Recombinant Arginine Deiminase from Levilactobacillus brevis Inhibits the Growth of Stomach Cancer Cells, Possibly by Activating the Intrinsic Apoptosis Pathway.

The anticancer potential of Levilactobacillus brevis KU15176 against the stomach cancer cell line AGS has been reported previously. In this study, we aimed to analyze the genome of L. brevis KU15176 and identify key genes that may have potential anticancer properties. Among potential anticancer molecules, the role of arginine deiminase (ADI) in conferring an antiproliferative functionality was confirmed. In vitro assay against AGS cell line confirmed that recombinant ADI from L. brevis KU15176 (ADI_br, 5 µg/mL), overexpressed in E. coli BL21 (DE3), exerted an inhibitory effect on AGS cell growth, resulting in a 65.32% reduction in cell viability. Moreover, the expression of apoptosis-related genes, such as bax, bad, caspase-7, and caspase-3, as well as the activity of caspase-9 in ADI_br-treated AGS cells, was higher than those in untreated (culture medium-only) cells. The cell-scattering behavior of ADI_br-treated cells showed characteristics of apoptosis. Flow cytometry analyses of AGS cells treated with ADI_br for 24 and 28 h revealed apoptotic rates of 11.87 and 24.09, respectively, indicating the progression of apoptosis in AGS cells after ADI_br treatment. This study highlights the potential of ADI_br as an effective enzyme for anticancer applications.

Safety Assessment of Levilactobacillus brevis KU15006: A Comprehensive Analysis of its Phenotypic and Genotypic Properties.

Levilactobacillus brevis KU15006, isolated from kimchi, exhibits pathogen-antagonistic and anti-diabetic activities; however, the safety of this strain has not been assessed. In the present study, L. brevis KU15006 was evaluated to elucidate its safety as a probiotic strain using phenotypic and genotypic analyses. Its safety was assessed using a minimum inhibitory concentration test comprising nine antibiotics, 26 antibiotic resistance genes, a single conjugative element, virulence gene analysis, hemolysis, cell cytotoxicity, mucin degradation, and toxic metabolite production. L. brevis KU15006 exhibited equal or lower minimum inhibitory concentration for the nine antibiotics than the cut-off value established by the European Food Safety Authority. It did not harbor antibiotic resistance and virulence genes. L. brevis KU15006 lacked ß-hemolysis, mucin degradation, cytotoxicity against Caco-2 cells, gelatin liquefaction, bile salt deconjugation, and toxic metabolite production abilities. Based on the results, L. brevis KU15006, which has antagonistic and anti-diabetic effects, could be marketed as a probiotic in the future.

Conversion of Host Cell Receptor into Virus Destructor by Immunodisc to Neutralize Diverse SARS-CoV-2 Variants.

The decreasing efficacy of antiviral drugs due to viral mutations highlights the challenge of developing a single agent targeting multiple strains. Using host cell viral receptors as competitive inhibitors is promising, but their low potency and membrane-bound nature have limited this strategy. In this study, the authors show that angiotensin-converting enzyme 2 (ACE2) in a planar membrane patch can effectively neutralize all tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that emerged during the COVID-19 pandemic. The ACE2-incorporated membrane patch implemented using nanodiscs replicated the spike-mediated membrane fusion process outside the host cell, resulting in virus lysis, extracellular RNA release, and potent antiviral activity. While neutralizing antibodies became ineffective as the SARS-CoV-2 evolved to better penetrate host cells the ACE2-incorporated nanodiscs became more potent, highlighting the advantages of using receptor-incorporated nanodiscs for antiviral purposes. ACE2-incorporated immunodisc, an Fc fusion nanodisc developed in this study, completely protected humanized mice infected with SARS-CoV-2 after prolonged retention in the airways. This study demonstrates that the incorporation of viral receptors into immunodisc transforms the entry gate into a potent virucide for all current and future variants, a concept that can be extended to different viruses.

A2 milk consumption and its health benefits: an update.

Milk is a widely consumed nutrient-rich food containing protein variants such as casein A2 and A1. A1 differs from A2 in an amino acid at position 67 (Pro67 to His67). The breakdown of ß-casein yields ß-casomorphins (BCM), among which BCM-7 is extensively studied for its effects on the human body. Animal studies have shown that A1 ß-casein milk increases digestive transit time and enhances myeloperoxidase activity. Individuals with lactose intolerance prefer A2 milk to conventional A1 milk, as BCM-7 in A1 milk can lead to inflammation and discomfort in sensitive individuals. A2 milk, which contains A2 ß-casein, is believed to be more easily digestible than A1 ß-casein. Its popularity has grown owing to reports linking A1 casein to diseases such as type 1 diabetes, heart disease, and autism. A2 milk has gained popularity as an alternative to A1 milk, primarily because of its potential benefits for individuals with certain diseases. This review aims to provide an updated understanding of A2 milk consumption and its health benefits. This review aims to provide an updated understanding of A2 milk consumption and its health benefits.

Heat-Treated Paraprobiotic Latilactobacillus sakei KU15041 and Latilactobacillus curvatus KU15003 Show an Antioxidant and Immunostimulatory Effect.

The lactic acid bacteria, including Latilactobacillus sakei and Latilactobacillus curvatus, have been widely studied for their preventive and therapeutic effects. In this study, the underlying mechanism of action for the antioxidant and immunostimulatory effects of two strains of heat-treated paraprobiotics was examined. Heat-treated L. sakei KU15041 and L. curvatus KU15003 showed higher radical scavenging activity in both the 2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assays than the commercial probiotic strain LGG. In addition, treatment with these two strains exhibited immunostimulatory effects in RAW 264.7 macrophages, with L. curvatus KU15003 showing a slightly higher effect. Additionally, they promoted phagocytosis and NO production in RAW 264.7 cells without any cytotoxicity. Moreover, the expression of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 was upregulated. These strains resulted in an increased expression of inducible nitric oxide synthase and cyclooxygenase-2. Moreover, the nuclear factor-κB and mitogen-activated protein kinase signaling pathways were stimulated by these strains. These findings suggest the potential of using L. sakei KU15041 and L. curvatus KU15003 in food or by themselves as probiotics with antioxidant and immune-enhancing properties.

Assessing the Anti-Obesity Potential of Lactococcus lactis subsp. lactis CAB701: Modulation of Adipocyte Differentiation and Lipid Metabolism in In Vitro and In Vivo Models.

In this study, the potential anti-obesity effects of Lactococcus lactis subsp. lactis CAB701, a probiotic strain isolated from cabbage, were investigated using in vitro and in vivo assays. L. lactis subsp. lactis CAB701 inhibited adipocyte differentiation of 3T3-L1 cells by 67%. In an in vivo model of high-fat diet-induced obese mice, treatment with L. lactis subsp. lactis CAB701 markedly reduced body weight and peri-epididymal fat mass, and significantly reduced serum total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels. Molecular analysis revealed a significant modulation of key genes and proteins involved in lipid metabolism and adipogenesis. Specifically, fatty acid synthase and peroxisome proliferator-activated receptor gamma were significantly downregulated in peri-epididymal adipose tissue, alluding to the molecular mechanism underlying the anti-obesity effects exerted by L. lactis subsp. lactis CAB701. Furthermore, histological examination revealed a significant reduction in adipocyte size in the treated group, indicating effective adipose tissue remodeling. Our findings suggest that L. lactis subsp. lactis CAB701 mediates anti-obesity effects through the modulation of critical molecular markers and lipid profiles. L. lactis subsp. lactis CAB701 thus represents a promising candidate for combating obesity and related metabolic disorders.

Paraprobiotics: definition, manufacturing methods, and functionality.

Probiotics are living microorganisms that are beneficial to the host, enhancing the immune response by promoting antibody production, regulating cytokine secretion, and stimulating T cells. However, probiotics have limitations in that they require viability control and have a short shelf life. Recently, the use of paraprobiotics has gained attention. These include dead bacterial cells, bacterial fractions, and cell lysate that have health benefits and are stable and safe for use. Paraprobiotics comprise molecules of bacterial cell wall compounds, such as peptidoglycans, teichoic acids, polysaccharides, and cell surface proteins. Paraprobiotics are manufactured by a diverse range of techniques, including thermal treatments, high pressure, ultraviolet rays, sonication, ionizing radiation, and pH modification. Their beneficial health effects include immunomodulatory, intestinal balancing, anticancer, and antimicrobial activities. Therefore, this review summarizes and discusses the manufacturing methods and bioavailability of paraprobiotics and suggests their potential health advantages.

Immunostimulatory Activity of Lactococcus lactis subsp. lactis CAB701 Isolated from Jeju Cabbage.

This study explored the potential of Lactococcus lactis subsp. lactis CAB701 as a probiotic strain, focusing on its immunostimulatory properties. Despite adverse conditions in the gastrointestinal environment, this strain exhibited remarkable survivability, as evidenced by its tolerance to acid, bile, and pancreatin, coupled with its impressive ability to adhere to Caco-2 cells. It also exhibited significant antioxidant activity, similar to the established probiotic Lacticaseibacillus rhamnosus GG (LGG). Our research elucidates the potent immunostimulatory effects of L. lactis subsp. lactis CAB701. This strain significantly enhanced nitric oxide production in RAW 264.7, far exceeding that obtained with LGG. An in-depth examination revealed elevated expression of key inflammatory mediators, including inducible nitric oxide synthase, tumor necrosis factor-alpha, cyclooxygenase-2, interleukin (IL)-1 beta, and IL-6. L. lactis subsp. lactis CAB701 increases the expression of critical signaling proteins in the mitogen-activated protein kinase pathway. This prompted a substantial increase in the expression of phosphorylated c-Jun N-terminal kinases and extracellular signal-regulated kinases, suggesting their role in modulating these immune-related pathways. Overall, these findings demonstrate the significant immunostimulatory capacity of L. lactis subsp. lactis CAB701, positioning it as a potential candidate for probiotic use, especially in applications that enhance immune responses.

The Radical Scavenging Activities and Anti-Wrinkle Effects of Soymilk Fractions Fermented with Lacticaseibacillus paracasei MK1 and Their Derived Peptides.

Soybean-derived peptides exert several beneficial effects in various experimental models. However, only a few studies have focused on the radical scavenging and anti-wrinkle effects of soymilk-derived peptides produced via different processes, such as fermentation, enzymatic treatment, and ultrafiltration. Therefore, in this study, we investigated the radical scavenging and antiwrinkle effects of soymilk fractions produced using these processes. We found that 50SFMKUF5, a 5 kDa ultrafiltration fraction fermented with Lacticaseibacillus paracasei MK1 after flavourzyme treatment, exhibited the highest radical scavenging activity using the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay as well as potent anti-wrinkle effects assessed by type 1 procollagen production and tumor necrosis factor-α production in ultraviolet B (UVB)-treated human dermal fibroblasts and HaCaT keratinocytes. To identify potential bioactive peptides, candidate peptides were synthesized, and their anti-wrinkle effects were assessed. APEFLKEAFGVN (APE), palmitoyl-APE, and QIVTVEGGLSVISPK peptides were synthesized and used to treat UVB-irradiated fibroblasts, HaCaT keratinocytes, and α-melanocyte-stimulating hormone-induced B16F1 melanoma cells. Among these peptides, Pal-APE exerted the strongest effect. Our results highlight the potential of soymilk peptides as anti-aging substances.

Synergistic Immunostimulatory Activities of Probiotic Strains, Leuconostoc lactis and Weissella cibaria, and the Prebiotic Oligosaccharides They Produce.

Synbiotics contain health-beneficial bacteria, i.e., probiotics and prebiotics selectively utilized by the probiotics. Herein, three probiotic strains, Leuconostoc lactis CCK940, L. lactis SBC001, and Weissella cibaria YRK005, and the oligosaccharides produced by these strains (CCK, SBC, and YRK, respectively) were used to prepare nine synbiotic combinations. Macrophages (RAW 264.7) were treated with these synbiotic combinations and the corresponding lactic acid bacteria and oligosaccharides alone to evaluate the treatments' immunostimulatory activities. The level of nitric oxide (NO) production was significantly higher in the macrophages treated with the synbiotics than in those treated with the corresponding probiotic strains and the oligosaccharide alone. The immunostimulatory activities of the synbiotics increased regardless of the probiotic strain and the type of oligosaccharide used. The expressions of tissue necrosis factor-α, interleukin-1ß, cyclooxygenase-2, inducible NO synthase genes, and extracellular-signal-regulated and c-Jun N-terminal kinases were significantly higher in the macrophages treated with the three synbiotics than in those treated with the corresponding strains or with the oligosaccharides alone. These results indicate that the synergistic immunostimulatory activities of probiotics and the prebiotics they produced in the studied synbiotic preparations resulted from the activation of the mitogen-activated protein-kinase-signaling pathway. This study suggests the combined use of these probiotics and prebiotics in the development of synbiotic preparations as health supplements.

Genome analysis revealed a repertoire of oligosaccharide utilizing CAZymes in Weissella confusa CCK931 and Weissella cibaria YRK005.

Weissella bacteria are gram-positive, anaerobic, fermentative, and have probiotic potential. This study aimed to compare the genomes of W. cibaria YRK005 and W. confusa CCK931 isolated from young radish and kimchi, respectively. The genomic size of W. cibaria YRK005 and W. confusa CCK931 with GC content is 2.36 Mb (45%) and 2.28 Mb (44.67%), respectively. The genome study identified 92 and 83 CAZymes genes, respectively, for W. cibaria YRK005 and W. confusa CCK931, that are responsible for 26 and 27 glycoside hydrolases (GH) and 21 and 27 glycosyl transferases. Both species have one gene for carbohydrate esterases and three genes for carbohydrate-binding modules. The primary CAZymes found in both species that are involved in oligosaccharide utilization are GH1, GH2, GH30, GH13_30, GH13_31, GH42, GH43, and GH65. The study also details the production pathways for glycogen and folate. Both strains include a unique repertoire of genes, including hypothetical proteins, showing adaptability to diverse ecological niches and evolution over time. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01232-7.

Beyond probiotics: a narrative review on an era of revolution.

Whether knowingly or unknowingly, humans have been consuming probiotic microorganisms through traditionally fermented foods for generations. Bacteria, like lactic acid bacteria, are generally thought to be harmless and produce many metabolites that are beneficial for human health. Probiotics offer a wide range of health benefits; however, their therapeutic usage is limited because they are living organisms. As a result, the focus on the health advantages of microbes has recently shifted from viable live probiotics to non-viable microbes made from probiotics. These newly emerging non-viable microbes include paraprobiotics, postbiotics, psychobiotics, nutribiotics, and gerobiotics. Their metabolites can boost physiological health and reveal the therapeutic effects of probiotics. This new terminology in microbes, their traits, and their applications are summarized in the present review.

Immunostimulatory activity of Lactococcus lactis LM1185 isolated from Hydrangea macrophylla.

The lactic acid bacteria, Lactococcus lactis subsp. lactis LM1185 was isolated from Hydrangea macrophylla. Strain LM1185 showed 50.5% of acid tolerance at pH 2.5 for 2 h and 30.4% of 0.3% (w/v) bile salt tolerance for 24 h. The antioxidant activity of this strain was measured at 99.4% of 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity. When RAW 264.7 macrophage cells were treated with strain LM1185, there was no observed cytotoxicity. This strain showed high nitric oxide production and mRNA expression levels of cytokines such as tumor necrosis factor-α and inducible nitric oxide synthase (iNOS). The nuclear factor-kB signaling pathway was activated by this strain resulting in the production of iNOS and cyclooxygenase-2 determined by western blotting. The present results indicated that L. lactis subsp. lactis LM1185 could be used as potential probiotics and may play a crucial role in the immunostimulatory effect on macrophages. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01199-5.

Bioinformatics and its role in the study of the evolution and probiotic potential of lactic acid bacteria.

Due to their numerous well-established applications in the food industry, there have been many studies regarding the adaptation and evolution of lactic acid bacteria (LAB) in a wide variety of hosts and environments. Progress in sequencing technology and continual decreases in its costs have led to the availability of LAB genome sequence data. Bioinformatics has been central to the extraction of valuable information from these raw genome sequence data. This paper presents the roles of bioinformatics tools and databases in understanding the adaptation and evolution of LAB, as well as the bioinformatics methods used in the initial screening of LAB for probiotic potential. Moreover, the advantages, challenges, and limitations of employing bioinformatics for these purposes are discussed.

Structural analysis and prebiotic activity of exopolysaccharide produced by probiotic strain Bifidobacterium bifidum EPS DA-LAIM.

Exopolysaccharide (EPS)-producing Bifidobacterium bifidum EPS DA-LAIM was isolated from healthy human feces, the structure of purified EPS from the strain was analyzed, and its prebiotic activity was evaluated. The EPS from B. bifidum EPS DA-LAIM is a glucomannan-type heteropolysaccharide with a molecular weight of 407-1007 kDa, and its structure comprises 2-mannosyl, 6-mannosyl, and 2,6-mannosyl residues. The purified EPS promoted the growth of representative lactic acid bacteria and bifidobacterial strains. Bifidobacterium bifidum EPS DA-LAIM increased nitric oxide production in RAW 264.7 macrophage cells, indicating its immunostimulatory activity. Bifidobacterium bifidum EPS DA-LAIM also exhibited high gastrointestinal tract tolerance, gut adhesion ability, and antioxidant activity. These results suggest that EPS from B. bifidum EPS DA-LAIM is a potentially useful prebiotic material, and B. bifidum EPS DA-LAIM could be applied as a probiotic candidate. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01213-w.

Production and characterization of lentivirus vector-based SARS-CoV-2 pseudoviruses with dual reporters: Evaluation of anti-SARS-CoV-2 viral effect of Korean Red Ginseng.

Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean Red Ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.

4,4'-Diaponeurosporene Production as C30 Carotenoid with Antioxidant Activity in Recombinant Escherichia coli.

Carotenoids, a group of isoprenoid pigments, are naturally synthesized by various microorganisms and plants, and are industrially used as ingredients in food, cosmetic, and pharmaceutical product formulations. Although several types of carotenoids and diverse microbial carotenoid producers have been reported, studies on lactic acid bacteria (LAB)-derived carotenoids are relatively insufficient. There is a notable lack of research focusing on C30 carotenoids, the functional characterizations of their biosynthetic genes and their mass production by genetically engineered microorganisms. In this study, the biosynthesis of 4,4'-diaponeurosporene in Escherichia coli harboring the core biosynthetic genes, dehydrosqualene synthase (crtM) and dehydrosqualene desaturase (crtN), from Lactiplantibacillus plantarum subsp. plantarum KCCP11226 was constructed to evaluate and enhance 4,4'-diaponeurosporene production and antioxidant activity. The production of 4,4'-diapophytoene, a substrate of 4,4'-diaponeurosporene, was confirmed in E. coli expressing only the crtM gene. In addition, recombinant E. coli carrying both C30 carotenoid biosynthesis genes (crtM and crtN) was confirmed to biosynthesize 4,4'-diaponeurosporene and exhibited a 6.1-fold increase in carotenoid production compared to the wild type and had a significantly higher antioxidant activity compared to synthetic antioxidant, butylated hydroxytoluene. This study presents the discovery of an important novel E. coli platform in consideration of the industrial applicability of carotenoids.

Potential Probiotic Properties of Exopolysaccharide-Producing Lacticaseibacillus paracasei EPS DA-BACS and Prebiotic Activity of Its Exopolysaccharide.

Exopolysaccharide (EPS)-producing Lacticaseibacillus paracasei EPS DA-BACS was isolated from healthy human feces and its probiotic properties, as well as the structure and prebiotic activity of the EPS from this strain were examined. EPS from L. paracasei EPS DA-BACS had a ropy phenotype, which is known to have potential health benefits and is identified as loosely cell-bounded glucomannan-type EPS with a molecular size of 3.7 × 106 Da. EPS promoted the acid tolerance of L. paracasei EPS DA-BACS and provided cells with tolerance to gastrointestinal stress. The purified EPS showed growth inhibitory activity against Clostridium difficile. L. paracasei EPS DA-BACS cells completely inhibited the growth of Bacillus subtilis, Pseudomonas aeruginosa, and Aspergillus brasiliensis, as well as showed high growth inhibitory activity against Staphylococcus aureus and Escherichia coli. Treatment of lipopolysaccharide-stimulated RAW 264.7 cells with heat-killed L. paracasei EPS DA-BACS cells led to a decrease in the production of nitric oxide, indicating the anti-inflammatory activity of L. paracasei EPS DA-BACS. Purified EPS promoted the growth of Lactobacillus gasseri, Bifidobacterium bifidum, B. animalis, and B. faecale which showed high prebiotic activity. L. paracasei EPS DA-BACS harbors no antibiotic resistance genes or virulence factors. Therefore, L. paracasei EPS DA-BACS exhibits anti-inflammatory and antimicrobial activities with high gut adhesion ability and gastrointestinal tolerance and can be used as a potential probiotic.

Comparative genome analysis of four Leuconostoc strains with a focus on carbohydrate-active enzymes and oligosaccharide utilization pathways.

Leuconostoc is mostly found in food, plants, and dairy products. Due to their innate genomic features, such as the presence of carbohydrate-active enzymes, bacteriocins, and plasmids, Leuconostoc spp. have great biotechnological potential. In this study, four strains were isolated and identified as Leuconostoc mesenteroides SG315 (LA), L. citreum SG255 (LB), L. lactis CCK940 (LC), and L. lactis SBC001 (LD). Comparative analysis was performed using their draft genome sequences. Differences among the four strains were analyzed using the average nucleotide identity, dot plot, and multiple alignments of conserved genomic sequences. Functional profiling revealed 2134, 1917, 1751, and 1816 open reading frames; 2023, 1823, 1655, and 1699 protein-coding genes; 60, 57, 83, and 82 RNA-coding genes; and GC content of 37.5 %, 38.8 %, 43.3 %, and 43.2 %, in LA, LB, LC, and LD, respectively. The total number of genes encoding carbohydrate-active enzymes was 76 (LA), 73 (LB), 57 (LC), and 67 (LD). These results indicate that the four strains shared a large number of genes, but their gene content is different. Furthermore, most genes with unknown functions were observed in the prophage regions of the genome. This study also elucidated the oligosaccharide utilization and folate biosynthesis pathways in Leuconostoc spp. Taken together, our findings provide useful information on the genomic diversity of CAZymes in the four Leuconostoc strains and suggest that these species could be used for potent exploitation.

The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors.

(1) Background: The N-methyl-D-aspartate receptors (NMDARs) mediate fast excitatory currents leading to depolarization. Postsynaptic NMDARs are ionotropic glutamate receptors that mediate excitatory glutamate or glycine signaling in the CNS and play a primary role in long-term potentiation, which is a major form of use-dependent synaptic plasticity. The overstimulation of NMDARs mediates excessive Ca2+ influx to postsynaptic neurons and facilitates more production of ROS, which induces neuronal apoptosis. (2) Methods: To confirm the induced inward currents by the coapplication of glutamate and ergotamine on NMDARs, a two-electrode voltage clamp (TEVC) was conducted. The ergotamine-mediated inhibitory effects of NR1a/NR2A subunits were explored among four different kinds of recombinant NMDA subunits. In silico docking modeling was performed to confirm the main binding site of ergotamine. (3) Results: The ergotamine-mediated inhibitory effect on the NR1a/NR2A subunits has concentration-dependent, reversible, and voltage-independent properties. The major binding sites were V169 of the NR1a subunit and N466 of the NR2A subunit. (4) Conclusion: Ergotamine effectively inhibited NR1a/NR2A subunit among the subtypes of NMDAR. This inhibition effect can prevent excessive Ca2+ influx, which prevents neuronal death.