Method for directionally screening probiotics capable of producing acetylcholine

Abstract

The invention discloses a method for directionally screening probiotics capable of producing acetylcholine, and belongs to the technical field of microorganisms. On the basis of bioinformatics analysis of a choline acetyltransferase related gene sequence and a conserved protein structural domain in an acetylcholine synthesis route, a protein structural domain Acetyltransf17 (IPR041380) which is not previously represented and possibly participates in synthesis of acetylcholine is excavated; the function of the gene is further verified through gene knockout and fermentation tests of the strain. The protein structural domain gene is used as a screening target, whether the strain has the capability of producing acetylcholine or not can be confirmed by analyzing the genome of a to-be-detected strain, and compared with a traditional screening method based on a fermentation test, the efficiency can be remarkably improved when the marker is applied to specific screening and identification of the strain.

Description

Technical Field

[0001] The invention relates to a method for directional screening of probiotics producing acetylcholine, belonging to the technical field of microorganisms. Background Art

[0002] Acetylcholine is an important neurotransmitter that is widely present in the central and peripheral nervous systems and is involved in the regulation of multiple physiological functions. It is synthesized from choline and acetyl-CoA through the catalysis of choline acetyltransferase, and binds to nicotinic and muscarinic acetylcholine receptors to play a role in neurotransmission, muscle contraction, and cardiovascular regulation. In addition to playing an important role in the central nervous system, acetylcholine's function in the enteric nervous system is equally critical. The enteric nervous system is called the "second brain" and can autonomously control intestinal function. Acetylcholine maintains intestinal health by regulating intestinal peristalsis and secretion release, and affects the overall physiological balance by regulating intestinal immune responses. Studies have shown that the level of acetylcholine in the intestine directly affects the function of the enteric nervous system, and probiotics may play a role in this process.

[0003] In recent years, more and more studies have shown that intestinal microorganisms play an important role in regulating the function of the host's enteric nervous system. Although intestinal microorganisms themselves do not directly synthesize neurotransmitters, they affect the synthesis and function of neurotransmitters such as γ-aminobutyric acid (GABA), serotonin, and dopamine by regulating host metabolism. For example, some lactic acid bacteria regulate intestinal motility by producing GABA through metabolism; intestinal microorganisms indirectly regulate the production of serotonin by affecting tryptophan metabolism; similarly, intestinal microorganisms regulate the synthesis of dopamine through metabolism, thereby affecting intestinal function and host behavior.

[0004] As the study of probiotics deepens, more and more evidence shows that some microorganisms not only have probiotic effects, but may also synthesize neuroactive substances such as acetylcholine. In 1947, scientists first detected acetylcholine in the fermentation broth of Lactobacillus plantarum, and then further confirmed its existence in a study in 1977. In 2022, researchers discovered acetylcholine again in the fermentation broth of the Blautiawexlerae strain, indicating that not only lactic acid bacteria, but other intestinal microorganisms may also have the ability to synthesize acetylcholine. This provides a new research direction for the potential role of probiotics in host physiological regulation. Nevertheless, the specific mechanism by which microorganisms synthesize acetylcholine is still unclear. In animal cells, the biosynthesis of acetylcholine is mainly carried out by choline and acetyl-CoA under the catalysis of choline acetyltransferase (ChAT). However, whether microorganisms have acetylcholine synthesis pathways similar to those of animal cells still needs further study. Preliminary evidence shows that some intestinal microorganisms may generate acetylcholine through different metabolic pathways using choline and acetyl-CoA or similar precursors. These findings suggest that microorganisms may have specific enzymes or metabolic networks that support their ability to synthesize acetylcholine.

[0005] Traditional screening methods for acetylcholine-producing probiotics usually include several key steps. First, the target strain is isolated and purified from a complex microbial community to obtain a pure culture of a single strain. Next, the obtained pure culture strain is fermented, and the supernatant in the fermentation broth is separated by centrifugation. In order to detect the acetylcholine content in the supernatant, the target substance in the supernatant needs to be evenly dispersed in an acetonitrile solvent to ensure the stability and uniformity of acetylcholine in the solution. Subsequently, the acetylcholine content in the fermentation supernatant is determined by constructing a quantitative curve of the acetylcholine standard and using ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS / MS) technology. Although this method has high sensitivity, its complex operating steps, high technical requirements and cost issues limit its popularity in large-scale screening of acetylcholine-producing strains. Therefore, future research may need to explore screening methods that are highly specific, simple and economical to improve screening efficiency and reduce costs. Summary of the invention

[0006] The present invention aims to solve the problems of low efficiency and high cost in the current screening process of acetylcholine-producing probiotics, and provides a method for directional screening of acetylcholine-producing probiotics. The method significantly improves the screening efficiency and reduces the cost through experimental verification and quantitative analysis.

[0007] The present invention provides the use of Acetyltransf_17 protein in identifying acetylcholine-producing microorganisms.

[0008] In one embodiment, the Acetyltransf_17 protein has the amino acid sequence shown in SEQ ID NO.1.

[0009] In one embodiment, the acetylcholine-producing microorganism has a gene encoding the Acetyltransf_17 protein.

[0010] In one embodiment, the gene has the nucleotide sequence shown in SEQ ID NO.2.

[0011] The present invention also provides a method for directed screening of acetylcholine-producing probiotics, wherein the method is to detect the genotype of the microorganism to be detected, and the strain with a marker gene is the acetylcholine-producing strain; the marker gene has a nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO.2.

[0012] In one embodiment, the PCR amplification is performed using the primers shown in SEQ ID NO.4 or SEQ ID NO.5.

[0013] In one embodiment, the PCR comprises colony PCR or PCR using extracted genomic DNA as a template.

[0014] In one embodiment, the PCR amplification reaction conditions are set as follows: pre-denaturation at 95°C for 4 minutes; 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds, for a total of 30 cycles; extension at 72°C for 10 minutes. Optionally, the amplified PCR product is stored at 4°C.

[0015] In one embodiment, the strain having the Acetyltransf_17 domain can amplify the electrophoresis band of 755 bp shown in SEQ ID NO.3.

[0016] In one embodiment, the method further comprises sequencing the amplified DNA fragments.

[0017] In one embodiment, the method further comprises: culturing the strain obtained by screening in a culture medium, and detecting the acetylcholine production in the fermentation broth.

[0018] In one embodiment, the detection is performed by liquid chromatography-mass spectrometry (LC-MS) for detection and quantitative analysis.

[0019] In one embodiment, the detection conditions of the liquid chromatography-mass spectrometry (LC-MS) detection are: the chromatographic column uses a Thermo Scientific Syncronis HILIC 1.7μm column; the mobile phase A is an 80% acetonitrile-water mixture; the mobile phase B is a 0.1% (v / v) formic acid-water solution, the flow rate is 0.3mL / min; the injection volume is 2μL; the column temperature is 30°C; and the electrospray ionization source (ESI) is in positive ion mode.

[0020] In one embodiment, the conditions for liquid chromatography-mass spectrometry (LC-MS) detection of acetylcholine are as follows:

[0021] The chromatographic column uses a Thermo Scientific Syncronis HILIC 1.7μm column to ensure good separation of acetylcholine from other compounds; the mobile phase A is a mixed solution of acetonitrile and water (80% acetonitrile, 20% water by volume), in which 0.1% formic acid is added to adjust the pH value; the phase B is pure water, which usually also contains 0.1% (v / v) formic acid; the flow rate is 0.3mL / min to ensure that the separation is completed in a shorter time; the injection is 2μL of sample each time, which effectively reduces the sample amount and improves the detection sensitivity. The column temperature is maintained at 30℃, which helps to maintain a stable separation effect. The detection mode uses an electrospray ionization source (ESI) in positive ion mode and detects in MS / MS mode. The parent ion (m / z 146.1) of acetylcholine and its characteristic fragment ion (m / z 87.0) are the main objects of detection.

[0022] In one embodiment, the culturing is carried out at 30-37° C. for at least 12 hours.

[0023] In one embodiment, the probiotics include but are not limited to Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus mucosus ferments, and Lactobacillus salivarius.

[0024] The present invention also provides products for identifying, detecting and / or screening acetylcholine-producing probiotics.

[0025] In one embodiment, the product contains the primer pair shown in SEQ ID NO.4 and SEQ ID NO.5.

[0026] In one embodiment, the product includes, but is not limited to, a kit.

[0027] In one embodiment, the kit further contains reagents commonly used in PCR, including but not limited to DNA polymerase, dNTP mixture, buffer, MgCl2, stabilizer or enhancer.

[0028] The present invention also provides products prepared by using the screened acetylcholine-producing probiotics, wherein the products include but are not limited to functional foods, fermented foods, nutritional supplements or medicines.

[0029] In one embodiment, the functional food includes yogurt, beverages, meal replacement powder, etc., and acetylcholine-producing probiotics are used to improve the health value of the food.

[0030] In one embodiment, the product can prevent and relieve intestinal problems such as constipation and irritable bowel syndrome by improving intestinal motility and regulating neurotransmitter levels.

[0031] Technical effects:

[0032] The present invention greatly improves the efficiency of screening acetylcholine-producing probiotics by combining genome comparison and PCR technology. Compared with conventional screening methods, the method of the present invention can significantly shorten the screening cycle and reduce experimental costs. Under the premise of known strain gene sequence, the PCR verification of this method can complete the screening within about 12 hours. The traditional method usually requires steps such as strain streaking separation, activation, passage, complete fermentation, extraction of fermentation broth and LC-MS detection, and the whole process takes at least 96 hours. In contrast, the method of the present invention shortens the screening cycle by about 84 hours and increases the efficiency by 8 times. In addition, the method of the present invention can process about 50 samples per unit time, which greatly improves the efficiency of screening. The screened probiotics have the ability to synthesize acetylcholine, can be used to develop functional foods and health products, and have the effect of improving intestinal function and regulating the nervous system. The method of the present invention can also provide new tools for studying the gut-brain axis, which helps to further reveal the role of intestinal microorganisms in the regulation of the nervous system. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the amino acid sequence of the protein domain Acetyltransf_17 (IPR041380) and its three-dimensional structure simulation; wherein, A~C are the RMSD scores of the spatial structure fitting of the protein domain in Breinasaccharomyces cerevisiae, Lactobacillus LSP Lp1 and Micrococcus and the protein domain Acetyltransf_17 (IPR041380), respectively, which evaluates its stability and similarity with the target protein; D is the three-dimensional structure model of the protein domain Acetyltransf_17 (IPR041380), showing its structural characteristics.

[0034] Figure 2Schematic diagram of the process of verifying the acetylcholine production ability of lactic acid bacteria through gene knockout and complementation experiments; A is a flowchart of the construction of the gene knockout vector, showing the upstream and downstream homologous arms and key restriction sites; B is the PCR identification diagram after the construction of the knockout strain, showing the band results of each sample; C is the PCR identification diagram of the complementation strain construction, verifying the success of gene complementation; D is the acetylcholine production of each strain in synthetic culture medium; E is the acetylcholine production of each strain in MRS culture medium; F is a comparison of the growth curves of the wild type and knockout strains of Lactobacillus plantarum CCFM1291, showing the effect of gene knockout on growth.

[0035] Figure 3 Figure 2 is the distribution of acetylase in fifty strains and the results of acetylcholine production determination; A is the distribution of acetylation domains in different strains; B is the mass spectrum of acetylcholine detected in the supernatant of MRS culture medium fermentation of Lactobacillus plantarum; C is the fermentation experiment results of 50 randomly selected strains of Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentans, and Lactobacillus salivarius.

[0036] Figure 4 It is a diagram of the experimental results of the specific application of the screening method; wherein A is the PCR gel electrophoresis result of the Acetyltransf_17 (IPR041380) protein domain in 14 randomly selected strains. Among these 14 strains, only three strains of Lactobacillus plantarum were detected with this protein domain; B is the detection result of the acetylcholine content in the fermentation supernatant of these strains. The results show that the production of acetylcholine was detected only in the fermentation supernatant of three strains of Lactobacillus plantarum. DETAILED DESCRIPTION

[0037] (I) Sequence information:

[0038] The amino acid sequence of Acetyltransf_17 (IPR041380) (SEQ ID NO.1):

[0039] MTTDYRLTSDADREAFYQLYQYAFNNHDTPARRRFFMDRYQHGWIYGRHDHGQLVSGL

[0040] YSLPLAVNFHGVTYRMNGIGDVMSAPEYAGQGGAGQLLTTALTEMAANHVTLSYLAPFA

[0041] YGYYRRFGYEHVFDHAHQVMAAPDLPRVKPTDWSGTITRYGNDGLAQINDFYARQPQN

[0042] QRGGLIRPAWWQHYLTLKHHWSVAIYRNAAANIEGYLIYERQATNFAIQEWVTSTPTAFE

[0043] RLANFITKHGTTFETFSYDSPSATNGLDLLADPYRLKVTVQPYMMARIVDLYDFIKRYPFL

[0044] TDIQPIRLAVSDATLPANQGIWSLTRHNGQVTLNRVSDQLTGPADIQLTIQQLTTACFGTQSLARAYQHGLITGDVNAINRLDTSLVKERPALIDYF;

[0045] Nucleotide sequence (SEQ ID NO.2) of the gene encoding Acetyltransf_17(IPR041380):

[0046] TTAGAAGTAGTCAATAAGTGCTGGTCGTTCTTTAACTAGACTAGTGTCAAGTCGGTT

[0047] GATCGCATTCACATCACCGGTGATTAATCCGTGTTGGTACGCCCGGGCTAAGCTCTGGG

[0048] TGCCAAAGCAAGCAGTGGTTAATTGCTGAATCGTTAACTGAATGTCAGCCGGTCCAGT

[0049] TAATTGGTCGCTAACCCGATTGAGCGTTACTTGCCCGTTGTGCCGCGTCAATGACCAGA

[0050] TTCCTTGATTAGCAGGGAGGGTAGCGTCACTGACTGCTAGGCGAATTGGTTGGATGTC

[0051] CGTGAGAAACGGATAGCGCTTGATAAAGTCATATAAATCAACAATGCGGGCCATCATGT

[0052] ACGGCTGGACAGTCACTTTTAAACGGTATGGGTCGGCTAGTAGGTCCAAGCCGTTGGT

[0053] CGCACTTGGGCTATCATAACTAAACGTTTCAAAGGTGGTGCCATGCTTAGTGATAAAGT

[0054] TAGCCAGTCGTTCGAAAGCGGTCGGCGTGCTAGTCACCCATTCTTGAATCGCAAAGTT

[0055] AGTGGCCTGCCGTTCATAGATCAAATAGCCCTCGATATTGGCAGCCGCATTGCGGTAAA

[0056] TTGCGACCGACCAATGATGTTTGAGAGTCAGGTAATGTTGCCACCAGGCTGGTCGAAT

[0057] CAGACCACCACGCTGATTTTGCGGTTGTCGTGCATAAAAATCGTTGATTTGGGCCAGC

[0058] CCGTCATTGCCATAGCGGGTGATCGTACCAGACCAGTCCGTCGGTTTGACACGTGGTA

[0059] GGTCAGAAGCGGCCATGACTTGGTGCGCGTGGTCGAAGACGTGTTCATAGCCAAAAC

[0060] GACGATAGTAACCGTACGCGAAGGGGGCAAGGTAGGACAGCGTCACGTGGTTGGCTT

[0061] CCATTTCTGTCAGCGCCGTCGTTAACAGCTGTCCCGCGCCACCCTGACCGGCATATTC

[0062] AGGCGCACTCATCACGTCGCCAATACCATTCATCCGGTAGGTGACGCCATGAAAGTTG

[0063] ACTGCTAGTGGTAGACTGTATAGCCCACTGACCAGTTGACCATGATCGTGACGGCCGT

[0064] AGATCCAGCCATGTTGGTAGCGATCCATGAAAAATCGCCGCCGCGCGGGGGTATCGTG

[0065] ATTATTGAACGCGTATTGATAAAGTTGGTAGAAAGCTTCGCGGTCAGCATCGCTAGTTAAACGGTAATCAGTTGTCAT;

[0066] Forward Primer (SEQ ID NO. 4): 5'-ACCAGATTCCTTGATTAGCA-3';

[0067] Reverse Primer (SEQ ID NO.5): 5'-CTATACAGTCTACCACTAGCA-3';

[0068] Forward primer GC content: 50%;

[0069] Reverse primer GC content: 50%;

[0070] Forward primer Tm value: 58°C;

[0071] Reverse primer Tm value: 58°C;

[0072] Amplification product length: 784bp.

[0073] Amplified nucleotide sequence (SEQ ID NO.3):

[0074] ACCAGATTCCTTGATTAGCAGGGAGGGTAGCGTCACTGACTGCTAGGCGAATTGGTTG

[0075] GATGTCCGTGAGAAACGGATAGCGCTTTGATAAAGTCATATAAATCAACAATGCGGGCC

[0076] ATCATGTACGGCTGGACAGTCACTTTTAAACGGTATGGGTCGGCTAGTAGGTCCAAGC

[0077] CGTTGGTCGCACTTGGGCTATCATAACTAAACGTTTCAAAGGTGGTGCCATGCTTAGT

[0078] GATAAAGTTAGCCAGTCGTTCGAAAGCGGTCGGCGTGCTAGTCACCCATTCTTGAATC

[0079] GCAAAGTTAGTGGCCTGCCGTTCATAGATCAAATAGCCCTCGATATTGGCAGCCGCATT

[0080] GCGGTAAATTGCGACCGACCAATGATGTTTGAGAGTCAGGTAATGTTGCCACCAGGCT

[0081] GGTCGAATCAGACCACCACGCTGATTTTGCGGTTGTCGTGCATAAAAATCGTTGATTT

[0082] GGGCCAGCCCGTCATTGCCATAGCGGGTGATCGTACCAGACCAGTCCGTCGGTTTGAC

[0083] ACGTGGTAGGTCAGAAGCGGCCATGACTTGGTGCGCGTGGTCGAAGACGTGTTCATA

[0084] GCCAAAACGACGATAGTAACCGTACGCGAAGGGGGCAAGGTAGGACAGCGTCACGT

[0085] GGTTGGCTTCCATTTCTGTCAGCGCCGTCGTTAACAGCTGTCCGCGCCACCCTGACC

[0086] GGCATATTCAGGCGCACTCATCACGTCGCCAATACATTCATCCGGTAGGTGACGCCATGAAAGTTGACTGCTAGTGGTAGACTGTATA.

[0087] (II) Culture medium

[0088] Synthetic culture medium: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, K2HPO4 2 g / L, MgSO4·7H2O 0.2 g / L.

[0089] Example 1 Characterization of the characteristic protein domain involved in acetylcholine biosynthesis in Lactobacillus plantarum

[0090] First, we retrieved and downloaded all Fasta sequences of choline acetyltransferase reported in bacteria from the NCBI database (3 in total) and saved these sequences to a local folder. Then, we annotated the domains of the full gene sequence of Lactobacillus plantarum, which produces high acetylcholine, through the protein and nucleotide conserved domain database of the NCBI database. Based on functional screening, the study narrowed the scope to domains related to acetylation function and found a protein domain Acetyltransf_17 (IPR041380) that has never been characterized in Lactobacillus plantarum.

[0091] Then, Swiss-model was used to perform 3D modeling on the amino acid sequences of all acetylation functional domains in Lactobacillus plantarum and the amino acid sequence of choline acetyltransferase in the NCBI database. After the model file was downloaded, the Superalign method of Pymol software in Python was used to align the domain functions at low amino acid sequence similarity and score them ( Figure 1 ). The results showed that the protein domain Acetyltransf_17 (IPR041380) had the lowest fitting root mean square error (RMSD) score with choline acetyltransferase in the protein spatial structure, indicating that their functional similarity was the highest. Further analysis showed that the protein domain Acetyltransf_17 (IPR041380) had a three-layer α / β / α structure mixed with a β-folded structure. This structure was previously only found in the enhanced intracellular survival protein (Eis) of mycobacteria and may be responsible for catalyzing the transfer of acetyl groups, but its specific function has not yet been identified.

[0092] Example 2 Verification of the necessity of protein domain Acetyltransf_17 (IPR041380) for acetylcholine synthesis by Lactobacillus plantarum

[0093] (1) Construction of Acetyltransf_17 (IPR041380) gene knockout strain:

[0094] Adopt the method for homologous recombination, protein domain Acetyltransf_17 (IPR041380) DNA sequence in plant lactobacillus and chloramphenicol resistance fragment DNA sequence on pNZ5319 plant lactobacillus knockout vector are carried out double exchange, build protein domain Acetyltransf_17 (IPR041380) gene knockout strain.First, design and synthesize specific primers, for increasing the upstream and downstream homology arms of Acetyltransf_17 gene, fragment length is about 500bp.Utilize the genomic DNA of plant lactobacillus CCFM1291 (bacterial strain is disclosed in the patent document that publication number is CN115786190B) as template, obtain upstream and downstream homology arms by pcr amplification.Subsequently, the upstream and downstream homology arms obtained by amplification and the chloramphenicol resistance gene fragment of pNZ5319 carrier are carried out double exchange, build homologous recombination vector. The constructed knockout plasmid was introduced into Lactobacillus plantarum CCFM1291 by electroporation, and after transformation, it was inoculated into a selective medium containing chloramphenicol to screen positive clones. The positive transformants screened were analyzed by PCR and DNA sequencing, and the results confirmed that the protein domain Acetyltransf_17 (IPR041380) gene in Lactobacillus plantarum was successfully knocked out, and the strain was named the knockout strain.

[0095] (2) The Acetyltransf_17 gene fragment was amplified and cloned into the upstream gene fragment of the chloramphenicol resistance fragment of the expression vector pNZ44, and then introduced into the knockout strain constructed in step (1) by electric shock method. The correct strain was verified and named as the complement strain.

[0096] (3) Comparison of biological characteristics of wild-type strains, knockout strains, and complemented strains

[0097] The wild-type strain (Lactobacillus plantarum CCFM1291) and the Acetyltransf_17 knockout strain were inoculated into MRS medium and cultured at 37°C for 24 hours. The OD 600 The growth curves were evaluated by RT-PCR and the results showed that there were no significant differences in growth rate and maximum growth volume between the two groups.

[0098] The wild-type strain (Lactobacillus plantarum CCFM1291), Acetyltransf_17 knockout strain and complement strain were fermented in MRS medium and synthetic medium, respectively, under 37°C constant temperature anaerobic incubator conditions, and the fermentation broth was collected after 12 hours of fermentation, and the acetylcholine content in the fermentation supernatant was detected. The acetylcholine content in the fermentation supernatant was detected using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). The UHPLC-MS detection conditions were Thermo Scientific SyncronisHILIC 1.7μm column, 0.1% formic acid aqueous solution and acetonitrile gradient elution as the mobile phase, and electrospray ionization source (ESI) and positive ion mode were used for mass spectrometry detection. The results showed that the synthesis of acetylcholine was significantly reduced when fermented in MRS medium and synthetic medium, while the Ach production of Lactobacillus plantarum CCFM1291 complement strain increased in MRS medium, but there was no obvious change in synthetic medium. Specifically, in MRS medium, the acetylcholine production of wild-type Lactobacillus plantarum CCFM1291 was 13.87 μg / mL, the production of the knockout strain was 3.87 μg / mL, which was about 70% lower than that of the wild type, and the production of the complement strain was 6.84 μg / mL, which was about 80% higher than that of the knockout strain. In synthetic medium, the acetylcholine production of wild-type Lactobacillus plantarum CCFM1291 was 0.22 μg / mL, and the production of the knockout strain was 0.12 μg / mL, which was about 50% lower than that of the wild type. The production of the complement strain was 0.13 μg / mL, which was almost no significant change compared to the knockout strain. The expression of the Acetyltransf_17 gene in the complement strain may not be as strong as that of the wild-type strain. Even if the gene itself is correctly complemented, differences in transcription and translation efficiency may lead to insufficient protein synthesis, thereby affecting the production of acetylcholine.

[0099] Example 3 Directed screening of probiotics producing acetylcholine

[0100] Fifty strains were randomly selected from Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum, and Lactobacillus salivarius, and the specific information is shown in Table 1. Directed screening efficiency verification was performed.

[0101] Table 1 Strain information

[0102]

[0103]

[0104] First, the whole genomes of these 50 strains were sequenced, and the CD-Search function in the NCBI online database was used to predict the domain structure based on the whole genome amino acid sequences of these strains, and the domain range was limited to the acetylation family. The results showed that the protein domain Acetyltransf_17 (IPR041380) gene was only present in Lactobacillus plantarum. Subsequently, fermentation experiments were carried out on these 50 strains, and the acetylcholine content in the fermentation supernatant was detected by LC-MS (chromatographic conditions). The results showed that acetylcholine was only detected in the fermentation supernatant of Lactobacillus plantarum, which is consistent with the screening results of bioinformatics analysis ( Figure 3 C).

[0105] Example 4: Specific application of the screening method

[0106] The following strains were randomly selected from the bacterial library of the Biotechnology Center of Jiangnan University for screening: 3 strains of Lactobacillus plantarum, 3 strains of Lactobacillus reuteri, 3 strains of Lactobacillus acidophilus, 3 strains of Lactobacillus rhamnosus and 2 strains of Lactobacillus helveticus.

[0107] The above strains were screened according to the following steps:

[0108] (1) Extract the genomic DNA of the strain to be tested and perform PCR amplification using the following primer pairs:

[0109] Forward Primer: 5'-ACCAGATTCCTTGATTAGCA-3';

[0110] Reverse Primer: 5'-CTATACAGTCTACCACTAGCA-3';

[0111] (2) The strain with a 784 bp band selected in step (1) was inoculated in MRS medium at a 2% inoculation rate and fermented for 24 hours, and the fermentation supernatant was taken for liquid chromatography-mass spectrometry (LC-MS) detection.

[0112] The screening result of step (1) shows that in 3 strains of plant lactobacillus to be tested, protein domain Acetyltransf_17 (IPR041380) is amplified, and the remaining strains do not amplify this target fragment. The LC-MS analysis result of step (2) shows that only plant lactobacillus has detected the generation of acetylcholine in the fermentation supernatant. The above results show that the screening method of the present invention can effectively and accurately screen out the probiotics of acetylcholine production, and for other strains not containing this domain, no false positive results occur in this screening method, which further proves the high specificity and accuracy of this screening method.

[0113] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 scope of protection of the present invention should be based on the definition of the claims.

Claims

1. The use of Acetyltransf_17 protein in identifying acetylcholine-producing microorganisms, characterized in that: The Acetyltransf_17 protein has the amino acid sequence shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The acetylcholine-producing microorganism has a gene encoding the Acetyltransf_17 protein.

3. A method for directional screening of acetylcholine-producing microorganisms, characterized in that: The method is to detect the genotype of the microorganism to be detected, and the strain with the marker gene is the acetylcholine-producing strain; the marker gene has the nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO.

2.

4. The method according to claim 3, characterized in that The PCR amplification is performed using the primers shown in SEQ ID NO.4 and SEQ ID NO.

5.

5. The method according to claim 4, characterized in that The method also includes sequencing the amplified DNA fragments.

6. The method according to claim 3 or 4, characterized in that: The specific steps of the method are: (1) Amplify the genomic DNA of the microorganism to be tested using the primers shown in SEQ ID NO.4 and SEQ ID NO.5, and detect the amplified DNA fragments by agarose gel electrophoresis. The target strain is the one that produces specific bands; (2) Cultivating the strain obtained by screening in step (1) in a culture medium and detecting the acetylcholine production in the fermentation broth.

7. The method according to claim 6, characterized in that The detection in step (2) is performed by liquid chromatography-mass spectrometry (LC-MS) for detection and quantitative analysis.

8. The method according to claim 6, characterized in that The culturing is carried out at 30-37° C. for at least 24 hours.

9. A product for identifying, detecting and / or screening acetylcholine-producing probiotics, characterized in that: Contains the primer pair shown by SEQ ID NO.4 and SEQ ID NO.

5.

10. Use of the method according to claims 3 to 8 or the product according to claim 9 in the breeding of acetylcholine-producing probiotics.

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