A rapid detection method for Lactobacillus fermentum resistant to bile salts and its application

Abstract

The present invention discloses a rapid detection method for bile salt-tolerant Lactobacillus fermentum and its application, belonging to the technical field of molecular biology. By comparative genomics of bile salt-tolerant and bile salt-sensitive strains of Lactobacillus fermentum, the present invention screened out potential bile salt-tolerant functional genes, and after verification, obtained the bile salt-tolerant functional gene sfcA, which can distinguish between bile salt-tolerant and bile salt-sensitive strains of Lactobacillus fermentum. This screening method is faster than the traditional evaluation of bile salt tolerance. Based on this gene fragment, primers were designed to amplify a target band of 1164 bp. The bile salt-tolerant gene of the present invention can be used for the rapid screening of bile salt-tolerant Lactobacillus fermentum.

Description

Technical Field

[0001] The present invention relates to a rapid detection method for Lactobacillus fermentum resistant to bile salts and its application, belonging to the technical field of molecular biology. Background Art

[0002] Bile acids are synthesized in hepatocytes from cholesterol as a precursor, stored in the gallbladder, and excreted into the proximal small intestine to dissolve fat-soluble nutrients when food is ingested. When they pass through the small intestine, most bile acids (>95%) are reabsorbed at the terminal ileum and undergo the enterohepatic circulation. Bile salts are sodium or potassium salts formed by the combination of bile acids secreted by hepatocytes through amide bonds with glycine or taurine. Bile salts are weak acids with emulsifier properties, and high concentrations of bile salts can disrupt the normal structure of cell membranes and even cause cell death. At the same time, bile salt stress can lead to protein folding or denaturation. Therefore, the ability to resist bile salts is an important evaluation criterion for probiotics.

[0003] Lactobacillus fermentum is a heterofermentative bacterial species that produces lactic acid, a large amount of acetic acid, ethanol, and CO2 after fermentation. This bacterium can ferment various sugars such as galactose, glucose, fructose, mannose, maltose, lactose, raffinose, sucrose, and trehalose to produce acid and has a strong acid resistance. However, there are significant inter-strain differences in the bile salt resistance of Lactobacillus fermentum. Based on the traditional method of simulating intestinal fluid, the survival rate of Lactobacillus under the condition of pH 8 and the presence of 0.3% bile salts can be evaluated. The viable cell count before and after 4 hours of bile salt treatment is measured by the gradient dilution plate counting method, and usually, 48 - 60 hours are required to obtain the bile salt resistance survival rate result (Patent Publication No. CN112111433A). On this basis, there is also a method of inoculating the strain into a medium containing / without bile salts to simulate the carbon source and nitrogen source of the gastrointestinal tract and calculating the growth rate of the strain under bile salt stress. This method also requires measuring the viable cell count of bacteria before and after tolerance (Patent Publication No. CN103571776A). Therefore, it is particularly important to develop a rapid detection method for the bile salt resistance of Lactobacillus fermentum to evaluate its potential as a probiotic.

[0004] In recent years, with the development of molecular biology, bacterial species identification can be carried out by 16S rDNA sequencing. However, based on 16S rDNA sequencing, different strains of the same bacterial species cannot be accurately distinguished, and there is a lack of molecular markers for screening bile salt-tolerant strains. Existing research reports cloned bsh1 and bsh2 of Lactobacillus fermentum MTCC8711 into the lactic acid bacteria expression plasmid pSLp111.3. After induced expression, it was found that only bsh2 had bile salt hydrolase activity (DOI: 10.1007 / s12010-014-1118-5). However, bile salt hydrolase was not found in the proteomic differential data of Lactobacillus fermentum NCDC400 after treatment with 1.2% bile salt, which may be due to the very low expression level of this protein under bile salt stress (DOI: 10.1016 / j.jprot.2017.08.008). These studies indicate that there is no direct correlation between bile salt hydrolase genes and bile salt resistance, and the bile salt tolerance functional genes of Lactobacillus fermentum remain to be explored. Therefore, it is urgent to explore potential bile salt tolerance genes as molecular markers for rapid screening of bile salt-tolerant Lactobacillus fermentum by PCR. Summary of the Invention

[0005] In view of the long screening cycle and poor result parallelism of current bile salt-tolerant strains, the purpose of the present invention is to screen bile salt tolerance functional genes through comparative genomics analysis of 10 Lactobacillus fermentum strains (5 bile salt-tolerant dominant strains and 5 bile salt-tolerant inferior strains). After verification by qPCR technology, the sfcA gene related to bile salt tolerance of Lactobacillus fermentum was selected as a screening marker, and primers were designed based on this gene sequence. The rapid screening of bile salt-tolerant Lactobacillus fermentum was carried out by PCR technology. This method can be used for rapid detection of bile salt-tolerant Lactobacillus fermentum strains.

[0006] The first object of the present invention is to provide a marker gene for identifying bile salt-tolerant Lactobacillus fermentum, and the nucleotide sequence of the marker gene is shown in SEQ ID NO.3.

[0007] The second object of the present invention is to provide a primer set for identifying bile salt-tolerant Lactobacillus fermentum, and the primer set includes an upstream primer with a nucleotide sequence shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown in SEQ ID NO.2.

[0008] The third object of the present invention is to provide a kit for identifying bile salt-tolerant Lactobacillus fermentum, and the kit contains the above primer set.

[0009] In one embodiment, the kit further contains PCR reaction reagents, qPCR reaction reagents or ddPCR reaction reagents.

[0010] The present invention provides the use of the above-mentioned marker gene, or the above-mentioned primer set, or the above-mentioned kit in screening Lactobacillus fermentum resistant to bile salts.

[0011] The present invention provides a method for rapidly identifying Lactobacillus fermentum resistant to bile salts. The method uses the sfcA gene fragment as a marker and the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 as primers, and the strain that amplifies the target fragment is Lactobacillus fermentum resistant to bile salts.

[0012] In one embodiment, the size of the target gene fragment is 1164bp.

[0013] In one embodiment, the amplification is performed on a sample containing the genome of Lactobacillus fermentum.

[0014] In one embodiment, the object includes genomic DNA extract, single colony or bacterial suspension.

[0015] In one embodiment, the genomic DNA includes, but is not limited to, that extracted from food or feces.

[0016] In one embodiment, the amplification is performed using the genomic DNA template containing the strain to be tested.

[0017] In one embodiment, the amplification is performed using a single colony as a template.

[0018] The present invention also provides the use of the above-mentioned marker gene or the above-mentioned primer set in the preparation of reagents or kits for identifying Lactobacillus fermentum resistant to bile salts.

[0019] Beneficial effects:

[0020] (1) The verification of the sfcA gene in Lactobacillus fermentum by the present invention shows that there is a certain relationship between the sfcA gene and the bile salt resistance of this species in Lactobacillus fermentum. Therefore, it can be used for the rapid screening of Lactobacillus fermentum strains resistant to bile salts.

[0021] (2) Compared with the traditional method, this method can rapidly identify Lactobacillus fermentum resistant to bile salts. In the present invention, PCR rapid identification can be achieved, and a large number of resistant strains can be screened for food, especially starters, without counting after culturing in simulated intestinal fluid for 4 hours. Description of the drawings

[0022] Figure 1 It is the evaluation of the bile salt tolerance ability of Lactobacillus fermentum.

[0023] Figure 2is the relative transcriptional level of bile salt tolerance functional genes in Lactobacillus fermentum 156 after treatment with 1.2% bile salts.

[0024] Figure 3 is the relative transcriptional level of the sfcA gene at different bile salt concentrations.

[0025] Figure 4 is the relative transcriptional level of the mtlR gene at different bile salt concentrations.

[0026] Figure 5 is the gel electrophoresis pattern of the sfcA gene.

[0027] Figure 6 is the growth rate of 12 Lactobacillus fermentum strains at a bile salt concentration of 1.2%.

[0028] Figure 7 is the survival rate of 12 Lactobacillus fermentum strains at a bile salt concentration of 1.2% based on the traditional bile salt tolerance method. Specific Embodiments

[0029] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings. However, the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available products or can be prepared by known methods.

[0030] The lysozyme involved in the following embodiments was purchased from Sangon Biotech (Shanghai) Co., Ltd., with an enzyme activity of ≥20000 U / mg;

[0031] MRS medium; 10 g of tryptone, 5 g of yeast extract, 10 g of beef extract, 20 g of glucose, 0.5 g of magnesium sulfate heptahydrate, 0.25 g of manganese sulfate monohydrate, 2 g of sodium acetate, 2 g of ammonium citrate dibasic, 2.6 g of dipotassium hydrogen phosphate trihydrate, 1 mL of Tween, 1000 mL of deionized water.

[0032] MRS medium containing 1.2% bile salts: 12 g of bile salts (Oxoid LP0055), 10 g of tryptone, 5 g of yeast extract, 10 g of beef extract, 20 g of glucose, 0.5 g of magnesium sulfate heptahydrate, 0.25 g of manganese sulfate monohydrate, 2 g of sodium acetate, 2 g of ammonium citrate dibasic, 2.6 g of dipotassium hydrogen phosphate trihydrate, 1 mL of Tween, 1000 mL of deionized water.

[0033] Example 1: Evaluation of the bile salt tolerance ability of Lactobacillus fermentum

[0034] Ten Lactobacillus fermentum strains were isolated and identified from fecal samples. After being activated for three generations in MRS liquid, the bacterial suspension was inoculated into MRS medium (control group) and MRS medium containing 1.2% bile salts (experimental group) at an inoculation amount of 2% (v / v), and cultured at 37 °C and 200 rpm for 12 h. The OD of the bacterial suspension in the stationary phase was measured. 600 The growth rate was calculated. Among them, the growth rate = OD of the experimental group 600 / OD of the control group 600 × 100%.

[0035] The experimental results are as Figure 1 shown. There were significant inter-strain differences in the bile salt tolerance of the ten Lactobacillus fermentum strains. Among them, the growth rate of Lactobacillus fermentum 156 was the highest at 77.4%, indicating a high tolerance to bile salts; the growth rate of Lactobacillus fermentum FSH101 was 1.61%, suggesting that its growth was completely inhibited.

[0036] Example 2: Screening of bile salt tolerance functional genes of Lactobacillus fermentum

[0037] Strains with a growth rate > 50% under 1.2% bile salt conditions were classified as dominant strains, and those with a growth rate < 25% were classified as inferior strains for comparative genomic analysis. The core gene database of five tolerant strains was constructed using the default parameters of OrthoMCL. Then, the whole genomes of the five inferior strains were aligned with the core gene database of the dominant strains using PGAP, and the genes missing in the inferior strains were screened according to the orthologous clusters.

[0038] The results are shown in Table 1. Twenty-five genes were missing in the five inferior strains. Among them, one was related to pyruvate metabolism, one was related to lipopolysaccharide biosynthesis, seven were related to the PTS system, eight were related to the two-component system, and two were related to ribosome composition. At the same time, three hypothetical proteins and three genes encoding glycosyltransferase, oxidoreductase, and gluconate reductase were also predicted.

[0039] Table 1 Bile salt tolerance functional genes of Lactobacillus fermentum

[0040]

[0041] Example 3: qPCR verification of bile salt tolerance functional genes of Lactobacillus fermentum

[0042] Select the bile salt-tolerant dominant strain Lactobacillus fermentum 156, and extract the strain RNA using the Trizol method. The control group was cultured with ordinary MRS for Lactobacillus fermentum 156, and the experimental group was cultured with MRS containing 1.2% bile salt. Add 1 mL of 20 mg / mL lysozyme to a 1.5 mL enzyme-free EP tube containing bacterial sludge, mix well, incubate at 37 °C for 30 min, centrifuge at 12,000 g at 4 °C for 2 min, and discard the supernatant. Add 1 mL of Trizol and pipette repeatedly, then let it stand at room temperature for 10 min. Add 200 μL of chloroform, shake manually for 15 s, then let it stand at room temperature for 5 min, centrifuge at 12,000 g at 4 °C for 15 min to separate the layers. Pipette 300 μL of the aqueous layer solution into a new 1.5 mL EP tube; add 300 μL of isopropanol, mix by inverting up and down, let it stand at 4 °C for 10 min, centrifuge at 12,000 g at 4 °C for 10 min, and discard the supernatant. Add 1 mL of 75% ethanol solution (prepared with DEPC water, freshly prepared) to wash the precipitate, centrifuge at 12,000 g at 4 °C for 5 min, discard the supernatant, and repeat once. After opening the lid to let the ethanol evaporate, add 20 μL of DEPC water, mix well, incubate at 60 °C for 10 min, and immediately place it on ice to obtain the extracted RNA solution. Reverse the RNA into cDNA (HiScript III RT SuperMix for qPCR (+gDNA wiper).

[0043] According to the functional genes screened in Example 2, select the genes with clear metabolic pathways on KEGG, design primers using Primer3.0, and the sequences are shown in Table 2. The 16srRNA gene is used as an internal reference, and qPCR amplification is performed with cDNA as a template. The amplification conditions are as follows:

[0044] ① The qPCR reaction system consists of: 5 μL of iTaq universal SYBR Green supermix (2×), 0.5 μL each of 10 μM forward and reverse primers, 1 μL of cDNA template, and 3 μL of water.

[0045] ② The qPCR reaction conditions are: pre-denaturation at 50 °C for 3 min, denaturation at 95 °C for 5 s, annealing at 55 °C for 30 s, 34 cycles, and a melting curve is added.

[0046] The results are as Figure 2As shown, the relative transcriptional levels of six genes (citXG, citF, citE, citD, citC, sfcA) related to the two-component system were up-regulated. Among the genes related to the bacterial phosphotransferase system (PTS), only mtlR, ulaA, ulaB, and ulaC were up-regulated after bile salt treatment, while pstA, pstC, and pstS were down-regulated. In addition, rpmG and rpmF related to ribosome composition were down-regulated, and the relative transcriptional levels of waaB related to lipopolysaccharide biosynthesis and ldhA related to pyruvate metabolism increased.

[0047] Table 2 Primers for different PCR reactions

[0048]

[0049]

[0050] Example 4: Effects of different bile salt concentrations on the sfcA gene of Lactobacillus fermentum

[0051] Bile salt tolerance is one of the screening criteria for probiotics. Finding genes related to probiotic bile salt tolerance as molecular markers has a positive effect on the rapid screening of bile salt-tolerant strains in the future. Based on the results of Example 3, the present invention found that the relative transcriptional levels of sfcA and mtlR genes were most significantly up-regulated under 1.2% bile salt stress. After activating Lactobacillus fermentum 156 for 3 generations in liquid MRS medium, it was simultaneously inoculated into ordinary MRS medium and MRS medium containing 0.3%, 0.6%, 0.9%, 1.2%, 1.5%, and 2% bile salts respectively. After culturing for 12 h, the cells were collected, and RNA was extracted and reverse transcribed into cDNA according to the method in Example 3. Using 16S rRNA as an internal reference, the relative transcriptional levels of sfcA and mtlR genes at different bile salt concentrations were relatively quantified using primer pairs.

[0052] The results are as Figure 3 and Figure 4 shown. As the bile salt concentration in the medium increased, the relative transcriptional level of the sfcA gene generally showed an upward trend, while the relative transcriptional level of the mtlR gene did not increase significantly. This indicates that there is a certain relationship between the sfcA gene in Lactobacillus fermentum 156 and the bile salt resistance of the cells.

[0053] Example 5: Verification of sfcA gene-specific primers in screening bile salt-tolerant Lactobacillus fermentum

[0054] Select the full-length 1164bp sfcA gene to design and synthesize specific primer sequences: forward primer (SEQ ID NO.1): 5’-AGCCTGAAAATGAAAACGATGAAGTTTTC-3’, reverse primer (SEQ ID NO.2): 5’-TTAGTGTTTTTGCTGGGCAAAGTCG-3’. Select 12 Lactobacillus fermentum strains with unknown bile salt tolerance. Streak the cryopreserved bacterial solution on MRS solid medium and culture at 37°C for 24 - 48h. After picking single colonies and activating them for 3 generations in MRS liquid medium, inoculate the bacterial solution at an inoculation amount of 2% (v / v) into ordinary MRS and MRS medium containing 1.2% bile salt respectively. After culturing for 12h, measure OD 600 , and calculate the growth rate. Meanwhile, take single colonies / bacterial solution for PCR amplification, and the amplification conditions are as follows:

[0055] The PCR amplification reaction system is: 2×Taq Plus MasterMix (Dye) 25μL, 1μL of bacterial solution template (single colony), 2μL each of 10μM forward and reverse primers, add ddH2O to 50μL.

[0056] The PCR amplification reaction conditions are: pre-denaturation at 94°C for 2min (bacterial solution) or pre-denaturation at 94°C for 3min (colony), denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, 34 cycles, and final extension at 72°C for 2min.

[0057] Take 30μL of the PCR product of each sample and load it into the wells of 1.0% agarose gel respectively, and perform electrophoresis at 120V for 30min. The first lane is 100bp Marker. As Figure 5 shown, obvious bands appear near 1000bp in lanes 1 - 5, and no specific bands appear in lanes 6 - 12. Meanwhile, as Figure 6 shown, according to the method described in Example 1, the growth rate of the 5 strains with obvious bands > 50%, and the growth rate of the strains without bands < 25%. The above data show that the method can quickly and effectively screen Lactobacillus fermentum strains with bile salt tolerance.

[0058] Comparative Example 1: Evaluation of the bile salt tolerance of Lactobacillus fermentum by the traditional simulated intestinal fluid method

[0059] Prepare simulated intestinal fluid: Dissolve trypsin in sterilized normal saline (0.9% w / v, adjust the pH to 8 with NaOH) to a final concentration of 1g / L, and add bile salt to a final concentration of 1.2%. Filter with a 0.22μm filter membrane and set aside.

[0060] The 12 strains of Lactobacillus fermentum with unknown bile salt tolerance preserved by freezing were streaked on an MRS solid plate and cultured at 37 °C for 24 - 48 h. After being activated for three generations in MRS liquid, the bacterial solution was inoculated into an MRS medium at an inoculation amount of 2% (v / v). The bacterial solution in the stationary phase was centrifuged at 5000 g for 5 min at 4 °C, the supernatant was discarded, and it was washed twice with physiological saline. After centrifuging and discarding the supernatant, an equal volume of simulated intestinal fluid was added. The bacterial solutions at 0 h and after culturing at 37 °C for 4 h were respectively taken for plate viable count.

[0061] As Figure 7 shown, the conclusion of evaluating the bile salt tolerance of Lactobacillus fermentum according to the traditional simulated intestinal fluid is consistent with the electrophoresis conclusion in Example 5. Plate viable count requires gradient dilution of the bacterial solution, culturing for 48 h after pouring, the operation is complex, and both the dilution volume and the temperature of the poured culture medium will affect the experimental results, resulting in poor parallelism. Therefore, the sfcA gene obtained in the present invention can be used for the rapid screening of bile salt-tolerant Lactobacillus fermentum.

[0062] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Use of a primer set for identifying Lactobacillus fermentum resistant to bile salts or a kit for identifying Lactobacillus fermentum resistant to bile salts in screening Lactobacillus fermentum resistant to bile salts, characterized in that, The primer set comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; the kit contains the primer set described above.

2. The application according to claim 1, wherein The kit also contains PCR reaction reagents, qPCR reaction reagents or ddPCR reaction reagents.

3. A method for rapidly identifying Lactobacillus fermentum resistant to bile salts, characterized in that, The method is to amplify a test sample with a primer set for identifying Lactobacillus fermentum resistant to bile salts, and the primer set comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.

2.

4. The method according to claim 3, characterized in that, The amplification is performed on a sample containing Lactobacillus fermentum.

5. The method according to claim 4, characterized in that The sample containing Lactobacillus fermentum includes genomic DNA extract, single colony or bacterial suspension.

6. Use of a primer set for identifying Lactobacillus fermentum resistant to bile salts in the preparation of a reagent or kit for identifying Lactobacillus fermentum resistant to bile salts, characterized in that, The primer set comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2.

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