Developing a High-Umami, Low-Salt Soy Sauce through Accelerated Moromi Fermentation with Corynebacterium and Lactiplantibacillus Strains.

The traditional fermentation process of soy sauce employs a hyperhaline model and has a long fermentation period. A hyperhaline model can improve fermentation speed, but easily leads to the contamination of miscellaneous bacteria and fermentation failure. In this study, after the conventional koji and moromi fermentation, the fermentation broth was pasteurized and diluted, and then inoculated with three selected microorganisms including Corynebacterium glutamicum, Corynebacterium ammoniagenes, and Lactiplantibacillus plantarum for secondary fermentation. During this ten-day fermentation, the pH, free amino acids, organic acids, nucleotide acids, fatty acids, and volatile compounds were analyzed. The fermentation group inoculated with C. glutamicum accumulated the high content of amino acid nitrogen of 0.92 g/100 mL and glutamic acid of 509.4 mg/100 mL. The C. ammoniagenes group and L. plantarum group were rich in nucleotide and organic acid, respectively. The fermentation group inoculated with three microorganisms exhibited the best sensory attributes, showing the potential to develop a suitable fermentation method. The brewing speed of the proposed process in this study was faster than that of the traditional method, and the umami substances could be significantly accumulated in this low-salt fermented model (7% w/v NaCl). This study provides a reference for the low-salt and rapid fermentation of seasoning.

Temperature effects on plasmalogen profile and quality characteristics in Pacific oyster (Crassostrea gigas) during depuration.

The quality of Pacific oyster (Crassostrea gigas) can be affected by many factors during depuration, in which temperature is the major element. In this study, we aim to determine the quality and plasmalogen changes in C. gigas depurated at different temperatures. The quality was significantly affected by temperature, represented by varying survival rate, glycogen content, total antioxidant capacity, alkaline phosphatase activity between control and stressed groups. Targeted MS analysis demonstrated that plasmalogen profile was significantly changed during depuration with PUFA-containing plasmalogen species being most affected by temperature. Proteomics analysis and gene expression assay further verified that plasmalogen metabolism is regulated by temperature, specifically, the plasmalogen synthesis enzyme EPT1 was significantly downregulated by high temperature and four plasmalogen-related genes (GPDH, PEDS, Pex11, and PLD1) were transcriptionally regulated. The positive correlations between the plasmalogen level and quality characteristics suggested plasmalogen could be regarded as a quality indicator of oysters during depuration.

Hydrogel/microcarrier cell scaffolds for rapid expansion of satellite cells from large yellow croakers: Differential analysis between 2D and 3D cell culture.

Cell culture meat is based on the scaled-up expansion of seed cells. The biological differences between seed cells from large yellow croakers in the two-dimensional (2D) and three-dimensional (3D) culture systems have not been explored. Here, satellite cells (SCs) from large yellow croakers (Larimichthys crocea) were grown on cell climbing slices, hydrogels, and microcarriers for five days to analyze the biological differences of SCs on different cell scaffolds. The results exhibited that SCs had different cell morphologies in 2D and 3D cultures. Cell adhesion receptors (Itgb1andsdc4) and adhesion spot markervclof the 3D cultures were markedly expressed. Furthermore, myogenic decision markers (Pax7andmyod) were significantly enhanced. However, the expression of myogenic differentiation marker (desmin) was significantly increased in the microcarrier group. Combined with the transcriptome data, this suggests that cell adhesion of SCs in 3D culture was related to the integrin signaling pathway. In contrast, the slight spontaneous differentiation of SCs on microcarriers was associated with rapid cell proliferation. This study is the first to report the biological differences between SCs in 2D and 3D cultures, providing new perspectives for the rapid expansion of cell culture meat-seeded cells and the development of customized scaffolds.

The α-linkage in funoran and agarose could be hydrolyzed by a GH96 family enzyme: Discovery of the α-funoranase.

Agarans represent a group of galactans extracted from red algae. Funoran and agarose are the two major types and commercially applied polysaccharides of agaran. Although the glycoside hydrolases targeting ß-glycosidic bonds of agaran have been widely investigated, those capable of degrading α-glycosidic bonds of agarose were limited, and the enzyme degrading α-linkages of funoran has not been reported till now. In this study, a GH96 family enzyme BiAF96A_Aq from a marine bacterium Aquimarina sp. AD1 was heterologously expressed in Escherichia coli. BiAF96A_Aq exhibited dual activities towards the characteristic structure of funoran and agarose, underscoring the multifunctionality of GH96 family members. Glycomics and NMR analysis revealed that BiAF96A_Aq hydrolyzed the α-1,3 glycosidic bonds between 3,6-anhydro-α-l-galactopyranose (LA) and ß-d-galactopyranose-6-sulfate (G6S) of funoran, as well as LA and ß-d-galactopyranose (G) of agarose, through an endo-acting manner. The end products of BiAF96A_Aq were majorly composed of disaccharides and tetrasaccharides. The identification of the activity of BiAF96A_Aq on funoran indicated the first discovery of the funoran hydrolase for α-1,3 linkage. Considering the novel catalytic reaction, we proposed to name this activity as "α-funoranase" and recommended the assignment of a dedicated EC number for its classification.

Fucoidan from Apostichopus japonicus ameliorates alcoholic liver disease by regulating gut-liver axis homeostasis.

Long-term and excessive alcohol consumption can lead to the development of alcoholic liver disease (ALD), characterized by oxidative damage, intestinal barrier injury, and disruption of intestinal microbiota. In this study, we extracted fucoidan (Aj-FUC) from Apostichopus japonicus using enzymatic methods and characterized its structure. The ALD model was established in male Balb/c mice using 56° Baijiu, with silymarin as a positive control. Mice were orally administered 100 mg/kg·bw and 300 mg/kg·bw of Aj-FUC for 28 days to evaluate its effects on liver injury in ALD mice and explore its potential role in modulating the gut-liver axis. The results showed significant improvements in histopathological changes and liver disease in the Aj-FUC group. Aj-FUC treatment significantly increased the levels of glutathione (GSH) and glutathione peroxidase (GSH-Px) while weakly reduced the elevation of malondialdehyde (MDA) induced by ALD. It also regulated the Nrf2/HO-1 signaling pathway, collectively alleviating hepatic oxidative stress. Aj-FUC intervention upregulated the expression of ZO-1 and Occludin, thus contributing to repair the intestinal barrier. Additionally, Aj-FUC increased the content of short-chain fatty acids (SCFAs) and regulated the imbalance in gut microbiota. These results suggested that Aj-FUC alleviates ALD by modulating the gut-liver axis homeostasis. It may prove to be a useful dietary supplement in the treatment of alcoholic liver damage.

A smartphone-assisted portable sensing hydrogel modules based on UCNPs and Co3O4 NPs for fluorescence quantitation of hypoxanthine in aquatic products.

Hypoxanthine is a promising index for evaluating the freshness of various aquatic products. Combined the hydrogels containing upconversion nanoparticles (UCNPs), Co3O4 NPs, and N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt/4-amino-antipyrine (TOPS/4-AAP) with a smartphone, a portable sensor was developed for the convenient, sensitive detection of hypoxanthine. With the H2O2 from xanthine oxidase (XOD)-catalyzed reactions of hypoxanthine, the fluorescence of UCNPs was effectively quenched by the purple product produced from the oxidization of TOPS/4-AAP catalyzed by Co3O4 NPs exhibiting peroxidase activity, among which the color change could be transformed into digital signals for quantification of hypoxanthine. The Green value in the RGB analysis of the fluorescence image was negatively proportional to hypoxanthine concentration in the range of 2.5-20 mg/L with a detection limit of 0.69 mg/L and a quantitation limit of 2.30 mg/L. Finally, this sensor was applied for hypoxanthine detection in real aquatic products, showing potential application for freshness evaluation of aquatic products.

Astaxanthin Ameliorates Skeletal Muscle Atrophy in Mice With Cancer Cachexia.

Astaxanthin (AST) is a natural marine carotenoid with a variety of biological activities. This study aimed to demonstrate the possible mechanisms by which AST improves skeletal muscle atrophy in cancer cachexia. In this study, the effects of different doses of AST (30 mg/kg b.w., 60 mg/kg b.w. and 120 mg/kg b.w.) on skeletal muscle functions were explored in mice with cancer cachexia. The results showed that AST (30, 60 and 120 mg/kg b.w.) could effectively protect cachexia mice from body weight and skeletal muscle loss. AST dose-dependently ameliorated the decrease in myofibres cross-sectional area and increased the expression of myosin heavy chain (MHC). AST treatment decreased both the serum and muscle level of IL-6 but not TNF-α in C26 tumor-bearing cachexia mice. Moreover, AST alleviated skeletal muscle atrophy by decreasing the expression of two muscle-specific E3 ligases MAFBx and MuRF-1. AST improved mitochondrial function by downregulating the levels of muscle Fis1, LC3B and Bax, upregulating the levels of muscle Mfn2 and Bcl-2. In conclusion, our study show that AST might be expected to be a nutritional supplement for cancer cachexia patients.

Biochemical characterization and cleavage specificities analyses of three endo-1,3-fucanases within glycoside hydrolase family 174.

Sulfated fucans have garnered extensive research interest in recent decades due to their varied bioactivity. Fucanases are important tools for investigating sulfated fucans. This study reported the bioinformatic analysis and biochemical properties of three GH174 family endo-1,3-fucanases. Wherein, Fun174Rm and Fun174Sb showed the highest optimal reaction temperature among the reported fucanases, and Fun174Sb possessed favorable thermostability and catalysis efficiency. Fun174Rm displayed a random endo-acting manner, while Fun174Ri and Fun174Sb hydrolyzed sulfated fucan in processive manners. UPLC-MS and NMR analyses confirmed that the three enzymes catalyze cleavage of the α(1 â†’ 3)-bonds between Fucp2S and Fucp2S in the sulfated fucan from Isostichopus badionotus. These enzymes demonstrated novel cleavage specificities, which could accept α-Fucp2S residues at subsites -1 and + 1. The acquiring of these biotechnological tools would be beneficial to the in-depth research of sulfated fucans.

Sea cucumber plasmalogen enhance lipophagy to alleviate abnormal lipid accumulation induced by high-fat diet.

Sea cucumber phospholipids, including the plasmalogen (PlsEtn) and plasmanylcholine (PakCho), have been shown to play a regulatory role in lipid metabolism disorders, but their mechanism of action remains unclear. Therefore, high-fat diet (HFD) and palmitic acid were used to establish lipid accumulation models in mice and HepG2 cells, respectively. Results showed that PlsEtn can reduce lipid deposition both in vivo and in vitro. HFD stimulation abnormally activated lipophagy through the phosphorylation of the AMPK/ULK1 pathway. The lipophagy flux monitor revealed abnormalities in the fusion stage of lipophagy. Of note, only PlsEtn stimulated the dynamic remodeling of the autophagosome membrane, which was indicated by the significantly decreased LC3 II/I ratio and p62 level. In all experiments, the effect of PlsEtn was significantly higher than that of PakCho. These findings elucidated the mechanism of PlsEtn in alleviating lipid accumulation, showed that it might be a lipophagy enhancer, and provided new insights into the high-value utilization of sea cucumber as an agricultural resource.

Analysis of unsaturated alginate oligosaccharides using high-performance anion exchange chromatography coupled with mass spectrometry.

Alginate is a commercially important polysaccharide composed of mannuronic acid and its C5 differential isomer guluronic acid. Comprehensive research on alginate and alginate lyases requires efficient and precise analytical methods for alginate oligosaccharides. In this research, high-performance anion exchange chromatography (HPAEC) in parallel with pulsed amperometric detection (PAD) and mass spectrometry (MS) was applied to the analysis of oligosaccharides obtained by alginate lyase. By optimizing the chromatographic conditions including mobile phase concentration, flow rate, and elution gradient, the analysis of a single sample could be completed in 30 min. Seven unsaturated alginate oligosaccharides were separated and identified through their analysis time observed with PAD, including all structurally different unsaturated disaccharides and trisaccharides. The quantitative analysis of seven oligosaccharides was performed based on the quantitative capability of PAD. The method exhibited adequate linearity and precision parameters. All the calibration curves showed good linearity at least in the concentration range of 0.002 to 0.1 mg/mL. The HPAEC-PAD/MS method provides a general and efficient online method to analyze alginate oligosaccharides.

A smartphone-assisted sensing hydrogels based on UCNPs@SiO2-phenol red nanoprobes for detecting the pH of aquatic products.

For some aquatic products, pH has been considered a useful index to reflect the changes in materials during the loss of freshness. Based on the inner filter effect (IFE) between deprotonated phenol red (PR) and upconversion nanoparticles (UCNPs), UCNPs coated with PR-doped SiO2 shell were embedded in agarose hydrogel to develop a smartphone-assisted method for pH sensing. With the enhancement of pH response using a phase transfer agent (i.e., tetra butyl ammonium hydroxide, TBAH), the proposed senor realized the colorimetric and fluorescence detection of pH in the range of pH 6.6-8 and pH 6-8, respectively. The sensor also showed satisfied reversibility when switched between pH 6 and 8 for at least 5 cycles. Moreover, this sensor displayed great sensitivity, stability, and portability in analyzing actual fish, shrimp, and shellfish samples, providing a new sight for evaluating the freshness of aquatic products.

Sea Cucumber Plasmalogen Regulates the Lipid Profile in High-Fat Diet Mouse Liver via Lipophagy.

The sea cucumber plasmalogen PlsEtn has been shown to be associated with various chronic diseases related to lipid metabolism. However, the mechanism is unclear. Therefore, the present study used the sea cucumber plasmanylcholine PakCho as a structural contrast to PlsEtn and assessed its effect in 8 week high-fat diet (HFD)-fed mice. The lipidomic approach based on high-resolution mass spectrometry combined with molecular biology techniques was used to evaluate the mechanism of PlsEtn. The results showed that both PlsEtn and PakCho significantly inhibited an increase in mouse body weight and liver total triglyceride and total cholesterol levels caused by HFD. In addition, oil red O staining demonstrated that lipid droplets stored in the liver were degraded. Meanwhile, untargeted lipidomic experiments revealed that total lipids (increased by 42.8 mmol/mg prot; p < 0.05), triglycerides (increased by 38.9 mmol/mg prot; p < 0.01), sphingolipids (increased by 1.5 mmol/mg prot; p < 0.0001), and phospholipids (increased by 2.5 mmol/mg prot; p < 0.05) were all significantly elevated under HFD. PlsEtn resolved lipid metabolism disorders by alleviating the abnormal expression of lipid subclasses. In addition, five lipid molecular species, PE (18:1/20:4), PE (18:1/20:3), PE (18:1/18:3), TG (16:0/16:0/17:0), and TG (15:0/16:0/18:1), were identified as the biomarkers of HFD-induced lipid metabolism disorders. Finally, lipophagy-associated protein expression analysis showed that HFD abnormally activated lipophagy via ULK1 phosphorylation and PlsEtn alleviated lipophagy disorder through lysosomal function promotion. In addition, PlsEtn performed better than PakCho. Taken together, the current study results unraveled the mechanism of PlsEtn in alleviating lipid metabolism disorder and offered a new theoretical foundation for the high-value development of sea cucumber.

The Discovery of a Multidomain Mannanase Containing Dual-Catalytic Domain of the Same Activity: Biochemical Properties and Synergistic Effect.

In recent years, the wide application of mannan has driven the demand for the exploration of mannanase. As one of the main components of hemicellulose, mannan is an important polysaccharide that ruminants need to degrade and utilize, making rumen a rich source of mannanases. In this study, gene mining of mannanases was performed using bioinformatics, and potential dual-catalytic domain mannanases were heterologously expressed to analyze their properties. The hydrolysis pattern and enzymatic products were identified by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). A dual-catalytic domain mannanase Man26/5 with the same function as the substrate was successfully mined from the genome of cattle rumen microbiota. Compared to the single-catalytic domain, its higher thermal stability (≤50 °C) and catalytic efficiency confirm the synergistic effect between the two catalytic domains. It exhibited a unique "crab-like" structure where the CBM located in the middle is responsible for binding, and the catalytic domains at both ends are responsible for cutting. The exploration of its multidomain structure and synergistic patterns could provide a reference for the artificial construction and molecular modification of enzymes.

Investigation of the Presence of Fibrillin in Sea Cucumber (Apostichopus japonicus) Body Wall by Utilizing Targeted Proteomics and Visualization Strategies.

Fibrillin is an important structural protein in connective tissues. The presence of fibrillin in sea cucumber Apostichopus japonicus is still poorly understood, which limits our understanding of the role of fibrillin in the A. japonicus microstructure. The aim of this study was to clarify the presence of fibrillin in the sea cucumber A. japonicus body wall. Herein, the presence of fibrillin in sea cucumber A. japonicus was investigated by utilizing targeted proteomics and visualization strategies. The contents of three different isoforms of fibrillin with high abundance in A. japonicus were determined to be 0.96, 2.54, and 0.15 µg/g (wet base), respectively. The amino acid sequence of fibrillin (GeneBank number: PIK56741.1) that started at position 631 and ended at position 921 was selected for cloning and expressing antigen. An anti-A. japonicus fibrillin antibody with a titer greater than 1:64 000 was successfully obtained. It was observed that the distribution of fibrillin in the A. japonicus body wall was scattered and dispersed in the form of fibril bundles at the microscale. It further observed that fibrillin was present near collagen fibrils and some entangled outside the collagen fibrils at the nanoscale. Moreover, the stoichiometry of the most dominant collagen and fibrillin molecules in A. japonicus was determined to be approximately 250:1. These results contribute to an understanding of the role of fibrillin in the sea cucumber microstructure.

Stability and programmed sequential release of Lactobacillus plantarum and curcumin encapsulated in bilayer-stabilized W1/O/W2 double emulsion: Effect of pectin as protective shell.

In order to overcome the problem that traditional W1/O/W2 double emulsions do not have targeted release performance, thereby better meeting the health needs of consumers, ovalbumin fibrils/pectin-based bilayer-stabilized double emulsion (OP-BDE) co-encapsulated with Lactobacillus plantarum and curcumin was constructed with pectin as the outer protective shell, which was expected to be used in the development of novel functional foods. The effects of pectin coating on the viability of Lactobacillus plantarum under conditions including storage, pasteurization, freeze-thaw cycles and in vitro simulated digestion were investigated. Results showed that pectin as protective shell could significantly enhance the tolerance of Lactobacillus plantarum to various environmental factors. Besides, the adsorption of pectin endowed OP-BDE with higher lipolysis and stronger protective effect on curcumin, remarkably improving the photostability and bioaccessibility of curcumin. In addition, in vitro simulated gastrointestinal release study indicated that OP-BDE possessed programmed sequential release property, allowing curcumin and Lactobacillus plantarum to be released in small intestine and colon, respectively. OP-BDE is the first reported co-delivery emulsion system with programmed release characteristic. This study provides new insights into OP-BDE in constructing co-delivery systems and programmed sequential release of active substances, and has potential reference and application value in actual food production.

Gut Microbiota Metabolite TMA May Mediate the Effects of TMAO on Glucose and Lipid Metabolism in C57BL/6J Mice.

SCOPE: Gut microbiota can convert a variety of alkaloids and TMAO into TMA, which is then transported by the blood to the liver, and converted into TMAO. In recent years, TMAO has attracted wide attention as a metabolic risk factor in cardiovascular disease, diabetes, and other diseases. However, it is still unclear about the role of gut microbial metabolite TMA in the adverse health impacts of TMAO. METHODS AND RESULTS: Male C57BL/6J is treated with intraperitoneal (i.p.) or oral TMAO for 8 weeks, the area under the OGTT curve of oral group is significantly increased by about 15% compared to the control and injection groups. Serum triglyceride levels in the oral group are significantly higher by 28.2% and 24.6% than those in the control and injection groups, respectively. Meanwhile, cholesterol content in serum is significantly elevated by 27.6% and 30.7%. Similarly, proinflammatory factors gene expressions are significantly increased with oral but not i.p. TMAO intervention. Furthermore, transformation in HepG2 cells shows that TMAO could not be converted into TMA by hepatocytes. CONCLUSION: The adverse effects of TMAO on glucose and lipid metabolism in C57BL/6J mice may act through gut microbiota metabolite TMA.

Integrated Omics Approach: Revealing the Mechanism of Auxenochlorella pyrenoidosa Protein Extract Replacing Fetal Bovine Serum for Fish Muscle Cell Culture.

The process of producing cell-cultured meat involves utilizing a significant amount of culture medium, including fetal bovine serum (FBS), which represents a considerable portion of production expense while also raising environmental and safety concerns. This study demonstrated that supplementation with Auxenochlorella pyrenoidosa protein extract (APE) under low-serum conditions substantially increased Carassius auratus muscle (CAM) cell proliferation and heightened the expression of Myf5 compared to the absence of APE. An integrated intracellular metabolomics and proteomics analysis revealed a total of 13 and 67 differentially expressed metabolites and proteins, respectively, after supplementation with APE in the medium containing 5%FBS, modulating specific metabolism and signaling pathways, which explained the application of APE for passage cell culture under low-serum conditions. Further analysis revealed that the bioactive factors in the APE were protein components. Moreover, CAM cells cultured in reconstructed serum-free media containing APE, l-ascorbic acid, insulin, transferrin, selenium, and ethanolamine exhibited significantly accelerated growth in a scale-up culture. These findings suggest a promising alternative to FBS for fish muscle cell culture that can help reduce production costs and environmental impact in the production of cultured meat.

Changes in Flavor-Related Biomarkers in Pacific Oysters (Crassostrea gigas) Following Microplastic Exposure.

Microplastics have been an emerging threat to filtering species and the ingestion and impacts of microplastics on oysters are a cause for concern. However, much remains unknown about the effects of microplastics on flavor-related biomarkers in oysters. Herein, a laboratory microplastic exposure with concentrations of 1, 10, and 100 mg/L for 15 days was performed to investigate the impacts of microplastics on the flavor parameters of oysters. Exposure to microplastics changed the odor characteristics of oysters. Microplastic exposure had minor effects on the fatty acid composition; however, significant alterations in free amino acids and nucleotides were observed under the 1 and 10 mg/L exposure groups, respectively. The overall results indicated 10 mg/L of microplastic exposure significantly increased the equivalent umami value of oysters. These findings stressed the effects of microplastics on oysters and would be an important reference for the assessment of the potential risks associated with microplastics in marine edible species.

Characterization and structural identification of a family 16 carbohydrate-binding module (CBM): First structural insights into porphyran-binding CBM.

Porphyran is a favorable functional polysaccharide widely distributed in Porphyra. It displays a linear structure majorly constituted by alternating 1,4-linked α-l-galactopyranose-6-sulfate (L6S) and 1,3-linked ß-d-galactopyranose (G) units. Carbohydrate-binding modules (CBMs) are desired tools for the investigation and application of polysaccharides, including in situ visualization, on site and specific assay, and functionalization of biomaterials. However, only one porphyran-binding CBM has been hitherto reported, and its structural knowledge is lacking. Herein, a novel CBM16 family domain from a marine bacterium Aquimarina sp. BL5 was discovered and expressed. The recombinant protein AmCBM16 exhibited the desired specificity for porphyran. Bio-layer interferometry assay revealed that the protein binds to porphyran tetrasaccharide (L6S-G)2 with an association constant of 1.3 × 103 M-1. The structure of AmCBM16 was resolved by the X-ray crystallography, which displays a ß-sandwich fold with two antiparallel ß-sheets constituted by 10 ß-strands. Site-directed mutagenesis analysis demonstrated that the residues Gly-30, Trp-31, Lys-88, Lys-123, Phe-125, and Phe-127 play dominant roles in AmCBM16 binding. This study provides the first structural insights into porphyran-binding CBM.

Establishment of a targeted analysis method for gangliosides in mouse tissues by HILIC-ESI-MS/MS.

Gangliosides play an imperative role in cell signaling, neuronal recovery, apoptosis, and other physiological processes. For example, GM3 can regulate hypothalamic leptin resistance and control energy homeostasis, GD3 can mediate cell proliferation and differentiation and induce apoptosis, and GQ1b can stimulate neurogenesis. Therefore, the present study sought to establish and optimize the targeted analysis method for ganglioside subclasses and their molecular species using hydrophilic interaction liquid chromatography-triple quadrupole-MS/MS (HILIC-QQQ-MS/MS). Additionally, the fragmentation pattern of different ganglioside subclasses and their retention time patterns were analyzed, providing more accurate qualitative results. The limit of quantitation (LOQ) was as low as 10-4 ng. Moreover, the molecular species of gangliosides in the liver, cortex, and hypothalamus of C57BL/6 mice were analyzed using the established method. A total of 23 ganglioside subclasses with 164 molecular species, including 40 O-acetylated ganglioside molecular species and 28 NeuGc ganglioside molecular species, were identified using the semi-quantitative analysis method of an external standard curve corrected by an internal standard. In addition to NeuGc gangliosides, the contents of ganglioside subclasses were more abundant in the mouse brain than those in the mouse liver; especially, the contents of unsaturated gangliosides in the hypothalamus were much higher than those in the liver. Among them, O-acetylated gangliosides were detected only in the cortex and hypothalamus at a concentration of up to 100 µg/mg protein (40 molecular species). Overall, the proposed method expanded the detectable number of ganglioside subclasses and molecular species in biological samples and provided more opportunities for further study of the biological functions of gangliosides.