of solution 1 according to its concentration and place it in a 1.5L glass beaker. Add 200ml of ultrapure water and finally dilute to 1000ml using a 1000ml volumetric flask to obtain solution 1.

[0038] 1.2 Prepare 100ml of solution 2 for later use; first, weigh about 1g of vitamin K1 using an analytical balance, add anhydrous ethanol to make the concentration reach 0.35-1.25g / L, then filter it with a 0.22um filter membrane to sterilize it, and place it in a refrigerator at 4℃ for later use.

[0039] 1.3 Prepare 100ml of solution 3 for later use; First, use an analytical balance to weigh about 0.5g of heme chloride and dissolve it in 1ml of 1mol / L sodium hydroxide solution. Add distilled water to 100ml to make the concentration reach 0.02-0.08g / L. Then filter it through a 0.22um filter membrane to remove bacteria and obtain solution 3. Store it in a refrigerator at 4℃ for later use.

[0040] 1.4 Prepare 10 ml of solution 4 for later use; accurately weigh each component of solution 4 using an analytical balance, dissolve it in 10 ml of distilled water, and then filter it through a 0.22 μm filter membrane to remove bacteria, thus obtaining solution 4, which is then stored in a 4°C refrigerator for later use.

[0041] 1.5 Adjust the pH of solution 1 prepared in step 1.1 to 5.10-6.60 using hydrochloric acid and sodium hydroxide solution (measured at 25℃);

[0042] Furthermore, if liquid culture medium is being prepared, no agar needs to be added; if solid culture medium is being prepared, 17.00-25.00 g / L of agar needs to be added.

[0043] 1.6. Autoclave the solution obtained in step 1.5 at 121°C for 20 minutes. When the temperature drops to 50-55°C, add approximately 0.5 ml of solution 2, approximately 2.5 ml of solution 3, and approximately 0.2 ml of solution 4 to obtain the directional screening culture medium.

[0044] Step 2: Weigh the feces and culture them using the culture medium from Step 1;

[0045] Specifically, 0.1g of fresh, healthy child's feces is dissolved in 1ml of sterile saline; then, 100ul of the fecal solution is inoculated into 10ml of prepared liquid directional selection medium using a pipette, and cultured in an anaerobic incubator at 37℃ for 72-96h (the gas in the anaerobic incubator is divided into two types: high-purity nitrogen (99.999%) and a mixed gas: nitrogen: hydrogen: carbon dioxide = 85%: 5%: 10%).

[0046] Step 3: The enriched fecal microbiota solution from healthy children is screened for Lacticaseibacillus paracasei strains using two methods: plate screening and high-throughput screening of intestinal strains; thus obtaining the Lacticaseibacillus paracasei strain.

[0047] Further steps include the following:

[0048] Step 4, strain identification: Extract DNA from the bacterial culture taken in step 3, amplify the 16S gene sequence by PCR, detect the amplified band by electrophoresis and send the sample for sequencing, check the quality of the sequencing peak using software, and compare the gene sequence through the NCBI website, and summarize and organize the comparison results.

[0049] Step 5, strain preservation: Based on the strain identification results obtained in Step 4, select the strains to be preserved by number, and preserve the bacterial solution cultured in the test tubes or 96-well plates described in Step 3 according to the corresponding number; at the same time, to ensure that the bacterial solution is free from environmental contamination, use a 1ul disposable inoculation loop to dip a portion of the preserved bacterial solution and streak it in three zones of the plate for verification.

[0050] The specific methods for plate screening and high-throughput screening of gut bacteria are as follows:

[0051] Plate screening method:

[0052] The enriched fecal microbiota solution from healthy children was serially diluted and spread onto Lacticaseibacillus paracasei culture medium for directional screening. The operation was carried out in an anaerobic workstation with two types of gas in the anaerobic chamber: (99.999% high-purity nitrogen and a mixed gas: nitrogen: hydrogen: carbon dioxide = 85%: 5%: 10%). After culturing for 2-4 days, single colonies were picked and streaked onto corresponding Lacticaseibacillus paracasei agar plates for three rounds of purification. The purified single colonies were then inoculated into Lacticaseibacillus paracaseis liquid culture medium for directional screening and anaerobically cultured at 37°C and 200 rpm for 24-48 hours.

[0053] High-throughput screening of gut bacteria:

[0054] The total bacterial count and viability of enriched healthy children's fecal samples were determined using flow cytometry. The total viable count was calculated, and the samples were subjected to extreme dilution. The diluted bacterial culture was then transferred to sterile 96-well deep-well plates, sealed with sealing film, and placed in an anaerobic incubator at 37°C (the anaerobic incubator used two types of gas: 99.999% high-purity nitrogen and a mixed gas of nitrogen, hydrogen, and carbon dioxide (85%, 5%, and 10%)). After 2-4 days, plates with suitable growth were selected for bacterial strain selection and transferred to sterile 96-well deep-well plates. 50-100 μL of bacterial culture was transferred from each well for DNA extraction. An appropriate amount of liquid culture medium was then added to the 96-well deep-well plates to continue culturing the remaining bacterial culture.

[0055] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0056] 1. This screening method can accurately screen for human intestinal bacteria strain Lacticaseibacillus paracasei, and more than 97.0% of all screened strains are Lacticaseibacillus paracasei strains.

[0057] 2. This screening method has a long culture time and is simple to operate during the enrichment and screening of strains. It can be carried out simultaneously for multiple samples and batches, thereby improving the efficiency of screening for intestinal probiotics Lacticaseibacillus paracasei.

[0058] 3. This screening method is less prone to contamination because compound antibiotics are added during the preparation of the culture medium, and the liquid and solid culture media have a long shelf life (1 month). Attached Figure Description

[0059] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0060] Figure 1 This is a statistical analysis of the strains isolated by the plate screening method for Lacticaseibacillus paracasei in Example 1 of the present invention.

[0061] Figure 2 This is a statistical representation of the strains obtained in high-throughput screening of intestinal strains in Example 1 of the present invention.

[0062] Figure 3 This is a statistical representation of the strains obtained by screening in the classic MRS medium in the comparative examples.

[0063] Figure 4 The percentage of Lacticaseibacillus paracasei strains obtained by different screening methods in the embodiments of the present invention is statistically analyzed.

[0064] Figure 5 The comparison results of Lacticaseibacillus paracasei in NCBI. Detailed Implementation

[0065] The embodiments of the present invention will be described in detail below with reference to the examples. These examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified can be commercially available conventional products.

[0066] The method for targeted screening of human-derived intestinal probiotic strains Lacticaseibacillus paracasei, as described in this invention, comprises the following steps:

[0067] 1. Prepare solid and liquid culture media for targeted screening of Lacticaseibacillus paracasei. The specific preparation process for the culture media for targeted screening of Lacticaseibacillus paracasei is as follows;

[0068] 1.1. Weigh each component of solution 1 according to its concentration and place it in a 1.5L glass beaker. Add 200ml of ultrapure water and finally dilute to 1000ml using a 1000ml volumetric flask to obtain solution 1.

[0069] 1.2 Prepare 100ml of solution 2 for later use; first, weigh about 1g of vitamin K1 using an analytical balance, add anhydrous ethanol to make the concentration reach 0.35-1.25g / L, then filter it with a 0.22um filter membrane to sterilize it, and obtain solution 2, which is then placed in a refrigerator at 4℃ for later use.

[0070] 1.3 Prepare 100ml of solution 3 for later use; First, use an analytical balance to weigh about 0.5g of heme chloride and dissolve it in 1ml of 1mol / L sodium hydroxide solution. Add distilled water to 100ml to make the concentration reach 0.02-0.08g / L. Then filter it with a 0.22um filter membrane to remove bacteria and obtain solution 3. Store it in a refrigerator at 4℃ for later use.

[0071] 1.4 Prepare 10 ml of solution 4 for later use; accurately weigh each component of solution 4 using an analytical balance, dissolve it in 10 ml of distilled water, and then filter it through a 0.22 μm filter membrane to remove bacteria, thus obtaining solution 4, which is then stored in a 4°C refrigerator for later use.

[0072] 1.5 Adjust the pH of solution 1 prepared in step 1.1 to 5.10-6.60 (measured at 25℃) using hydrochloric acid and sodium hydroxide solution; if preparing liquid culture medium, no agar needs to be added, if preparing solid culture medium, 17.00-25.00 g / L of agar needs to be added.

[0073] 1.6. Autoclave the solution obtained in step 1.5 at 121°C for 20 minutes. When the temperature drops to 50-55°C, add approximately 0.5 ml of solution 2, approximately 2.5 ml of solution 3, and approximately 0.2 ml of solution 4 to obtain the directional screening culture medium.

[0074] The culture medium formulation used is expressed in terms of concentration and mainly includes the following components:

[0075] Solution 1:

[0076] 1) Peptone 10.00-15.50g / L

[0077] 2) Glucose 3.50-6.50 g / L

[0078] 3) Yeast powder 8.50-10.70g / L

[0079] 4) Sodium chloride 0.04-0.90 g / L

[0080] 5) Cysteine ​​hydrochloride 0.30-0.80 g / L

[0081] 6) Calcium chloride 0.004-0.009 g / L

[0082] 7) Magnesium sulfate 0.005-0.100 g / L

[0083] 8) Dipotassium hydrogen phosphate 0.02-0.08 g / L

[0084] 9) Potassium dihydrogen phosphate 0.04-0.09 g / L

[0085] 10) Sodium bicarbonate 0.25-0.75 g / L

[0086] 11) Tween 80 0.80-1.80 ml / L

[0087] 12) Rhazora 0.06-0.15 mg / L

[0088] Solution 2:

[0089] Vitamin K1 solution 0.35-1.25 g / L

[0090] Solution 3:

[0091] Heme chloride solution 0.02-0.08 g / L

[0092] Solution 4:

[0093] antibiotic solution

[0094] Vancomycin 2.0-7.0 mg / ml

[0095] Gentamicin 5.0-11.0 mg / ml

[0096] Kanamycin 6.0-14.0 mg / ml

[0097] pH value (measured at 25℃): 5.10-6.60

[0098] Solid culture medium (agar): 17.00-25.00 g / L

[0099] Prepare classic MRS solid and liquid culture media for screening gut bacteria. MRS culture medium formula: Casein digest 10 g / L; beef extract 10 g / L; triammonium citrate 4 g / L; sodium acetate 5 g / L; magnesium sulfate heptahydrate 0.2 g / L; manganese sulfate tetrahydrate 0.05 g / L; dipotassium hydrogen phosphate 2 g / L; glucose 20 g / L; Tween-80 1.08 g / L; final pH 5.7 ± 0.2, sterilized at 115℃ for 25 min.

[0100] The classic MRS medium is the most widely used and effective medium for screening Lacticaseibacillus paracasei probiotics.

[0101] 2. Classic MRS screening of Lacticaseibacillus paracasei probiotics: Fresh fecal samples from the same healthy child were added to physiological saline at a ratio of 1g:1000μl and shaken to mix. The samples were then diluted 10-fold with sterile physiological saline and inoculated into human intestinal flora serum culture flasks for enrichment culture. On days 3 and 6, the enriched culture was aspirated and serially diluted with sterile physiological saline, then plated onto MRS agar plates. After incubation at 37°C for 24-72 hours in an anaerobic incubator, single colonies were picked and streaked onto the corresponding agar plates for purification (3 rounds). The purified single colonies were then inoculated into MRS liquid medium and incubated at 37°C and 200rpm for 24-48 hours.

[0102] 3. Targeted screening of human-derived gut probiotic Lacticaseibacillus paracasei:

[0103] Plate screening: The culture medium enriched using the liquid medium for directional screening of *Lacticaseibacillus paracasei* was serially diluted with sterile physiological saline and plated onto solid medium for directional screening of *Lacticaseibacillus paracasei*. After incubation in an anaerobic incubator at 37°C for 7-14 days, single colonies were picked and streaked onto corresponding directional screening *Lacticaseibacillus paracasei* agar plates for three rounds of purification. The purified single colonies were inoculated into liquid medium for directional screening of *Lacticaseibacillus paracasei* and anaerobically cultured at 37°C and 200 rpm for 48-72 hours. Genomic DNA was extracted from the bacterial culture in the shaker using a bacterial genomic DNA rapid extraction kit (T5 Direct PCR Kit) and used as a template.

[0104] High-throughput screening of intestinal bacteria: The total bacterial count and viability of healthy children's fecal samples enriched with liquid culture medium for targeted screening using Lacticaseibacillus paracasei were determined using flow cytometry. The total viable count was calculated, and the samples were subjected to extreme dilutions. The diluted bacterial culture was then transferred to sterile 96-well deep-well plates, sealed with sealing film, and placed in an anaerobic incubator at 37°C (the anaerobic incubator used two types of gas: high-purity nitrogen (99.999%) and a mixed gas: nitrogen: hydrogen: carbon dioxide = 85%: 5%: 10%). After 2-4 days, plates with suitable growth were selected for bacterial selection, and the strains were transferred to sterile 96-well deep-well plates for further culture. 50-100 μL of bacterial culture was transferred from each well for bacterial DNA extraction. An appropriate amount of liquid culture medium was then added to the 96-well deep-well plates to continue culturing the remaining bacterial culture.

[0105] 4. Genomic DNA was extracted from the bacterial culture in the shaker using a bacterial genomic DNA rapid extraction kit (T5 Direct PCR Kit), and the extracted genomic DNA was used as a template.

[0106] To prepare the PCR reaction system for amplifying the 16S DNA sequence of the bacterial strain, use 10.6 μl of 2×Taq Plus Master Mix (Qingke Biotechnology, China), 0.2 μl of upstream primer 27F (5'AGAGTTTGATCCTGGCTCAG 3'), 0.2 μl of downstream primer 1492R (5'TACGGCTACCTTGTTACGACTT 3'), 2 μl of bacterial DNA template, and add water to a final volume of 30 μl. Place the system in a PCR instrument for pre-denaturation at 95℃ for 3 min, followed by 28 cycles (95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 30 s), and a final extension at 72℃ for 5 min.

[0107] Take 2 μl of the PCR product for electrophoresis and check if the bands are the expected size. After purifying the PCR product, perform first-generation sequencing and enter the sequencing results into the 16S ribosomal RNA sequences (Bacteria and Archaea) database on the National Center for Biotechnology Information (NCBI) website for sequence alignment.

[0108] 5. Preserve the new bacterial strains and potential probiotics obtained from the comparison.

[0109] Intestinal strains were isolated and cultured from fecal samples of two children using targeted selection medium (plate screening and high-throughput screening) for *Lacticaseibacillus paracasei* and classical MRS, resulting in a total of 186 strains. Among them, 60 strains of 5 different intestinal strains were isolated using the targeted selection method (plate screening) for *Lacticaseibacillus paracasei*. Figure 1 The Lacticaseibacillus paracasei targeted screening method (high-throughput screening of intestinal strains) was used to isolate 60 strains of 5 different intestinal strains, such as... Figure 1 The classic GAM medium screening method was used to isolate 67 strains of 10 different intestinal bacteria, such as... Figure 3It includes gut microbiota species reported in existing literature, such as: Enterococcus gallinarum, Enterococcus lactis, Bifidobacterium pseudocatenulatum, Bifidobacterium faecale, Lacticaseibacillus paracasei, Streptococcus salivarius, Brachybacterium rhamnosum, Lacticaseibacillus paracasei subsp. Tolerans, Bifidobacterium bifidum, and Enterococcus avium.

[0110] Data analysis was performed on the bacterial strains obtained from two fecal samples using two different screening methods. The plate screening method for *Lacticaseibacillus paracasei* yielded 60 strains: 53 *Lacticaseibacillus paracasei*, 2 *Bifidobacterium pseudocatenulatum*, 2 *Enterococcus faecalis*, 2 *Bifidobacterium longum*, and 1 *Parabacteroides distasonis*, achieving the desired targeted screening effect. The percentage of *Lacticaseibacillus paracasei* strains was 88.33%. The high-throughput screening method for *Lacticaseibacillus paracasei* yielded 79 strains: 77 *Lacticaseibacillus paracasei* and 2 *Lacticaseibacillus paracasei* subsp. *Tolerans*, also achieving the desired targeted screening effect. The percentage of *Lacticaseibacillus paracasei* strains was 97.47%; *Lacticaseibacillus paracasei* obtained from the classic GAM screening medium accounted for 8.96% of all screened strains. The proportions of the target strain *Lacticaseibacillus paracasei* obtained by the directional screening method of this invention were 88.33%, 97.47%, and 8.96%, respectively. Figure 4 As shown.

[0111] The sequence alignment results of *Lacticaseibacillus paracasei* strains obtained through targeted screening of the intestinal strain *Lacticaseibacillus paracasei* in the 16S ribosomal RNA sequences (Bacteria and Archaea) database on the National Center for Biotechnology Information (NCBI) website are as follows: Figure 5 As shown in the figure, the bacterium is a strain of *Lacticaseibacillus paracasei*, and its 16S rDNA sequence is shown in SEQ ID No. 1. The colonies are milky white, opaque, round, raised, smooth, and have regular edges.

[0112] SEQ ID No. 1:

[0113] GCAAGTCGACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAAC

[0114] ATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTA

[0115] AGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAG

[0116] AACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATG

[0117] GACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGA

[0118] TGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGAC

[0119] ACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGG

[0120] ACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGT

[0121] CGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTC

[0122] GGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAG

[0123] CCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAA

[0124] AGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAAC

[0125] CGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAG

[0126] TGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACC

[0127] AGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAG

[0128] CATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACG

[0129] ATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACG

[0130] CATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAG

[0131] GAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAA

[0132] GCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAG

[0133] ATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGT

[0134] CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC

[0135] TTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGT

[0136] GACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTAT

[0137] GACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGA

[0138] CCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGG

[0139] CTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGC

[0140] ACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC

[0141] ATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCTAA.

[0142] In summary, this targeted screening method for human-derived probiotics *Lacticaseibacillus paracasei* can accurately screen for probiotics that inhibit *Desulfovibrio* and / or *Enterobacter* spp. in the gut, inhibit *Helicobacter pylori* and / or *Escherichia coli*-*Shigella* spp., and possess potential for lowering blood pressure, anti-tumor activity, anti-oxidation, and cholesterol reduction. A key advantage of this method is its ease of obtaining human-derived probiotics *Lacticaseibacillus paracasei*. It enriches the probiotic strain resource library of human-derived probiotics, achieving the goal of accurately screening *Lacticaseibacillus paracasei*, saving significant manpower and resources, and improving screening efficiency. This provides abundant probiotic resources for the food and pharmaceutical applications of *Lacticaseibacillus paracasei*.

[0143] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for targeted screening of human intestinal Lacticaseibacillus paracasei strains, characterized in that... Includes the following steps: Step 1: Preparation of directional solid and liquid culture media; The culture medium formulation used is expressed in terms of concentration and includes the following components: Solution 1: Solution 2: Vitamin K1 solution 0.35-1.25 g / L Solution 3: Heme chloride solution 0.02-0.08 g / L Solution 4: antibiotic solution Prepare the above culture media separately and set aside for later use; Step 2: Weigh the feces and culture them using the culture medium from Step 1; Step 3: The bacterial solution obtained in Step 2 after enrichment is screened for Lacticaseibacillus paracasei strains to obtain Lacticaseibacillus paracasei strains; the screened bacterial solution is cultured in 96-well deep-well plates. Step 4, strain identification: Extract DNA from the bacterial culture taken in step 3, amplify the 16S gene sequence by PCR, detect the amplified band by electrophoresis and send the sample for sequencing, check the quality of the sequencing peak using software, and compare the gene sequence through the NCBI website, and summarize and organize the comparison results. Step 5, strain preservation: Based on the strain identification results obtained in Step 4, select the strains to be preserved and preserve the bacterial culture in the 96-well deep plate described in Step 3 according to the corresponding numbers; at the same time, to ensure that the bacterial culture is free from environmental contamination, use a 1ul disposable inoculation loop to dip a portion of the preserved bacterial culture and streak it in three zones of the plate for verification.

2. The method for targeted screening of human intestinal Lacticaseibacillus paracasei strains according to claim 1, characterized in that... In step 1, the preparation of the above culture medium includes the following steps; 1.

1. Weigh each component of solution 1 according to its concentration and place it in a 1.5L glass beaker. Add 200ml of ultrapure water and finally dilute to 1000ml using a 1000ml volumetric flask to obtain solution 1. 1.2 Prepare 100ml of solution 2 for later use; first, weigh 1g of vitamin K1 using an analytical balance, add anhydrous ethanol to make the concentration reach 0.35-1.25g / L; then filter it with a 0.22um filter membrane to sterilize it, and obtain solution 2, which is then placed in a refrigerator at 4℃ for later use. 1.3 Prepare 100ml of solution 3 for later use; First, use an analytical balance to weigh 0.5g of heme chloride and dissolve it in 1ml of 1mol / L sodium hydroxide solution. Add distilled water to 100ml to make the concentration reach 0.02-0.08g / L. Then filter it with a 0.22um filter membrane to remove bacteria and obtain solution 3. Store it in a refrigerator at 4℃ for later use. 1.4 Prepare 10 ml of solution 4 for later use; accurately weigh each component of solution 4 using an analytical balance, dissolve it in 10 ml of distilled water, and then filter it through a 0.22 μm filter membrane to remove bacteria, thus obtaining solution 4, which is then stored in a 4°C refrigerator for later use. 1.5 Adjust the pH of solution 1 prepared in step 1.1 to 5.10-6.60 using hydrochloric acid and sodium hydroxide solution, and measure at 25℃; 1.

6. Autoclave the solution obtained in step 1.5 at 121°C for 20 minutes. When the temperature drops to 50-55°C, add 0.5 ml of solution 2, 2.5 ml of solution 3, and 0.2 ml of solution 4 to obtain the directional screening culture medium.

3. The method for targeted screening of human intestinal Lacticaseibacillus paracasei strains according to claim 1, characterized in that... In step 1.5, no agar needs to be added when preparing liquid culture medium, but 17.00-25.00 g / L of agar needs to be added when preparing solid culture medium.

4. The method for targeted screening of human intestinal Lacticaseibacillus paracasei strains according to claim 1, characterized in that... In step 2, accurately dissolve 0.1g of fresh, healthy child's feces in 1ml of sterile saline; then use a pipette to measure 100ul of the fecal solution and inoculate it into 10ml of prepared liquid directional selection medium, and incubate in an anaerobic incubator at 37℃ for 72-96h.

5. The method for targeted screening of human intestinal Lacticaseibacillus paracasei strains according to claim 1, characterized in that... In step 3, the screening includes plate screening and high-throughput screening of gut strains. The specific methods for plate screening and high-throughput screening of gut strains are as follows: Plate screening method: The enriched fecal microbiota solution from healthy children was serially diluted and spread onto Lacticaseibacillus paracasei culture medium for targeted screening. The operation was carried out in an anaerobic workstation. After 2-4 days of incubation, single colonies were picked and streaked onto corresponding Lacticaseibacillus paracasei agar plates for three rounds of purification. The purified single colonies were then inoculated into Lacticaseibacillus paracaseis liquid culture medium for targeted screening and anaerobically cultured at 37°C and 200 rpm on a shaker for 24-48 hours. High-throughput screening of gut bacteria: The total bacterial count and viability of enriched healthy children's fecal samples were determined using flow cytometry. The total viable count was calculated, and the samples were subjected to extreme dilution. The diluted bacterial culture was then transferred to sterile 96-well deep-well plates, sealed with sealing film, and incubated in an anaerobic incubator at 37°C. After 2-4 days, plates with suitable growth were selected for bacterial strain selection, and the strains were transferred to sterile 96-well deep-well plates for further incubation. 50-100 μL of bacterial culture was transferred from each well for bacterial DNA extraction. An appropriate amount of liquid culture medium was then added to the 96-well deep-well plates to continue culturing the remaining bacterial culture.

Images