Novel insight into the formation and improvement mechanism of physical property in fermented tilapia sausage by cooperative fermentation of newly isolated lactic acid bacteria based on microbial contribution.

Single starter can hardly elevate the gel property of fermented freshwater fish sausage. In this work, in order to improve the physical properties of tilapia sausage, two newly isolated strains of lactic acid bacteria (LAB), Latilactobacillus sakei and Pediococcus acidilactici were used for cooperative fermentation of tilapia sausage, followed by the revelation of their formation mechanisms during cooperative fermentation and their improvement mechanisms after comparison with natural fermentation. Both strains, especially L. sakei possessed good growth, acidification ability, and salt tolerance. The gel strength, hardness, springiness, chewiness, whiteness, acidification, and total plate count significantly elevated during cooperative fermentation with starters. Pediococcus, Acinetobacter, and Macrococcus were abundant before fermentation, while Latilactobacillus quickly occupied the dominant position after fermentation for 18-45 h with the relative abundance over 51.5 %. The influence of each genus on the physical properties was calculated through the time-dimension and group-dimension correlation networks. The results suggested that the increase of Latilactobacillus due to the good growth and metabolism of L. sakei contributed the most to the formation and improvement of gel strength, texture properties, color, acidification, and food safety of tilapia sausage after cooperative fermentation. This study provides a novel analysis method to quantitatively evaluate the microbial contribution on the changes of various properties. The cooperative fermentation of LAB can be used for tilapia sausage fermentation to improve its physical properties.

Metabolomics analysis of physicochemical properties associated with freshness degradation in frozen Antarctic krill (Euphausia superba).

This study aimed to determine the effect of different frozen temperatures during storage on the quality of Antarctic krill (Euphausia superba) and assess the change at the metabolite level via a combination of physicochemical property analysis, liquid chromatography-tandem mass spectrometry (LC-MS) based non-targeted metabolomics profiling. Regarding samples stored at -20 °C, the expressions of 7055 metabolites were elevated, while 2313 were downregulated. Lipids and lipid molecules had the highest proportion of differential metabolites. A total of 432 discriminatory metabolites with Kyoto Encyclopedia of Genes and Genomes (KEGG) IDs was obtained. We also observed that the concentrations of differential bitter free amino acids (FAAs) and oxidation products of arachidonic and linoleic acid increased. Moreover, as the storage temperature increased, the freshness, umami, and sweetness components were considerably reduced. Furthermore, results indicated that the color, pH and water-holding capacity (WHC) were potential indicators of quality deterioration, while inosinic acid was a probable biomarker for umami degradation of frozen Antarctic krill. In conclusion, this study demonstrates that storage at lower temperatures can be beneficial for maintaining the freshness of Antarctic krill from macro and micro perspectives.

Correlation analysis of phosphorylation of myofibrillar protein and muscle quality of tilapia during storage in ice.

In this study, the correlation between protein phosphorylation and deterioration in the quality of tilapia during storage in ice was examined by assessing changes in texture, water-holding capacity (WHC), and biochemical characteristics of myofibrillar protein throughout 7 days of storage. The hardness significantly decreased from 471.50 to 252.17 g, whereas cooking and drip losses significantly increased from 26.5% to 32.6% and 2.9% to 9.1%, respectively (P < 0.05). Myofibril fragmentation increased, while myofibrillar protein sulfhydryl content and Ca2+-ATPase activity decreased from 119.33 to 89.29 µmol/g prot and 0.85 to 0.46 µmolPi/mg prot/h, respectively (P < 0.05). Correlation analysis revealed that the myofibrillar protein phosphorylation level was positively correlated with hardness and Ca2+-ATPase activity but negatively correlated with WHC. Myofibrillar protein phosphorylation affects muscle contraction by influencing the dissociation of actomyosin, thereby regulating hardness and WHC. This study provides novel insights for the establishment of quality control strategies for tilapia storage based on protein phosphorylation.

[Identification of Cervi Cornu (Cervus elaphus) and its formula granules based on PCR-RFLP].

Cervi Cornu is the ossified antler, or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C. nippon, as a precious traditional Chinese medicine, has been recognized for its medicinal value and widely used in clinical practice. However, the origins of Cervi Cornu are miscellaneous, and Cervi Cornu is even mixed with adulterants in the market. Currently, there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cornu. So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants. In this study, the differences in the mitochondrial barcode cytochrome b(Cytb) gene sequences of C. elaphus, C. nippon and their related species were compared and the specific single nucleotide polymorphism(SNP) sites on the Cytb sequences of Cervi Cornu were screened out. According to the screened SNPs, Cervi Cornu-specific primers dishmy-F and dishmy-R were designed. The PCR system was established and optimized, and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated. The amplification products of C. elaphus and C. nippon were digested using the restriction enzyme MseⅠ. The results showed that after electrophoresis of the product from PCR with the annealing temperature of 56 ℃ and 35 cycles, a single specific band at about 100 bp was observed for C. elaphus samples, and the product of C. elaphus samples was 60 bp shorter than that of C. nippon samples. There was no band for adulterants from other similar species such as Alces alces, Rangifer tarandus, Odocoileus virginianus, O. hemionus, Cap-reolus pygargus, Przewalskium albirostis and negative controls. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method established in this study can quickly and accurately identify Cervi Cornu originated from C. elaphus in crude drugs, standard decoctions, and formula granules, and distinguish the origins of Cervi Cornu products, i.e., C. nippon and similar species. This study can be a reference for other studies on the quality standard of other formula granules of traditional Chinese medicines.

Improvement mechanism of volatile flavor in fermented tilapia surimi by cooperative fermentation of Pediococcus acidilactici and Latilactobacillus sakei: Quantization of microbial contribution through influence of genus.

Single starter can hardly improve the volatile flavor of fermented fish surimi. In this study, the changes of volatile compounds (VCs) and microbial composition during cooperative fermentation of Latilactobacillus sakei and Pediococcus acidilactici were studied by headspace solid-phase microextraction gas chromatography-mass spectrometry and 16S rRNA gene high-throughput sequencing. During cooperative fermentation, most VCs and the abundance of Latilactobacillus and Lactococcus significantly increased, while Pediococcus, Acinetobacter, and Macrococcus obviously decreased. After evaluation of correlation and abundance of each genus, Latilactobacillus and Lactococcus possessed the highest influence on the formation of volatile flavor during cooperative fermentation. Compared with the natural fermentation, cooperative fermentation with starters significantly enhanced most of pleasant core VCs (odor activity value≥1), but inhibited the production of trimethylamine and methanethiol, mainly resulting from the absolutely highest influence of Latilactobacillus. Cooperative fermentation of starters is an effective method to improve the volatile flavor in the fermented tilapia surimi.

Association of individual and combined exposures of 10 metals with periodontitis: Results from a large population-based study.

AIMS: To systematically investigate the association between individual and combined metal exposure and periodontitis. METHODS: Data encompassing complete periodontal examinations and metal detection in blood and urine samples were procured from the National Health and Nutrition Examination Survey (NHANES) 2011-2014. Three statistical methods, namely weighted logistic regression, Bayesian kernel machine regression (BKMR), and weighted quantile sum (WQS) regression, were used to evaluate the independent and combined associations between metals and periodontitis. RESULTS: Elevated concentrations of blood cadmium (odds ratio [OR]: 1.73, 95% confidence interval [CI]: 1.15-2.61) and blood lead (OR: 1.17, 95 %CI: 1.02-1.34) exhibited a positive association with periodontitis, even after adjusting for potential confounding factors. The BKMR and WQS regression suggested that the co-exposure of metals was also positively associated with periodontitis. Moreover, estradiol and albumin were identified as potential mediators in the relationship between the WQS index of the 10 metals in blood and periodontitis explaining 25.36% and 2.02% of the relationship, respectively. Furthermore, generally consistent patterns of associations between metals and periodontitis and mediating roles of estrogen and albumin were observed after a series of sensitivity analyses. CONCLUSION: This study provides evidence of positive associations between elevated levels of cadmium, lead or metal mixture and periodontitis, which may be partially mediated by sex hormones and oxidative stress indicators.

In-depth discovery and taste presentation mechanism studies on umami peptides derived from fermented sea bass based on peptidomics and machine learning.

Umami peptides originating from fermented sea bass impart a distinctive flavor to food. Nevertheless, large-scale and rapid screening for umami peptides using conventional techniques is challenging because of problems such as prolonged duration and complicated operation. Therefore, we aimed to screen fermented sea bass using peptidomics and machine learning approaches. The taste presentation mechanism of umami peptides was assessed by molecular docking of T1R1/T1R3. Seventy umami peptides identified in fermented sea bass predominantly originated from 28 precursor proteins, including troponin, myosin, motor protein, and creatine kinase. Six umami peptides with the lowest energies formed stable complexes by binding to T1R3. SER170, SER147, GLN389, and HIS145 are critical binding sites for T1R1/T1R3. Four dominant interacting surface forces were identified: aromatic interactions, hydrogen bonding, hydrophilic bonds, and solvent-accessible surfaces. Our study unveils a method to screen umami peptides efficiently, providing a basis for further exploration of their flavor in fermented sea bass.

Integrated 4D label-free proteomics and data mining to elucidate the effects of thermal processing on crisp grass carp protein profiles.

The crisp grass carp (CGC; Ctenopharyngodon idellus C. et V.), known for its unique texture and flavour, is a culinary delicacy whose quality is significantly influenced by thermal processing. This study employed 4D label-free proteomics and data mining techniques to investigate the proteomic changes in CGC muscle tissue induced by various heating temperatures. CGC samples were subjected to a series of heat treatments at increasing temperatures from 20 °C to 90 °C. Proteins were extracted, digested, and analysed using high-resolution mass spectrometry. The proteomic data were then subjected to extensive bioinformatics analysis, including GO and KEGG pathway enrichment. We identified a total of 1085 proteins, 516 of which were shared across all the temperature treatments, indicating a core proteome responsible for CGC textural properties. Differential expression analysis revealed temperature-dependent changes, with significant alterations observed at 90 °C, suggesting denaturation or aggregation of proteins at higher temperatures. Functional enrichment analysis indicated that proteins involved in amino acid metabolism, glutathione metabolism, and nucleotide metabolism were particularly affected by heat. Textural analysis correlated these proteomic changes with alterations in CGC quality attributes, pinpointing 70 °C as the optimum temperature for maintaining the desired texture. A strong positive correlation between specific upregulated proteins was identified, such as the tubulin alpha chain and collagen alpha-1(IV) chain, and the improved textural properties of CGC during thermal processing, suggesting their potential as the potential biomarkers. This study offers a comprehensive proteomic view of the thermal stability and functionality of CGC proteins, delivering invaluable insights for both the culinary processing and scientific management of CGC. Our findings not only deepen the understanding of the molecular mechanisms underpinning the textural alterations in CGC during thermal processing but also furnish practical insights for the aquaculture industry. These insights could be leveraged to optimize cooking techniques, thereby enhancing the quality and consumer appeal of CGC products.

Enrofloxacin pharmacokinetics in yellow catfish (Pelteobagrus fulvidraco): A comparative analysis of oral, intramuscular, and bath administration.

Enrofloxacin (ENR) residues in yellow catfish (Pelteobagrus fulvidraco) often exceed the standard due to excessive use. This study explored the pharmacokinetics of ENR and its metabolite ciprofloxacin (CIP) in yellow catfish following a single dose of 10 mg/kg body weight via intramuscular injection (IM), oral gavage (PO), or a 5-h drug bath at 10 mg/L and 25°C. High-performance liquid chromatography-mass spectrometry was used to determine the ENR and CIP concentrations in various tissues. The highest ENR concentration occurred with IM administration, peaking at 4.124 mg/L in the plasma, 8.359 mg/kg in the kidney, 6.272 mg/kg in the liver, and 5.192 mg/kg in the muscle. However, PO administration resulted in the longest metabolic time, with elimination half-lives of 56.47 h in plasma, 86.43 h in the kidney, 76.25 h in the liver, and 64.75 h in muscle. Additionally, the area under the concentration-time curve values for IM, PO, and bath administration in yellow catfish plasma were 108.36, 88.96, and 22.08 mg·h/L, respectively. These results indicate the effectiveness of all three administration methods in treating bacterial diseases in yellow catfish. The selection of an appropriate administration method depends on the minimal inhibitory concentration of ENR against pathogenic bacteria. Yellow catfish subjected to PO and IM administration require longer resting periods before they can be marketed than those receiving drug bath administration.

Formation and improvement mechanism of physical property and volatile flavor of fermented tilapia surimi by newly isolated lactic acid bacteria based on two dimensional correlation networks.

Fermentation is an effective way to improve the gel properties of freshwater fish surimi. In this study, two newly isolated Lactiplantibacillus plantarum H30-2 and Pediococcus acidilactici H30-21 were used to improve the physical properties and volatile flavor of fermented tilapia surimi. L. plantarum H30-2 quickly improved the whiteness, gel strength, hardness, and chewiness within 18 h. Among 172 volatile compounds analyzed by HS-SPME-GC-MS, most pleasant core flavor compounds (OAV ≥ 1) were improved by L. plantarum H30-2. L. plantarum H30-2 could always adapt to the surimi environment while P. acidilactici H30-21 could not. Two dimensional correlation networks showed that Lactiplantibacillus and Lactococcus were responsible for the quality formation in surimi during natural fermentation or with starters, while the quality improvement after L. plantarum H30-2 addition mainly resulted from the increasing Lactiplantibacillus and its higher acetic acid production. L. plantarum H30-2 can be developed as a special starter using for tilapia surimi fermentation.

Elucidating the potential of chlorogenic acid for controlling Morganella psychrotolerans growth and histamine formation.

AIM: To investigate the inhibitory impact of chlorogenic acid (CGA) on the growth of Morganella psychrotolerans and its ability to form histamine. METHODS AND RESULTS: The antimicrobial effect of CGA on M. psychrotolerans was evaluated using the minimum inhibitory concentration (MIC) method, revealing an MIC value of 10 mg ml-1. The alkaline phosphatase (AKP) activity, cell membrane potential, and scanning electron microscopy images revealed that CGA treatment disrupted cell structure and cell membrane. Moreover, CGA treatment led to a dose-dependent decrease in crude histidine decarboxylase (HDC) activity and gene expression of histidine decarboxylase (hdc). Molecular docking analysis demonstrated that CGA interacted with HDC through hydrogen bonds. Furthermore, in situ investigation confirmed the efficacy of CGA in controlling the growth of M. psychrotolerans and significantly reducing histamine formation in raw tuna. CONCLUSION: CGA had good activity in controlling the growth of M. psychrotolerans and histamine formation.

Comparison of the taste mechanisms of umami and bitter peptides from fermented mandarin fish (Chouguiyu) based on molecular docking and electronic tongue technology.

Unclear taste mechanisms of peptides limit rapid screening of taste peptides with high intensity. In this study, the taste mechanisms of umami and bitter peptides from Chouguiyu were compared. After molecular docking of core umami (NWDDMEK, WFKDEEF, EEEKPKF, DFDDIQK, and DGEKVDF) and bitter (VQDVLKL, VELLKLE, LVVDGVK, VVDLTVR, and VVDGVKL) peptides with T1R1/T1R3 and TASR14, respectively, salt bridges and conventional hydrogen bonds were the main interactions in all taste peptides, in which acidic amino acid residues contributed to the interaction with their receptors. The taste intensity of peptides after solid-phase synthesis was further verified using electronic tongue technology. Spearman correlation analysis showed that docking energy was an important factor for the intensity of taste peptides, while interaction energy and the distance between the binding unit (BU) and the stimulating unit (SU) were also responsible for the bitter intensity. This study provides a theoretical basis to screen novel taste peptides with high taste intensity in fermented foods.

Ultrasound improves the low-sodium salt curing of sea bass: Insights into the effects of ultrasound on texture, microstructure, and flavor characteristics.

The purpose of this study was to evaluate the effects of ultrasonic pretreatment on the quality of cured sea bass. Compared to static marination, ultrasonication significantly increased the rate of NaCl transfer, reduced the hardness and chewiness of fish, and improved water retention. Microstructural observations revealed that the sea bass muscle fibers were severely fragmented, with their borders becoming increasingly blurred with increasing ultrasonic intensity. In addition, ultrasound-assisted marination significantly increased the degradation of proteins, total free amino acid levels, and relative levels of volatile flavor substances such as aldehydes and esters. Therefore, the use of an appropriate ultrasound treatment for the salt curing of fish has a positive effect on the textural and flavor characteristics of sea bass, with the most optimal approach being 300.W ultrasound treatment for 60 min. Overall, the results of this study provide technical evidence for improving the quality of lightly cured low-salt content seafood.

Transforming the fermented fish landscape: Microbiota enable novel, safe, flavorful, and healthy products for modern consumers.

Regular consumption of fish promotes sustainable health while reducing negative environmental impacts. Fermentation has long been used for preserving perishable foods, including fish. Fermented fish products are popular consumer foods of historical and cultural significance owing to their abundant essential nutrients and distinct flavor. This review discusses the recent scientific progress on fermented fish, especially the involved flavor formation processes, microbial metabolic activities, and interconnected biochemical pathways (e.g., enzymatic/non-enzymatic reactions associated with lipids, proteins, and their interactions). The multiple roles of fermentation in preservation of fish, development of desirable flavors, and production of health-promoting nutrients and bioactive substances are also discussed. Finally, prospects for further studies on fermented fish are proposed, including the need of monitoring microorganisms, along with the precise control of a fermentation process to transform the traditional fermented fish to novel, flavorful, healthy, and affordable products for modern consumers. Microbial-enabled innovative fermented fish products that consider both flavor and health benefits are expected to become a significant segment in global food markets. The integration of multi-omics technologies, biotechnology-based approaches (including synthetic biology and metabolic engineering) and sensory and consumer sciences, is crucial for technological innovations related to fermented fish. The findings of this review will provide guidance on future development of new or improved fermented fish products through regulating microbial metabolic processes and enzymatic activities.

Exploring plant polyphenols as anti-allergic functional products to manage the growing incidence of food allergy.

The active substances derived from plants have received increasing attention owing to their wide range of pharmacological applications, including anti-tumor, anti-allergic, anti-viral, and anti-oxidative activities. The allergy epidemic is a growing global public health problem that threatens human health and safety. Polyphenols from plants have significant anti-allergic effects and are an important source of anti-allergic drug research and development. Here, we describe recent advances in the anti-allergic efficacy of plant polyphenols, including their comprehensive effects on cellular or animal models. The current issues and directions for future development in this field are discussed to provide a theoretical basis for the development and utilization of these active substances as anti-allergic products.

BmKK2, a thermostable Kv1.3 blocker from Buthus martensii Karsch (BmK) scorpion, inhibits the activation of macrophages via Kv1.3-NF-κB- NLRP3 axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Inflammation plays pivotal role in the development of chronic diseases. Reducing chronic inflammation is an important strategy for preventing and managing many chronic diseases. In traditional Chinese medicine, the processed Buthus martensii Karsch (BmK) scorpion (also called "Quanxie") has been used to treat chronic inflammatory arthritis and spondylitis for hundreds of years suggests that "Quanxie" could potentially be utilized as a resource for identifying new anti-inflammatory compounds. However, the molecular basis and the underline mechanism for the anti-inflammatory effect of processed BmK scorpion are still unclear. AIM OF THE STUDY: The study aims to determine the potential involvement of macrophage-expressed Kv1.3 in the anti-inflammatory effect of processed BmK scorpion venom, as well as to identify new Kv1.3 blockers derived from processed BmK scorpion. MATERIALS AND METHODS: In this study, the in vivo and in vitro anti-inflammatory activities were determined using carrageenan-induced paw edema, LPS-induced sepsis mouse models and LPS-induced macrophage activation model respectively. The effect of processed BmK scorpion water extract, processed BmK venom and BmKK2 on different potassium channels were detected by whole-cell voltage-clamp recordings on transfected HEK293 cells or mouse BMDMs. The cytokines were detected using Q-PCR and competitive enzyme-linked immunosorbent assay. High performance liquid chromatography, SDS-PAGE and peptide Mass Spectrometry analysis were used to isolate and identify the BmKK2. SiRNA, western blotting and flow cytometry were used to analysis the anti-inflammatory mechanism of BmKK2. RESULTS: Here we demonstrate that BmKK2, a thermostable toxin targeting Kv1.3 is the critical anti-inflammatory component in the processed BmK scorpion. BmKK2 inhibits inflammation by targeting and inhibiting the activity of macrophage Kv1.3, thereby inhibiting the activation of NF-κB-NLRP3 pathway and the subsequent release of inflammatory factors. CONCLUSIONS: These findings provide new insights into the molecular basis of the anti-inflammatory effects of "Quanxie" and highlight the importance of targeting Kv1.3 expressed on macrophages as an anti-inflammatory approach.

Cooperative combination of non-targeted metabolomics and targeted taste analysis for elucidating the taste metabolite profile and pathways of traditional fermented golden pompano.

Fermentation plays a key role in taste formation in traditional fermented golden pompano and involves a series of complex metabolic reactions. Indeed, the taste profile of fermented golden pompano exhibits remarkable variation during early fermentation. Herein, nutritional fingerprinting (proteins, amino acids, lipids, etc.) was applied to discriminate the various biomolecular changes involved in golden pompano fermentation. Among the differential metabolites, amino acids, small peptides, lipids, and nucleotides were considered taste-related compounds. An increase in the amino acid content was observed during fermentation, while the peptide content decreased. Glutamic acid, alanine, and lysine had the highest taste activity values and were the main contributors to taste formation. Metabolic pathway enrichment analysis revealed that taste formation was primarily associated with alanine, aspartate, and glutamate metabolism. These findings provide a deeper understanding of taste mechanisms and establish a basis for the targeted regulation of taste formation in the fermented fish industry.

A novel antioxidant protein of non-phycobiliprotein family derived from marine red alga Porphyra haitanensis.

BACKGROUND: Oxidation has been reported as the one of the deterioration reactions of proteins in aquatic products. Searching for new bioactive substances from marine algae has been one of the main areas in food science and additives. RESULTS: In this study, a novel protein from the red alga Porphyra haitanensis was determined after ammonium sulfate precipitation and gel filtration chromatography. It closely corresponded to the antioxidant activity and was identified as an uncharacterized protein with a molecular mass of 43 kDa, designated Ph43. Bioinformatic analysis revealed that Ph43 is a novel protein of non-phycobiliprotein family with putative chordin domains and rich in α-helical conformation. Recombinant protein (rPh43) was expressed in Escherichia coli as a Hig-tagged protein using a pET-22b vector system and purified by affinity high-performance liquid chromatography. Spectroscopy analysis revealed that there were no structural differences between rPh43 and natural recovered Ph43. Moreover, rPh43 showed equal/higher antioxidant activity compared with Ph43. rPh43 has the potential for application as a natural antioxidant for food stabilization. CONCLUSION: Our results identified a novel antioxidant protein with molecular mass of 43 kDa derived from Porphyra haitanensis that belongs to the non-phycobiliprotein family. © 2023 Society of Chemical Industry.

Identification and application of species-specific peptide biomarkers from soft-shelled turtles (Pelodiscus sinensis) using post-translational modification detection-based peptidomics analysis.

The soft-shelled turtle is a commercially aquatic species in Asian countries, which serves as an important source of collagen with high nutritional and medicinal value, so it is of great significance to distinguish soft-shelled turtle derived collagen from others or adulterations. In this work, peptidomics analysis based on post-translational modification (PTM) assay was used to discover specific peptide biomarkers of soft-shelled turtle gelatin (STG). In total eight specific sequences and 74 peptides with different PTM types were screened out, and seven peptides with good signal responses and STG specificity were selected and validated as STG-specific peptide biomarkers. These peptide biomarkers could be used for distinguishing STG from other animal gelatins, and applied for ensuring the quality of collagens or gelatins from soft-shelled turtle with authenticity and traceability.

A novel strategy to identify the species-specific peptide biomarkers in Pheretima aspergillum (E. Perrier) based on enzymatic digestion followed by LC-MS/MS methods.

Guang Dilong [P. aspergillum (E. Perrier)], is an animal-derived traditional Chinese medicine made from the dried body of Pheretima aspergillum (E. Perrier) (TCM). Due to its widely application and high medical values, preparations of P. aspergillum (E. Perrier) may be adulterated by four other species, including three crucial Pheretima species [P. vulgaris (Chen), P. pectinifera (Mkhaeken), and P. guillemi (Michaelsen)] and one considerable adulteration [Metaphire magna (Chen)]. This study developed a novel and effective strategy for analyzing and authenticating Guang Dilong based on enzymatic digestion of protein. The nanoLC-MS/MS technique used to evaluate complete peptidomics profiles of trypsin-digested samples, resulting in the identification of species-specific peptide biomarkers in P. aspergillum (E. Perrier). The significance of different samples and peptides in the target species set was then investigated using mathematical set theory. Consequently, seven peptides were chosen as prospective biomarkers. Finally, five specific peptide biomarkers for differentiating Guang Dilong with other species were confirmed and validated using UFLC-MS/MS and MRM mode. The suggested technique may also be beneficial in evaluating the quality of other animal-derived goods for safety issues in order to avoid misidentification.