Acquisition method and application of lactobacillus crispatus specific metabolic substrate and application of specific CAZzyme gene and PUL
By systematically mining the CAZyme gene differences between *Lactobacillus curvaturei* and *Lactobacillus indolentus*, specific metabolic substrates were screened out, solving the problem of increasing the abundance of *Lactobacillus curvaturei* in existing strategies, and achieving improvement in vaginal flora structure and increased ART success rate.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-30
- Publication Date
- 2026-05-12
AI Technical Summary
Existing strategies for improving vaginal microecology are difficult to specifically increase the relative abundance of *Lactobacillus curvature* in the complex vaginal microenvironment, have limited inhibitory effects on *Lactobacillus indolentus*, lack a systematic understanding of the selective interactions between specific substrates and specific species, and lack verifiable evidence chains.
By systematically mining the CAZyme gene differences between *Lactobacillus curvature* and *Lactobacillus indolentus*, constructing a feature matrix and heatmap, we screened out CAZyme genes and PULs that are unique to *Lactobacillus curvature* but not present in *Lactobacillus indolentus*. Combined with the KEGG metabolic pathway map, we verified the metabolic capacity of candidate carbohydrate substrates, prepared specific culture media for culture and verification, and finally determined the specific metabolic substrates of *Lactobacillus curvature*.
It enhances the growth and reproduction of Lactobacillus curvature in the vagina, improves the vaginal flora structure, and increases the success rate of assisted reproductive technology (ART).
Smart Images
Figure CN122024864A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of bioengineering and microbiome technology, specifically relating to a method for obtaining specific metabolic substrates of Lactobacillus curvature, their application, and the application of the specific CAZyme gene and PUL. Background Technology
[0002] Assisted reproductive technology (ART) is a medical procedure to help infertile couples conceive. Studies have shown that the vaginal microecology plays a crucial role in women's reproductive health and ART outcomes. A vaginal flora dominated by *Lactobacillus crispatus* is typically associated with lower vaginal pH, less bacterial vaginosis (BV), and higher pregnancy success rates. Conversely, because *Lactobacillus iners* provides weaker protection to the host epithelium, a flora dominated by this species is more likely to be associated with recurrent BV and an increased risk of sexually transmitted infections, and is also associated with adverse outcomes such as miscarriage and ART implantation failure. It is generally believed that metabolic enzymes, such as carbohydrate-active enzymes, play a vital role in bacterial growth and reproduction. Adding specific *Lactobacillus crispatus* metabolic substrates to selectively promote its growth may improve the effectiveness and success rate of ART. Therefore, selectively promoting the colonization and growth of *Lactobacillus crispatus* in the vaginal environment has become an important direction in current microecological regulation.
[0003] In recent years, carbohydrate-active enzymes (CAZymes) and their associated polysaccharide utilization loci (PULs) have been shown to determine the ability of microorganisms to utilize different carbohydrate substrates. The CAZymes of vaginal lactobacilli produce monosaccharides by breaking down substrates such as glycogen, providing them with essential energy and potentially generating lactic acid and short-chain fatty acids to maintain the acidic environment of the vagina. Therefore, if specific carbohydrate substrates can be obtained that are preferentially utilized by *Lactobacillus curvature* but not readily or easily utilized by *Lactobacillus indolentus*, it is hoped that supplementing with these substrates can selectively promote the growth and colonization of *Lactobacillus curvature*, thereby improving vaginal flora structure and enhancing ART outcomes. Systematic research into the differences between *Lactobacillus indolentus* and *Lactobacillus curvature* at the CAZyme gene level is crucial for screening prebiotics required for *Lactobacillus curvature*-specific metabolic pathways.
[0004] Existing strategies for improving vaginal microecology mainly include exogenous lactobacillus supplementation and broad-spectrum prebiotic supplementation. However, these approaches often fail to specifically increase the relative abundance of *Lactobacillus curvatureensis* in the complex vaginal microenvironment, and have limited inhibitory effects on "non-ideal" lactobacilli such as *Lactobacillus indolentus*. More importantly, existing strategies usually cannot provide a verifiable chain of evidence that "which substrate or type of substrate can be selectively utilized by *Lactobacillus curvatureensis*", and lack a systematic understanding of the selective interaction relationship between specific substrates and specific bacterial species.
[0005] The development of bioinformatics has provided new approaches to addressing these issues, with several gene function annotation tools available for analyzing the metabolic characteristics of Lactobacillus. Among them, dbCAN (carbohydrate enzyme gene annotation software) is specifically designed to identify carbohydrate-active enzymes (CAZymes) in the genome, annotating protein sequences and PULs based on characteristic structural domains. eggNOG gene annotation software is a commonly used homology alignment and functional annotation tool, capable of functionally annotating the genome based on existing homologous genes, and its annotation results can be mapped to KEGG metabolic pathways, thus placing specific genes within the macroscopic context of metabolic pathway networks to understand their functions.
[0006] However, at the genomic analysis level, software such as dbCAN and eggNOG offer fragmented and generalized functions, each providing annotation information for only one dimension. They lack standardized and systematic professional analytical procedures for comparing differences in carbohydrate metabolism among different lactobacillus species. Currently, no research has systematically explored the CAZyme gene differences between *Lactobacillus curlis* and *Lactobacillus indolentus*, and there is a lack of a complete method to integrate CAZyme differences with substrate screening and apply it to "promoting *Lactobacillus curlis*, improving the female vaginal microecology, and ART outcomes." Summary of the Invention
[0007] In view of this, the present invention provides a method for obtaining specific metabolic substrates of Lactobacillus curvature, comprising the following steps:
[0008] Step 1: Obtain genomic data of multiple strains of two types of lactobacillus, Lactobacillus curlis and Lactobacillus inertis, from public databases and perform preprocessing;
[0009] Step 2: Gene annotation is performed on the preprocessed genomic data, including annotation of carbohydrate-active enzyme (CAZyme) genes and polysaccharide utilization loci (PUL); and whole-genome functional annotation is performed on the genome; the PUL is a gene cluster in the bacterial genome related to the metabolism of a specific sugar, and the annotation results include the sugar substrates that the PUL can metabolize;
[0010] Step 3: Based on the annotation information of carbohydrate-active enzyme (CAZyme) genes and polysaccharide utilization sites (PULs) described in Step 2, a feature matrix representing the genetic background differences between *Lactobacillus curvatureii* and *Lactobacillus indolentus* is constructed. An intuitive genetic background difference heatmap is generated based on this feature matrix. Subsequently, the feature matrix is trained using a random forest algorithm, and the random forest model generates a ranking of the feature importance of the differential CAZyme genes and PULs between the two lactobacilli. Combining the feature importance ranking with the genetic background difference heatmap, multiple CAZyme genes or PULs are screened. If the screening criteria are met, this CAZyme gene or PUL is considered a CAZyme gene or PUL unique to *Lactobacillus curvatureii* but not present in *Lactobacillus indolentus*. Furthermore, based on the results of whole-genome functional annotation of the genomes of *Lactobacillus curvatureii* and *Lactobacillus indolentus* in Step 2, the whole-genome genes of the two bacteria are classified according to the KEGG metabolic pathways in which the genes reside, and different colors are used to represent different bacterial species and mapped onto the KEGG metabolic pathway map to identify genes and KEGG metabolic pathways unique to *Lactobacillus curvatureii* but not present in *Lactobacillus indolentus*.
[0011] Step 4: Based on the CAZyme gene, PUL, and KEGG metabolic pathway unique to Lactobacillus curvature and not found in Lactobacillus in Step 3, a set of candidate carbohydrate substrates that can be specifically metabolized by Lactobacillus curvature were screened.
[0012] Step 5: Construct a culture medium containing the specific carbohydrate substrates mentioned above, and set up standard MRS medium and sugar-free medium as controls. Inoculate *Lactobacillus curvature* and control strains, using inert *Lactobacillus*, into different media and culture them at 37℃ under anaerobic conditions. Measure the OD value of the bacterial solution at regular intervals and plot growth curves to verify the metabolic capacity of *Lactobacillus curvature* for the specific substrates. If *Lactobacillus curvature* is in a stable phase in the specific substrate medium and its OD value is significantly higher than its OD value in the sugar-free medium (P < 0.05), it indicates that the substrate can be effectively metabolized by *Lactobacillus curvature*. This updates the candidate carbohydrate substrate set that *Lactobacillus curvature* can specifically metabolize, resulting in the final metabolic substrate set. A higher OD value indicates more bacteria in the culture dish, higher light occlusion, and better bacterial growth, indicating that the bacteria can utilize the corresponding substrate to accelerate growth and reproduction.
[0013] Furthermore, the public database includes the NCBI Genome Database; the preprocessing in step 1 includes removing low-quality, incomplete genome sequences.
[0014] Furthermore, the genome was annotated with carbohydrate-active enzyme (CAZyme) genes and polysaccharide utilization sites (PULs). A combination of HMMER and DIAMOND tools was used for analysis, with annotation databases including dbCAN HMMdb, CAZyDB, and dbCAN-sub to ensure the accuracy of CAZyme gene and PUL annotations. Sequence alignment was performed between the bacterial genome and the database genes to obtain the KEGG ortholog (KO) group number, enzyme classification number (EC), and KEGG reaction number. This information will be used to map genes specific to a single Lactobacillus species and genes common to multiple Lactobacillus species to the KEGG metabolic pathway.
[0015] Furthermore, in step 3, when constructing the feature matrix, each CAZyme family (such as GH1, GH36, GT101, etc.) and PUL (such as PUL0048, PUL0088, etc.) are used as feature units, and each strain of Lactobacillus curvature and Lactobacillus inertia are used as sample units. In the genome of each strain of Lactobacillus curvature and Lactobacillus inertia, the presence of the feature is recorded as 1, and its absence is recorded as 0. The feature matrix here is equivalent to a grid diagram, with the horizontal axis representing the strains of each bacterium and the vertical axis representing each gene. Each cell in the grid diagram has a corresponding strain and gene. If the genome of this strain contains the gene, it is recorded as "1", and if it does not contain the gene, it is recorded as "0". The subsequent heatmap generation is actually the generation of a grid map; when training the random forest algorithm with the feature matrix, the number of decision trees is set to 100-200, and the algorithm parameters are optimized through 5-fold cross-validation; when screening for CAZyme genes or PULs that are specific to Lactobacillus curvature but not to Lactobacillus indolentus, if a certain CAZyme gene or PUL is present in more than 70% of the Lactobacillus curvature strain genomes and not present in more than 90% of the Lactobacillus indolentus strain genomes, then this gene or PUL is considered a CAZyme gene or PUL that is specific to Lactobacillus curvature but not to Lactobacillus indolentus.
[0016] Further: In step 3, the KEGG metabolic pathway maps are image files of target metabolic pathway maps obtained directly from the KEGG website, including but not limited to map00010 glycolysis / gluconeogenesis, map00500 starch and sucrose metabolism, and map02010 ABC transporter pathway maps. Based on the results of whole-genome functional annotation of the two bacterial genomes in step 2, each KEGG metabolic pathway map is labeled according to whether *Lactobacillus curvature* and *Lactobacillus indolent* have the corresponding genes. Genes specific to *Lactobacillus curvature* are marked in green, genes specific to *Lactobacillus indolent* are marked in blue, and genes shared by both bacteria are marked in two colors to visually distinguish and clarify the unique genes and specific metabolic pathways of *Lactobacillus curvature*. The above labeling steps in the KEGG metabolic pathway maps are used to identify key metabolic nodes specific to or enriched in *Lactobacillus curvature* at the metabolic pathway level and associate them with CAZyme genes and PUL differential characteristics to support subsequent candidate substrate screening and culture validation.
[0017] Further: In step 5, the substrate concentration for preparing the specific sugar substrate was set at 5, 10, and 20 g / L. The basal medium was MRS medium (with the original carbon source removed). During the culture process, the OD value of the bacterial solution in the medium was measured using an enzyme-linked immunosorbent assay (ELISA) reader to reflect the growth and reproduction of the bacteria. The measurement wavelength was 600 nm, the culture time was 24 hours, and the measurement interval was 6 hours. The growth curve was plotted using GraphPad Prism software. By comparing the growth retardation time, logarithmic growth rate, and stationary phase OD value of *Lactobacillus curvature* and *Lactobacillus inertia* in the specific substrate medium and the control medium, their metabolic capacity for the substrate and their growth capacity in this substrate or the inhibitory capacity of the substrate on the growth of this bacterium were determined. If *Lactobacillus curvature* was in the stationary phase in the specific substrate medium and its OD value was significantly higher than its OD value in the sugar-free medium (P < 0.05), it indicated that the substrate could be effectively metabolized by *Lactobacillus curvature*.
[0018] According to another aspect of the present invention, an application of *Lactobacillus curvature*-specific CAZyme gene and PUL is provided, wherein substrates that *Lactobacillus curvature* can specifically metabolize are screened based on the gene or PUL, and the substrates are used to prepare products that improve vaginal microecology; the *Lactobacillus curvature*-specific CAZyme gene and PUL are identified by the above method; the specific CAZyme gene and PUL include, but are not limited to, GH36 (glycoside hydrolase family 36) gene (aga, 3.2.1.22), GH13_29 (glycoside hydrolase family 13_29) gene (treC, 3.2.1.93), GH1 (glycoside hydrolase family 1) gene (pbg4, pbg6, 3.2.1.86), PUL0048 (containing GH13_29), PUL0088 (containing GH36), and metabolic co-preparations containing the enzymes corresponding to the above genes.
[0019] This invention also provides an application of a specific metabolic substrate of *Lactobacillus curvaturei* for preparing a product that improves the success rate of assisted reproductive technology. The specific metabolic substrate of *Lactobacillus curvaturei* is obtained by any of the methods described in claims 1-6. The product is a vaginal microecological regulator containing the above substrate, which promotes the growth of *Lactobacillus curvaturei* in the vagina and inhibits the growth of *Lactobacillus inertia* by supplementing the substrate, thereby improving the vaginal flora structure and thus improving the success rate of ART.
[0020] The advantage of the method described in this invention lies in its ability to systematically screen carbohydrate metabolism-related features of *Lactobacillus curvature* and *Lactobacillus indolent* based on multi-strain-scale genomic information and multi-source annotation results. This information is then correlated with metabolic pathway information to form a candidate substrate set. Further, culture validation is used to form a closed loop, ultimately identifying *Lactobacillus curvature*-specific metabolic substrates, thereby improving the accuracy and reliability of substrate acquisition. The ultimate goal of screening for substrates specifically metabolized by *Lactobacillus curvature* is to promote the growth and reproduction of *Lactobacillus curvature* through these substrates, increasing its proportion in the vaginal microecology, thereby protecting the vagina and reducing the proliferation of other harmful bacteria. This improves the success rate of assisted reproductive technology. Attached Figure Description
[0021] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.
[0022] 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. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0023] Figure 1This is a flowchart illustrating the gene annotation of the genomes of *Lactobacillus curvaturei* and *Lactobacillus indolenti* according to the present invention.
[0024] Figure 2 This is a flowchart illustrating how the present invention converts dbCAN annotations into heatmaps and uses a random forest model to generate differential gene sorting.
[0025] Figure 3 This is a flowchart illustrating how the whole genome annotation is represented in text form and mapped to the KEGG metabolic pathway map in this invention;
[0026] Figure 4 This is a flowchart illustrating the experimental evaluation of the growth of bacteria such as Lactobacillus curvature in a culture medium for the present invention. Detailed Implementation
[0027] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numerals in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely examples of systems consistent with some aspects of the invention as detailed in the appended claims.
[0028] refer to Figure 1-4 This invention provides a method for obtaining specific metabolic substrates of *Lactobacillus curvatureensis*. The specific implementation of this invention includes five main stages: data preparation, gene annotation, data integration, substrate screening, and experimental verification. Specifically,
[0029] Step 1: First, obtain the genomic data of Lactobacillus crispatus and Lactobacillus iners from the NCBI database.
[0030] Computers preprocessed the genomes of a large number of Lactobacillus curvature and Lactobacillus inertia strains from NCBI, such as removing low-quality and incomplete genome sequences, to ensure the breadth and representativeness of the results.
[0031] Step 2: Gene annotation is performed on the preprocessed genomic data, including annotation of carbohydrate-active enzyme (CAZyme) genes and polysaccharide utilization sites (PULs) in the genome; and whole-genome functional annotation of the genome. Each of these gene annotation processes generates a tab-delimited value file representing information such as the genes and their functions corresponding to each sequence segment in the bacterial genome. The PULs are gene clusters in the bacterial genome related to the metabolism of a certain type of carbohydrate, and the annotation results include the carbohydrate substrates that the PULs can metabolize.
[0032] Step 3: Based on the annotation information of carbohydrate-active enzyme (CAZyme) genes and polysaccharide utilization sites (PULs) described in Step 2, construct a feature matrix representing the genetic background differences between *Lactobacillus curvatureii* and *Lactobacillus indolentus*. Gene background difference heatmaps are generated based on this feature matrix to allow direct visual observation of differentially expressed genes. The feature matrix is trained using a random forest algorithm, and the random forest model generates a ranking of the feature importance of differentially expressed CAZyme genes and PULs between the two lactobacilli. Since the feature importance ranking represents all genes of the two lactobacilli studied, screening is required. Therefore, comparing the gene background difference heatmaps, if a certain CAZyme gene or PUL is present in more than 70% of the *Lactobacillus curvatureii* strain genomes and not present in more than 90% of the *Lactobacillus indolentus* strain genomes, then this gene or PUL is considered a CAZyme unique to *Lactobacillus curvatureii* and not present in *Lactobacillus indolentus*. Genes and PUL were used to evaluate the ability of differentially expressed genes selected by the random forest algorithm to distinguish strains. Based on the results of whole-genome functional annotation of Lactobacillus curvature and Lactobacillus inertia in step 2, the whole-genome genes of the two bacteria were classified according to the KEGG metabolic pathways in which the genes are located, and different colors were used to represent different bacterial species and mapped to the KEGG metabolic pathway map to identify genes and metabolic pathways that are unique to Lactobacillus curvature but not to Lactobacillus inertia.
[0033] Step 4: Based on the CAZyme gene, PUL and KEGG metabolic pathways that are unique to Lactobacillus curvature and not found in Lactobacillus in Step 3, a set of candidate carbohydrate substrates that can be specifically metabolized by Lactobacillus curvature were screened.
[0034] Step 5: Construct a culture medium containing the specific carbohydrate substrates mentioned above, and set up standard MRS medium and sugar-free medium as controls; inoculate *Lactobacillus curvaturei* and control strains (inert *Lactobacillus*) into different media, and culture them at 37℃ under anaerobic conditions. Measure the OD value of the bacterial solution at regular intervals, and plot growth curves to verify the metabolic capacity of *Lactobacillus curvaturei* for the specific substrates. If *Lactobacillus curvaturei* is in a stable phase in the specific substrate medium and its OD value is significantly higher than its OD value in the sugar-free medium (P < 0.05), it indicates that the substrate can be effectively metabolized by *Lactobacillus curvaturei*. This updates the candidate carbohydrate substrate set that *Lactobacillus curvaturei* can specifically metabolize, resulting in the final set of metabolic substrates. A higher OD value indicates more bacteria in the culture dish, higher light shading, and more bacteria, indicating better bacterial growth and that the bacteria can utilize the corresponding substrate to accelerate growth and reproduction. As an improvement to the scheme, the public database mentioned includes the NCBI Genome Database; the preprocessing in Step 1 includes removing low-quality and incomplete genomic sequences.
[0035] As an improvement to the protocol, carbohydrate-active enzyme (CAZyme) genes and polysaccharide utilization sites (PULs) were annotated in the genome. The HMMER and DIAMOND tools were used in combination for analysis, and the annotation databases included dbCAN HMMdb, CAZyDB, and dbCAN-sub to ensure the accuracy of CAZyme gene and PUL annotations. Sequence alignment was performed between the bacterial genome and the database genes to obtain the KEGG ortholog (KO) group number, enzyme classification number (EC), and KEGG reaction number of the genes. This information will be used to map genes specific to a single Lactobacillus species and genes common to multiple Lactobacillus species to the KEGG metabolic pathway.
[0036] As an improvement to the scheme, in step 3, when constructing the feature matrix, each CAZyme family (such as GH1, GH36, GT101, etc.) and PUL (such as PUL0048, PUL0088, etc.) are used as feature units, and each strain of *Lactobacillus curvatureii* and *Lactobacillus indolentus* is used as a sample unit. In the genome of each strain of *Lactobacillus curvatureii* and *Lactobacillus indolentus*, the presence of the feature is recorded as 1, and its absence is recorded as 0. The feature matrix here is equivalent to a grid diagram, with the horizontal axis representing the bacterial strains and the vertical axis representing the genes. Each cell in the grid diagram corresponds to a strain and a gene. If the strain's genome contains the gene, it is recorded as "1", and if it does not contain the gene, it is recorded as "0". The subsequent generation of the heatmap is actually the generation of a grid diagram. When training the random forest algorithm with the feature matrix, the number of decision trees is set to 100-200, and the algorithm parameters are optimized through 5-fold cross-validation.
[0037] As an improvement to the scheme: In step 3, the KEGG metabolic pathway maps are image files of target metabolic pathway maps obtained directly from the KEGG website, including but not limited to map00010 glycolysis / gluconeogenesis, map00500 starch and sucrose metabolism, and map02010 ABC transporter pathway maps; and based on the results of whole-genome functional annotation of the two bacterial genomes in step 2, each KEGG metabolic pathway map is labeled according to whether Lactobacillus curvature and Lactobacillus indolent have the corresponding genes. Genes specific to Lactobacillus curvature are marked in green, genes specific to Lactobacillus indolent are marked in blue, and genes shared by the two bacteria are marked in two colors, so as to visually distinguish and clarify the unique genes and specific metabolic pathways of Lactobacillus curvature. The above labeling steps in the KEGG metabolic pathway maps are used to identify key metabolic nodes that are unique to or enriched in Lactobacillus curvature at the metabolic pathway level and associate them with CAZyme genes and PUL differential features to support subsequent candidate substrate screening and culture validation.
[0038] As an improvement to the protocol: In step 5, the substrate concentration for preparing the culture medium containing the specific sugar substrate was set to three levels: 5, 10, and 20 g / L. The basal medium was MRS medium (with the original carbon source removed). During the culture process, the OD value of the bacterial culture was measured using an enzyme-linked immunosorbent assay (ELISA) reader to reflect the growth and reproduction of the bacteria. The measurement wavelength was 600 nm, the culture time was 24 hours, and the measurement interval was 6 hours. The growth curve was plotted using GraphPad Prism software. By comparing the growth retardation time, logarithmic growth rate, and stationary phase OD value of *Lactobacillus curvature* and *Lactobacillus inertia* in the specific substrate medium and the control medium, their metabolic capacity for the substrate and their growth capacity in this substrate or the inhibitory capacity of the substrate on the bacteria were determined. If *Lactobacillus curvature* was in the stationary phase in the specific substrate medium and its OD value was significantly higher than its OD value in the sugar-free medium (P < 0.05), it indicated that the substrate could be effectively metabolized by *Lactobacillus curvature*.
[0039] According to another aspect of the present invention, an application of *Lactobacillus curvature*-specific CAZyme gene and PUL is provided, wherein substrates that *Lactobacillus curvature* can specifically metabolize are screened based on the gene or PUL, and the substrates are used to prepare products that improve vaginal microecology; the *Lactobacillus curvature*-specific CAZyme gene and PUL are identified by the above method; the specific CAZyme gene and PUL include, but are not limited to, GH36 (glycoside hydrolase family 36) gene (aga, 3.2.1.22), GH13_29 (glycoside hydrolase family 13_29) gene (treC, 3.2.1.93), GH1 (glycoside hydrolase family 1) gene (pbg4, pbg6, 3.2.1.86), PUL0048 (containing GH13_29), PUL0088 (containing GH36), and metabolic co-preparations containing the enzymes corresponding to the above genes.
[0040] This invention also provides an application of a specific metabolic substrate of *Lactobacillus curvaturei* for preparing a product that improves the success rate of assisted reproductive technology. The specific metabolic substrate of *Lactobacillus curvaturei* is obtained by any of the methods described above. The product is a vaginal microecological regulator containing the substrate, which promotes the growth of *Lactobacillus curvaturei* in the vagina and inhibits the growth of *Lactobacillus inertia* by supplementing the substrate, thereby improving the vaginal flora structure and thus improving the success rate of ART.
[0041] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these changes and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for obtaining specific metabolic substrates of Lactobacillus curvature, characterized in that, Includes the following steps, Step 1: Obtain genomic data of multiple strains of two types of lactobacillus, Lactobacillus curlis and Lactobacillus inertis, from public databases and perform preprocessing; Step 2: Gene annotation is performed on the preprocessed genomic data, including annotation of the CAZyme gene (a carbohydrate-active enzyme) and polysaccharide utilization sites (PULs) in the genome; and whole-genome functional annotation is performed on the genome; the PULs are gene clusters in the bacterial genome that are related to the metabolism of a specific type of carbohydrate, and the annotation results include the carbohydrate substrates that the PULs can metabolize. Step 3: Based on the annotation information of the carbohydrate-active enzyme CAZyme gene and polysaccharide utilization site PUL described in Step 2, construct a feature matrix representing the genetic background differences between *Lactobacillus curvatureii* and *Lactobacillus indolentii*; generate a genetic background difference heatmap based on this feature matrix; then train the feature matrix using the random forest algorithm, and use the random forest model to generate a ranking of the feature importance of the difference CAZyme gene and PUL between the two lactobacilli. Combining feature importance ranking with the gene background difference heatmap, multiple CAZyme genes or PULs were screened. If the screening criteria were met, the CAZyme gene or PUL was considered to be a CAZyme gene or PUL unique to Lactobacillus curvature and not present in Lactobacillus indolentus. Based on the results of whole-genome functional annotation of Lactobacillus curvature and Lactobacillus indolentus in step 2, the whole-genome genes of the two bacteria were classified according to the KEGG metabolic pathways in which the genes are located, and different colors were used to represent different bacterial species and mapped to the KEGG metabolic pathway map to identify genes and KEGG metabolic pathways unique to Lactobacillus curvature and not present in Lactobacillus indolentus. Step 4: Based on the CAZyme gene, PUL, and KEGG metabolic pathway unique to Lactobacillus curvature and not found in Lactobacillus in Step 3, a set of candidate carbohydrate substrates that can be specifically metabolized by Lactobacillus curvature were screened. Step 5: Construct a culture medium containing the specific sugar substrates mentioned above, and set up standard MRS medium and sugar-free medium as controls; inoculate *Lactobacillus curvaturei* and inert *Lactobacillus* as controls into different culture media, and culture them at 37°C under anaerobic conditions. Measure the OD value of the bacterial solution at regular intervals, and plot the growth curve to verify the metabolic capacity of *Lactobacillus curvaturei* for the specific substrate; if *Lactobacillus curvaturei* is in the stationary phase in the specific substrate medium and its OD value is significantly higher than its OD value in the sugar-free medium (P < 0.05), it indicates that the substrate can be effectively metabolized by *Lactobacillus curvaturei*. This updates the set of candidate sugar substrates that *Lactobacillus curvaturei* can specifically metabolize, and obtains the final set of metabolic substrates.
2. The method as described in claim 1, characterized in that: The public database mentioned includes the NCBI Genome Database; the preprocessing in step 1 includes removing low-quality, incomplete genome sequences.
3. The method as described in claim 1, characterized in that, The genome was annotated with the CAZyme gene (a carbohydrate-active enzyme) and the PUL (polysaccharide utilization site). The HMMER and DIAMOND tools were used in combination for analysis. Annotation databases included dbCAN HMMdb, CAZyDB, and dbCAN-sub. Sequence alignment was performed between the bacterial genome and the database genes to obtain the KEGG ortholog group (KO) number, enzyme classification number (EC), and KEGG reaction number. This information will be used to map genes specific to a single Lactobacillus species and genes common to multiple Lactobacillus species to the KEGG metabolic pathway.
4. The method as described in claim 1, characterized in that: In step 3, when constructing the feature matrix, each CAZyme family and PUL is used as a feature unit, and each strain of *Lactobacillus curvaturei* and *Lactobacillus indolenti* is used as a sample unit. In the genome of each strain of *Lactobacillus curvaturei* and *Lactobacillus indolenti*, the presence of the feature is recorded as 1, and its absence is recorded as 0. The feature matrix here is equivalent to a grid diagram, with the horizontal axis representing the strains of each bacterium and the vertical axis representing each gene. Each cell in the grid diagram corresponds to a strain and a gene. If the genome of this strain contains the gene, it is recorded as 1, and if it does not contain the gene, it is recorded as 0. When screening for CAZyme genes or PULs that are specific to *Lactobacillus curvaturei* but not present in *Lactobacillus indolenti*, if a certain CAZyme gene or PUL is present in more than 70% of the genomes of *Lactobacillus curvaturei* strains and is not present in the genomes of more than 90% of the *Lactobacillus indolenti* strains, then this gene or PUL is considered a CAZyme gene or PUL that is specific to *Lactobacillus curvaturei* but not present in *Lactobacillus indolenti*.
5. The method as described in claim 1, characterized in that: In step 3, the KEGG metabolic pathway maps are image files of the target metabolic pathway maps obtained directly from the KEGG website, including but not limited to map00010 glycolysis / gluconeogenesis, map00500 starch and sucrose metabolism, and map02010 ABC transporter pathway maps. Based on the results of whole-genome functional annotation of the two bacterial genomes in step 2, each KEGG metabolic pathway map is labeled according to whether Lactobacillus curvature and Lactobacillus indolent have the corresponding genes. Genes specific to Lactobacillus curvature are marked in green, genes specific to Lactobacillus indolent are marked in blue, and genes shared by the two bacteria are marked in two colors, so as to visually distinguish and clarify the unique genes and specific metabolic pathways of Lactobacillus curvature. The above steps of labeling in the KEGG metabolic pathway map are used to identify key metabolic nodes that are unique to or enriched in Lactobacillus curvature at the metabolic pathway level, and to associate them with CAZyme genes and PUL differential characteristics to support subsequent candidate substrate screening and culture validation.
6. The method as described in claim 1, characterized in that: In step 5, the substrate concentration for preparing the specific sugar substrate was set at 5, 10, and 20 g / L. The basal medium was MRS medium (with the original carbon source removed). During the culture process, the OD value of the bacterial culture was measured using an enzyme-linked immunosorbent assay (ELISA) reader to reflect the growth and reproduction of the bacteria. The measurement wavelength was 600 nm, the culture time was 24 hours, and the measurement interval was 6 hours. The growth curve was plotted using GraphPad Prism software. By comparing the growth retardation time, logarithmic growth rate, and stationary phase OD value of *Lactobacillus curvature* and *Lactobacillus inertia* in the specific substrate medium and the control medium, their metabolic capacity for the substrate and their growth capacity in this substrate or the inhibitory capacity of the substrate on the growth of this bacterium were determined. If *Lactobacillus curvature* was in the stationary phase in the specific substrate medium and its OD value was significantly higher than its OD value in the sugar-free medium (P < 0.05), it indicated that the substrate could be effectively metabolized by *Lactobacillus curvature*.
7. A specific CAZyme gene for Lactobacillus curvature and its application in PUL, characterized in that, Based on the screening of substrates that *Lactobacillus curvature* can specifically metabolize, the substrates are used to prepare products that improve vaginal microecology; the *Lactobacillus curvature*-specific CAZyme gene and PUL are identified by the method in step 3 of claim 1; the specific CAZyme gene and PUL include, but are not limited to, GH36 (glycoside hydrolase family 36) gene (aga, 3.2.1.22), GH13_29 (glycoside hydrolase family 13_29) gene (treC, 3.2.1.93), GH1 (glycoside hydrolase family 1) gene (pbg4, pbg6, 3.2.1.86), PUL0048 (containing GH13_29), PUL0088 (containing GH36), and metabolic co-preparations containing the enzymes corresponding to the above genes.
8. The application of a specific metabolic substrate of Lactobacillus curvature, characterized in that, For the preparation of products that improve the success rate of assisted reproductive technology, the specific metabolic substrate of *Lactobacillus curvaturei* is obtained by any of the methods described in claims 1-6; the product is a vaginal microecological regulator containing the above substrate, which promotes the growth of *Lactobacillus curvaturei* in the vagina and inhibits the growth of *Lactobacillus inertia* by supplementing the substrate, thereby improving the vaginal flora structure and thus improving the success rate of ART.