Investigating the Role of ß-Disodium Glycerophosphate and Urea in Promoting Growth of Streptococcus thermophilus from Omics-Integrated Genome-Scale Models.

This study investigates the impact of urea and ß-GP on the growth of Streptococcus thermophilus S-3, a bacterium commonly used in industrial fermentation processes. Through a series of growth experiments, transcriptome, metabolome, and omics-based analyses, the research demonstrates that both urea and ß-GP can enhance the biomass of S. thermophilus, with urea showing a more significant effect. The optimal urea concentration for growth was determined to be 3 g/L in M17 medium. The study also highlights the metabolic pathways influenced by urea and ß-GP, particularly the galactose metabolism pathway, which is crucial for cell growth when lactose is the substrate. The integration of omics data into the genome-scale metabolic model of S. thermophilus, iCH502, allowed for a more accurate prediction of metabolic fluxes and growth rates. The study concludes that urea can serve as a viable substitute for ß-GP in the cultivation of S. thermophilus, offering potential cost and efficiency benefits in industrial fermentation processes. The findings are supported by validation experiments with 11 additional strains of S. thermophilus, which showed increased biomass in UM17 medium.

Understanding Ligilactobacillus salivarius from Probiotic Properties to Omics Technology: A Review.

Ligilactobacillus salivarius (basonym: Lactobacillus salivarius, L. salivarius) is a type of lactic acid bacteria (LAB) commonly found in the oropharyngeal-gastrointestinal tract (OGT). It has gained significant attention due to its probiotic and functional properties as well as its various health-promoting roles. L. salivarius strains exhibit strong resistance and adhesion in the OGT along with outstanding antioxidant and antimicrobial properties. Additionally, numerous L. salivarius strains have the ability to produce bacteriocins with antagonistic activity. These probiotic characteristics of L. salivarius indicate its remarkable potential in promoting favorable effects on human health. It has also been observed that L. salivarius has a positive effect on the composition of intestinal microbiota, thereby improving the metabolic profiling of intestinal microbiota, promoting a healthy and balanced internal environment. In recent years, multi-omics technologies such as genomics, transcriptomics, proteomics and metabolomics have been employed to gain a deeper understanding of the roles and mechanisms of L. salivarius associated with its functional properties. This review aims to provide an overview of the probiotic characteristics of L. salivarius, containing its specific interactions with the host microflora, as well as insights from omics studies.

Preparation of type 3 rice resistant starch using high-pressure homogenous coenzyme treatment and investigating its potential therapeutic effects on blood glucose and intestinal flora in db/db mice.

Resistant starch from rice was prepared using high-pressure homogenization and branched chain amylase treatment. The yield, starch external structure, thermal properties, and crystal structure of rice-resistant starch prepared in different ways were investigated. The results showed that the optimum homogenizing pressure was 90 MPa, the optimum digestion time was 4 h, the optimum concentration of branched-chain amylase was 50 U/g and the yield of resistant starch was 38.58 %. Scanning electron microscopy results showed a rougher surface and more complete debranching of the homogenized coenzyme rice-resistant starch granules. FT-IR and X-ray diffraction results showed that the homogenization treatment exhibited a spiral downward trend on rice starch relative crystallinity and a spiral upward trend on starch debranching and recrystallization. The 4-week dietary intervention in db/db type 2 diabetic mice showed that homogeneous coenzyme rice-resistant starch had a better glycemic modulating effect than normal debranched starch and had a tendency to interfere with the index of liver damage in T2DM mice. Additionally, homogeneous coenzyme rice-resistant starch proved more effective in improving intestinal flora disorders and enhancing the abundance of probiotics in T2DM mice.

Characterization of a Panel of Constitutive Promoters from Lactococcus cremoris for Fine-Tuning Gene Expression.

Lactococcus cremoris (homotypic synonym: Lactococcus lactis) is receiving increasing attention as a prominent vehicle for the delivery of live vaccines. This can hardly be achieved without developing tools for the genetic manipulation of L. cremoris, and the paucity of studies on L. cremoris endogenous promoters has attracted our attention. Here, we report the discovery and characterization of 29 candidate promoters identified from L. cremoris subsp. cremoris NZ9000 by RNA sequencing analysis. Furthermore, 18 possible constitutive promoters were obtained by RT-qPCR screening from these 29 candidate promoters. Then, these 18 promoters were cloned and characterized by a reporter gene, gusA, encoding ß-glucuronidase. Eventually, eight endogenous constitutive promoters of L. cremoris were obtained, which can be applied to genetic manipulation of lactic acid bacteria.

Fabrication of zein-tamarind seed polysaccharide-curcumin nanocomplexes: their characterization and impact on alleviating colitis and gut microbiota dysbiosis in mice.

In this work, a zein-tamarind seed polysaccharide (TSP) co-delivery system was fabricated using an anti-solvent precipitation method. The formation mechanism, characterization, and effect on alleviating colitis and gut microbiota dysbiosis in mice of zein-TSP-curcumin (Z/T-Cur) nanocomplexes were investigated. Hydrogen bonding and the hydrophobic effect played a key role in the formation of Z/T-Cur nanocomplexes, and the interactions were spontaneous and driven by enthalpy. The encapsulation efficiency, loading capacity, and bioavailability increased from 60.8% (Zein-Cur) to 91.7% (Z/T-Cur1:1), from 6.1% (Zein-Cur) to 18.3% (Z/T-Cur1:1), and from 4.7% (Zein-Cur) to 20.0% (Z/T-Cur1:1), respectively. The Z/T-Cur significantly alleviated colitis symptoms in DSS-treated mice. Additionally, the prepared nanocomplexes rebalanced the gut microbiota composition of colitis mice by increasing the abundance of Akkermansia. Odoribacter and Monoglobus were rich in the Z-T-Cur treatment group, and Turicibacter and Bifidobacterium were rich in the zein-TSP treatment group. This study demonstrated that the TSP could be helpful in the targeted drug delivery system.

Kisspeptin-10 binding to Gpr54 in osteoclasts prevents bone loss by activating Dusp18-mediated dephosphorylation of Src.

Osteoclasts are over-activated as we age, which results in bone loss. Src deficiency in mice leads to severe osteopetrosis due to a functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCRs) are the targets for ∼35% of approved drugs but it is still unclear how GPCRs regulate Src kinase activity. Here, we reveal that GPR54 activation by its natural ligand Kisspeptin-10 (Kp-10) causes Dusp18 to dephosphorylate Src at Tyr 416. Mechanistically, Gpr54 recruits both active Src and the Dusp18 phosphatase at its proline/arginine-rich motif in its C terminus. We show that Kp-10 binding to Gpr54 leads to the up-regulation of Dusp18. Kiss1, Gpr54 and Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss, and Kp-10 abrogated bone loss by suppressing osteoclast activity in vivo. Therefore, Kp-10/Gpr54 is a promising therapeutic target to abrogate bone resorption by Dusp18-mediated Src dephosphorylation.

Genome-scale reconstruction of the metabolic network in Streptococcus thermophilus S-3 and assess urea metabolism.

BACKGROUND: Streptococcus thermophilus is an important strain widely used in dairy fermentation, with distinct urea metabolism characteristics compared to other lactic acid bacteria. The conversion of urea by S. thermophilus has been shown to affect the flavor and acidification characteristics of milk. Additionally, urea metabolism has been found to significantly increase the number of cells and reduce cell damage under acidic pH conditions, resulting in higher activity. However, the physiological role of urea metabolism in S. thermophilus has not been fully evaluated. A deep understanding of this metabolic feature is of great significance for its production and application. Genome-scale metabolic network models (GEMs) are effective tools for investigating the metabolic network of organisms using computational biology methods. Constructing an organism-specific GEM can assist us in comprehending its characteristic metabolism at a systemic level. RESULTS: In the present study, we reconstructed a high-quality GEM of S. thermophilus S-3 (iCH492), which contains 492 genes, 608 metabolites and 642 reactions. Growth phenotyping experiments were employed to validate the model both qualitatively and quantitatively, yielding satisfactory predictive accuracy (95.83%), sensitivity (93.33%) and specificity (100%). Subsequently, a systematic evaluation of urea metabolism in S. thermophilus was performed using iCH492. The results showed that urea metabolism reduces intracellular hydrogen ions and creates membrane potential by producing and transporting ammonium ions. This activation of glycolytic fluxes and ATP synthase produces more ATP for biomass synthesis. The regulation of fluxes of reactions involving NAD(P)H by urea metabolism improves redox balance. CONCLUSION: Model iCH492 represents the most comprehensive knowledge-base of S. thermophilus to date, serving as a potent tool. The evaluation of urea metabolism led to novel insights regarding the role of urease. © 2023 Society of Chemical Industry.

Software-based screening for efficient sgRNAs in Lactococcus lactis.

BACKGROUND: The two essential editing elements in the clustered regularly interspaced short palindromic repeats (CRISPR) editing system are promoter and single-guide RNA (sgRNA), the latter of which determines whether Cas protein can precisely target a specific location to edit the targeted gene. Therefore, the selection of sgRNA is crucial to the efficiency of the CRISPR editing system. Various online prediction tools for sgRNA are currently available. These tools can predict all possible sgRNAs of the targeted gene and rank sgRNAs according to certain scoring criteria according to the demands of the user. RESULTS: We designed sgRNAs for Lactococcus lactis NZ9000 LLNZ_RS02020 (ldh) and LLNZ_RS10925 (upp) individually using online prediction software - CRISPOR - and successfully constructed a series of knockout strains to allow comparison of the knockout efficiency of each sgRNA and analyze the differences between software predictions and actual experimental results. CONCLUSION: Our experimental results showed that the actual editing efficiency of the screened sgRNAs did not match the predicted results - a phenomenon that suggests that established findings from eukaryotic studies are not universally applicable to prokaryotes. Software prediction can still be used as a tool for the initial screening of sgRNAs before further selection of suitable sgRNAs through experimental experience. © 2023 Society of Chemical Industry.

Insights into microbial communities and metabolic profiles in the traditional production of the two representative Hongqu rice wines fermented with Gutian Qu and Wuyi Qu based on single-molecule real-time sequencing.

Hongqu rice wine, a famous traditional fermented alcoholic beverage, is brewed with traditional Hongqu (mainly including Gutian Qu and Wuyi Qu). This study aimed to compare the microbial communities and metabolic profiles in the traditional brewing of Hongqu rice wines fermented with Gutian Qu and Wuyi Qu. Compared with Hongqu rice wine fermented with Wuyi Qu (WY), Hongqu rice wine fermented with Gutian Qu (GT) exhibited higher levels of biogenic amines. The composition of volatile flavor components of Hongqu rice wine brewed by different fermentation starters (Gutian Qu and Wuyi Qu) was obviously different. Among them, ethyl acetate, isobutanol, 3-methylbutan-1-ol, ethyl decanoate, ethyl palmitate, ethyl oleate, nonanoic acid, 4-ethylguaiacol, 5-pentyldihydro-2(3H)-furanone, ethyl acetate, n-decanoic acid etc. were identified as the characteristic aroma-active compounds between GT and WY. Microbiome analysis based on high-throughput sequencing of full-length 16S rDNA/ITS-5.8S rDNA amplicons revealed that Lactococcus, Leuconostoc, Pseudomonas, Serratia, Enterobacter, Weissella, Saccharomyces, Monascus and Candida were the predominant microbial genera during the traditional production of GT, while Lactococcus, Lactobacillus, Leuconostoc, Enterobacter, Kozakia, Weissella, Klebsiella, Cronobacter, Saccharomyces, Millerozyma, Monascus, Talaromyces and Meyerozyma were the predominant microbial genera in the traditional fermentation of WY. Correlation analysis revealed that Lactobacillus showed significant positive correlations with most of the characteristic volatile flavor components and biogenic amines. Furthermore, bioinformatical analysis based on PICRUSt revealed that microbial enzymes related to biogenic amines synthesis were more abundant in GT than those in WY, and the enzymes responsible for the degradation of biogenic amines were less abundant in GT than those in WY. Collectively, this study provides important scientific data for enhancing the flavor quality of Hongqu rice wine, and lays a solid foundation for the healthy and sustainable development of Hongqu rice wine industry.

Identification of new reference genes for colony counting by reverse-transcription quantitative PCR in Bifidobacterium animalis.

Bifidobacterium animalis, one of the predominant bacteria in the intestines of humans and other mammals, is widely added to dairy products. We employed RNA sequencing to analyze gene expression variance on a genome-wide scale and found stable reference genes (RG) in B. animalis. A total of 1,665 genes were identified by analyzing the data from the transcriptome under 4 different conditions, and 13 probable candidate RG with variation coefficient values <0.1 were validated using reverse-transcription quantitative PCR (RT-qPCR). The amplification efficiency of candidate RG were ranging from 94.16% to 126.25%. We integrated the analysis results of BestKeeper, geNorm, NormFinder, and RefFinder algorithms and revealed that rplD and atpA comprehensive ranked 1.68 and 2.82, respectively, which were more stable than traditional RG. Compared with plate count (1.58 × 106 cfu/mL), the concentrations of B. animalis AR668 by RT-qPCR using rplD, atpA, and 16S rRNA as RG were 2.27 × 106, 2.24 × 106, and 6.66 × 106 cfu/mL, respectively, after 10 h of fermentation in fermented skim milk. It suggested that rplD and atpA as RG can be accurate for colony counting of B. animalis. Our study provides the foundation for more accurate analysis of colony counting by RT-qPCR of B. animalis in dairy foods.

Unraveling the difference in flavor characteristics of Huangjiu fermented with different rice varieties using dynamic sensory evaluation and comprehensive two-dimensional gas chromatography-quadrupole mass spectrometry.

To investigate the specific differences in flavor characteristics of Huangjiu fermented with different rice varieties, dynamic sensory evaluation, comprehensive two-dimensional gas chromatography-quadrupole mass spectrometry (GC × GC-qMS) and multivariate statistical analysis were employed. Dynamic sensory evaluation methods including temporal dominance of sensations (TDS) and temporal check all that apply (TCATA) were applied to explore the differences and variations in sensory attributes. The sensory results showed that the intensity of astringency and post-bitterness in the Huangjiu fermented with glutinous rice was weaker while ester and alcoholic aroma were more prominent than the one fermented with japonica rice. The results of free amino acids and aroma compounds analysis indicated that the amino acids were mainly sweet and bitter amino acids, and some key aroma compounds were predominant in the Huangjiu fermented with glutinous rice, such as ethyl butyrate (OAV: 38-59), 3-methylthiopropionaldehyde (OAV: 47-96), ethyl caprylate (OAV: 30-38), while nonanal, phenyl acetaldehyde and vanillin contributed significantly to the Huangjiu fermented with japonica rice. The multivariate statistical analysis further confirmed that 17 compounds (VIP > 1 and p < 0.05) could be supposed to be the key compouns that cause significant flavor differences in Huangjiu samples fermented with different brewing rice. Moreover, partial least-squares analysis revealed that most compounds (ethyl butyrate, 3-penten-2-one, isoamyl acetate, and so on) correlated with ester and alcoholic aroma. The results could provide basic data and theoretical basis for the selection of raw materials in Huangjiu.

bsh1 Gene of Lactobacillus plantarum AR113 Plays an Important Role in Ameliorating Western Diet-Aggravated Colitis.

Western diet is thought to increase susceptibility to inflammatory bowel disease (IBD), and probiotics are a potential therapeutic agent for IBD. This study revealed the effects of Lactobacillus plantarum AR113 and L. plantarum AR113Δbsh1 on a dextran sulfate sodium (DSS)-induced colitis mouse model under the Western diet (WD). After four weeks of WD and low-sugar and low-fat diet (LD) intervention, induction with 3% DSS, and intragastric administration of probiotics, we found that L. plantarum AR113 could regulate blood glucose and lipid levels and have a certain protective effect on hepatocytes. Our results suggested that the L. plantarum AR113 alleviated DSS-induced colitis under the Western diet by improving dyslipidemia, repairing intestinal barrier dysfunction, and inhibiting the TLR4/Myd88/TRAF-6/NF-κB inflammatory pathway. However, these changes were not demonstrated in the L. plantarum AR113Δbsh1, and therefore, we reasoned that the presence of bsh1 may play a crucial role in the L. plantarum AR113 exerting its anti-inflammatory function. The relationship between bile salt hydrolase (BSH) and colitis was worthy of further exploration.

Establishment of CRISPR-Cas9 system in Bifidobacteria animalis AR668.

Bifidobacteria are representative intestinal probiotics that have extremely high application value in the food and medical fields. However, the lack of molecular biology tools limits the research on functional genes and mechanisms of bifidobacteria. The application of an accurate and efficient CRISPR system to genome engineering can fill the gap in efficient genetic tools for bifidobacteria. In this study, CRISPR system of B. animalis AR668 was established, which successfully knocked out gene 0348 and gene 0208. The influence of different homology arms and fragments on the knockout effect of the system was explored. In addition, the inducible plasmid curing system of bifidobacteria was innovatively established. This study contributes to the genetic modification and functional mechanism analysis of bifidobacteria.

Effects of tamarind seed polysaccharides on physicochemical characteristics of frozen dough: structure-function relationship.

BACKGROUND: Recently, frozen dough has become more popular because of its ability to be quickly transformed into freshly baked foods. During the storage and transport process, frozen dough can suffer some degree of damage caused by ice crystallization and recrystallization. Adding polysaccharides to frozen dough is a good way to solve this problem. Tamarind seed polysaccharide (TSP) has excellent ice crystal steady ability and has also been widely used in frozen foods. However, there is no study on the use of TSP in frozen dough. RESULTS: TSP can stabilize the bound water content, inhibit the freezable water content, and increase elasticity. However, the dough with different structures of TSP added was less firm after 30 days of freezing compared to the dough without TSP, and the porosity and stomatal density of the prepared steamed bread gradually decreased. The addition of TSP reduced gluten deterioration during the freezing process, thus decreasing the collapse and uneven porosity of the steamed bread. CONCLUSIONS: The results could provide new insights into the structure of TSP and its effect on the quality characteristics of frozen dough. © 2023 Society of Chemical Industry.

Characterization of structural and functional properties of soy protein isolate and sodium alginate interpenetrating polymer network hydrogels.

BACKGROUND: This study used enzymatic and Ca2+ cross-linking methods to prepare edible soy protein isolate (SPI) and sodium alginate (SA) interpenetrating polymer network hydrogels to overcome the disadvantages of traditional interpenetrating polymer network (IPN) hydrogels, such as poor performance, high toxicity, and inedibility. The influence of changes in SPI and SA mass ratio on the performance of SPI-SA IPN hydrogels was investigated. RESULTS: Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were used to characterize the structure of the hydrogels. Texture profile analysis (TPA), rheological properties, swelling rate, and Cell Counting Kit-8 (CCK-8) were used to evaluate physical and chemical properties and safety. The results showed that, compared with SPI hydrogel, IPN hydrogels had better gel properties and structural stability. As the mass ratio of SPI-SA IPN changed from 1:0.2 to 1:1, the gel network structure of hydrogels also tended to be dense and uniform. The water retention and mechanical properties of these hydrogels, such as storage modulus (G'), loss modulus (G"), and gel hardness increased significantly and were greater than those of the SPI hydrogel. Cytotoxicity tests were also performed. The biocompatibility of these hydrogels was good. CONCLUSIONS: This study proposes a new method to prepare food-grade IPN hydrogels with mechanical properties of SPI and SA, which may have strong potential for the development of new foods. © 2023 Society of Chemical Industry.

Bile salt hydrolase of Lactiplantibacillus plantarum plays important roles in amelioration of DSS-induced colitis.

Bile salt hydrolases are thought to be the gatekeepers of bile acid metabolism. To study the role of BSH in colitis, we investigated the ameliorative effects of different BSH-knockout strains of Lactiplantibacillus plantarum AR113. The results showed that L. plantarum Δbsh 1 and Δbsh 3 treatments did not improve body weight and alleviate the hyperactivated myeloperoxidase activity to the DSS group. However, the findings for L. plantarum AR113, L. plantarum Δbsh 2 and Δbsh 4 treatments were completely opposite. The double and triple bsh knockout strains further confirmed that BSH 1 and BSH 3 are critical for the ameliorative effects of L. plantarum AR113. In addition, L. plantarum Δbsh 1 and Δbsh 3 did not significantly inhibit the increase in pro-inflammatory cytokines or the decrease in an anti-inflammatory cytokine. These results suggest that BSH 1 and BSH 3 in L. plantarum play important roles in alleviating enteritis symptoms.

Dynamic analysis of volatile metabolites and microbial community and their correlations during the fermentation process of traditional Huangjiu (Chinese rice wine) produced around Winter Solstice.

Traditional Huangjiu produced around Winter Solstice has higher quality and a more harmonious aroma. To investigate the variations of volatile metabolites and microbial communities during fermentation, gas chromatography-ion migration chromatography (GC-IMS), gas chromatography-mass spectroscopy (GC-MS) and high-throughput sequencing were employed. Aroma compounds results showed that alcohols and phenols increased before 45 days of fermentation and then decreased after 45 days, while esters gradually increased. Fungal genera Saccharomyces, Aspergillu, and Rhizomucor were dominant, whereas Staphylococcus, Pediococcus and Weissella were the dominant bacterial genera in the late stage. In addition, 11 genera such as Lactobacillus, Saccharopolyspora and Aspergillus (|r| > 0.6, p < 0.05) may contributed to traditional Huangjiu ecosystem stability. Moreover, correlation analysis indicated the dominant microorganisms (Saccharopolyspora, Staphylococcus, Lactobacillus, Saccharomyces and Aspergillus) were positively correlated with key compounds. These results provided theoretical guidance for further study on the flavor regulation of traditional Huangjiu via microbial community level and microbial augmentation.

Understanding the Connection between Gut Homeostasis and Psychological Stress.

Long-term exposure to adverse life events that provoke acute or chronic psychological stress (hereinafter "stress") can negatively affect physical health and even increase susceptibility to psychological illnesses, such as anxiety and depression. As a part of the hypothalamic-pituitary-adrenal axis, corticotropin-releasing factor (CRF) released from the hypothalamus is primarily responsible for the stress response. Typically, CRF disrupts the gastrointestinal system and leads to gut microbiota dysbiosis, thereby increasing risk of functional gastrointestinal diseases, such as irritable bowel syndrome. Furthermore, CRF increases oxidative damage to the colon and triggers immune responses involving mast cells, neutrophils, and monocytes. CRF even affects the differentiation of intestinal stem cells (ISCs), causing enterochromaffin cells to secrete excessive amounts of 5-hydroxytryptamine (5-HT). Therefore, stress is often accompanied by damage to the intestinal epithelial barrier function, followed by increased intestinal permeability and bacterial translocation. There are multi-network interactions between the gut microbiota and stress, and gut microbiota may relieve the effects of stress on the body. Dietary intake of probiotics can provide energy for ISCs through glycolysis, thereby alleviating the disruption to homeostasis caused by stress, and it significantly bolsters the intestinal barrier, alleviates intestinal inflammation, and maintains endocrine homeostasis. Gut microbiota also directly affect the synthesis of hormones and neurotransmitters, such as CRF, 5-HT, dopamine, and norepinephrine. Moreover, the Mediterranean diet enhances the stress resistance to some extent by regulating the intestinal flora. This article reviews recent research on how stress damages the gut and microbiota, how the gut microbiota can improve gut health by modulating injury due to stress, and how the diet relieves stress injury by interfering with intestinal microflora. This review gives insight into the potential role of the gut and its microbiota in relieving the effects of stress via the gut-brain axis.

Potential therapeutic drug targets and pathways prediction for premature ovarian insufficiency -Based on network pharmacologic method.

ETHNOPHARMACOLOGICAL RELEVANCE: The incidence of premature ovarian insufficiency (POI) is gradually increasing, the proportion is rising especially in female infertility patients. The risk of death of POI patients with cardiovascular disease also increases significantly. The cause of POI is complex and unclear, and clinical treatment is still in the exploratory stage, are two major constraints of treating POI. Traditional Chinese medicine (TCM) is widely used in the treatment of POI, and it is a good way to combine the development of modern new drugs with the help of TCM to predict the therapeutic targets. AIM OF THE STUDY: In this study, four herbs commonly used in clinical treatment of POI, namely Radix Paeoniae, Polygonatum sibiricum, Rehmannia glutinosa and Eucommia ulmoides were selected to predict their mechanism in the treatment of POI, using network pharmacology methods. Then verify the predicted targets by animal test. Aim to find more effective POI potential core treatment targets and main pathways. MATERIALS AND METHODS: We screened the active ingredients of drugs from the TCM System Pharmacology Analysis Platform (TCMSP), Performed target prediction of active ingredients from databases such as SwissTargetPrediction and compare and analyze the POI-related targets retrieved from them to obtain potential targets for drug treatment of POI. Used STRING database to construct a protein interaction network, Cytoscape 3.7.2 software to construct an active ingredient-target-pathway network, and DAVID database to conduct the Kyoto Encyclopedia of Genes and Genomes (KEGG) on the intersection targets and gene ontology (GO) enrichment analysis. RESULTS: The result is: there were 25 key targets for the treatment of POI with Radix Paeoniae Alba, 31 for the treatment of POI by Eucommia ulmoides, 28 for the treatment of POI by Polygonatum sibiricum, and 8 key targets for the treatment of Rehmannia glutinosa. The intersection targets of four herbs were defined as the core targets, which are CYP19A1, EGF, ESR1, ESR2, MDM2, AR, PCYP17A1, PPARG. Four Chinese herbs treat POI mainly through HIF-1 signaling pathway, PI3K-Akt signaling pathway, FoxO signaling pathway, Estrogen signaling pathway etc. A mouse model of POI was constructed based on the results of network pharmacology to verify the predicted targets. The results showed that the protein expression of the core target changed, and the estrogen level was increased by reducing the expression of peroxisome proliferator-activated receptor gamma (PPARG). CONCLUSIONS: This study predicts the mechanism of multiple herbs in the treatment of POI, screens out more potential therapeutic drug targets and main pathways of POI treatment and provides new ideas for the subsequent development of POI therapeutic drugs.