Effect of Protamine on Microorganism Presence and Biogenic Amine Accumulation During Esox lucius Storage Under Refrigerated and Frozen Conditions

TL;DR

This study shows that protamine, extracted from Esox lucius byproducts, can effectively inhibit microbial growth and biogenic amine accumulation during fish storage.

Contribution

The study introduces a natural preservative from Esox lucius byproducts and establishes a method for biogenic amine detection in fish.

Findings

Protamine treatment significantly delayed microbial growth and succession during storage.

The 1% protamine treatment most effectively inhibited biogenic amine accumulation (p < 0.05).

An analytical method for detecting eight biogenic amines in fish muscle was successfully established.

Abstract

The Esox lucius is a high-quality fish species endemic to northern Xinjiang, having developed into a regional specialty industry with significant market value. However, during storage, it is prone to microbial growth that elevates biogenic amine levels, posing potential food safety risks. Therefore, this study systematically evaluated the effects of protamine—extracted from Esox lucius byproducts and used as a natural preservative—on the succession of microbial communities and biogenic amine accumulation in fish muscle under storage conditions of 4 °C, −3 °C, and −18 °C. A detection method for biogenic amines was also established. Results revealed characteristic changes in fish muscle microbial community α-diversity over storage time. Protamine treatment significantly delayed increases in total colony counts and microbial succession processes without altering the final dominant microbial community composition. By optimizing ultrasonic-assisted extraction and derivatization steps, an analytical method suitable for detecting eight biogenic amines in fish muscle matrices was established. Results indicate that protamine effectively inhibits the accumulation of all eight biogenic amines, with the 1% treatment group showing the most significant effect (p < 0.05). This study not only provides basis for the precise application of protamine in seafood preservation but also offers guidance for the resource utilization of aquatic by-products.

Figures

Full text

1. Introduction

Esox lucius, a significant commercial fish species widely distributed in cold waters across Eurasia and North America, is primarily concentrated in China’s northern Xinjiang region, including the Erqis River, Gili Lake, and Ulungur Lake basins. This species commands high commercial value and market demand due to its nutritional advantages: tender flesh, minimal intermuscular bones, high protein content, and low fat. However, during processing and storage, Esox lucius is highly susceptible to muscle softening and spoilage, severely limiting its quality, stability, and shelf life. The high-water content and protein richness of fish tissue provide an ideal substrate for microbial growth. Research indicates that even under low-temperature storage conditions, certain psychrophilic spoilage bacteria can maintain metabolic activity and proliferate extensively [1]. The extracellular proteases and lipases secreted by these microorganisms can specifically hydrolyze myofibrillar proteins and connective tissue proteins [2,3], disrupting muscle ultrastructure and significantly reducing water-holding capacity [4]. This manifests as pronounced texture softening and juice loss. Concurrently, specific enzyme systems produced by microbial metabolism, such as nucleoside phosphorylases and phosphatases, can synergize with endogenous muscle enzymes. This accelerates the degradation pathway of adenosine triphosphate (ATP) via inosine monophosphate (IMP) to hypoxanthine (Hx), leading to a significant increase in freshness deterioration indicators, such as the K value and Hx content. This, in turn, accelerates the deterioration of meat flavor, color, and edible safety [5]. Beyond direct enzymatic action, microorganisms can also catalyze the conversion of free amino acids into biogenic amines, such as histamine, tyramine, cadaverine, and putrescine, via decarboxylation [6]. These substances not only possess potential neurotoxicity but may also react with lipid oxidation products to form carcinogens, such as nitrosamines, posing food safety risks [7]. Notably, the accumulation of basic biogenic amines such as cadaverine and putrescine may help neutralize the acidic environment generated by glycolysis in post-slaughter fish muscle, thereby leading to a rebound in muscle tissue pH during later storage stages. This pH shift creates more favorable conditions for endogenous cathepsins, alleviating acid-induced inhibition of their activity. It may also indirectly disrupt the regulatory balance of the calpain system, thereby indirectly accelerating muscle protein degradation [8]. More importantly, the small nitrogenous molecules produced by protein hydrolysis provide additional growth substrates for surface microorganisms, further promoting microbial proliferation and biogenic amine synthesis. This creates a synergistic cycle of microbial activity, biogenic amines, and endogenous enzymes that accelerates the deterioration of quality [9]. In summary, microorganisms and their metabolic activities are among the core factors contributing to quality decline in fish, such as Esox lucius, during storage. They not only directly degrade nutrients and disrupt tissue structure but also induce chemical hazards and accelerate physical deterioration by producing secondary metabolites such as biogenic amines. Therefore, systematically controlling microbial growth and biogenic amine activity during the processing and storage of Esox lucius has significant theoretical and practical value for ensuring product safety and enhancing resource utilization efficiency.

Protamine is a low-molecular-weight, arginine-rich cationic short peptide primarily extracted from mature fish testes. Its molecules typically consist of approximately 30 to 50 amino acid residues, with arginine content accounting for over two-thirds of the total amino acids [10]. As a naturally occurring polycationic peptide, protamine ‘s antibacterial activity primarily stems from its high-density positive charge. It binds to negatively charged phospholipid molecules or lipopolysaccharides on bacterial cell membranes via electrostatic interactions, disrupting membrane integrity and permeability. This causes intracellular ion imbalance and leakage of contents, thereby inhibiting bacterial proliferation [11]. Furthermore, protamine can penetrate cell walls to enter the cytoplasm, where it binds to nucleic acids (DNA, RNA) or key enzyme proteins. This interferes with the transfer of microbial genetic information, energy metabolism, and biosynthetic processes, thereby inhibiting a wide range of microorganisms [12]. Existing research has confirmed its broad-spectrum inhibitory effects against Gram-positive and Gram-negative bacteria, as well as certain fungi [13]. Although the in vitro antibacterial properties of protamine have been extensively reported, for instance, Guo et al. found that a composite formulation of chitosan lactic acid, protamine, and glycerol monolaurate exhibited synergistic inhibitory effects against Pantoea agglomerans and Enterococcus faecalis in fresh noodles [14]; Hata et al. confirmed that protamine hydrochloride exhibits excellent antifungal efficacy in sugar-rich food matrices [15]. However, existing studies predominantly focus on the inhibitory effects of protamine against specific spoilage bacterial genera, with few investigations systematically elucidating its action patterns in real food matrices from a holistic perspective of microbial community succession. Within complex food systems, particularly in fish muscle matrices characterized by high moisture and protein content alongside diverse microbial ecosystems, the regulatory role of protamine in modulating microbial biomass fluctuations and community succession dynamics during storage remains poorly understood. More importantly, systematic and in-depth studies on the regulatory effects of protamine treatment on biogenic amine metabolism during low-temperature storage of fish muscle, including their concentration effects, remain lacking. The selective inhibitory effects of protamine on different types of biogenic amines are also unclear. This unresolved key scientific issue constrains the precise application of protamine as a green biological preservative in fish products. Therefore, this study used Esox lucius as the experimental subject, treating it with protamine during refrigeration and freezing. Breaking through the limitations of existing research that primarily concentrated on total microbial count measurements, it systematically revealed the regulatory patterns of protamine on the dynamic succession of microbial communities in Esox lucius under refrigerated and frozen storage conditions, elucidating its effects on microbial community structure. Simultaneously, targeting biogenic amines as a critical food safety risk indicator, we aim to establish a trace analysis method for biogenic amines in fish-based matrices. This will enable systematic elucidation of the influence characteristics and concentration-dependent effects of protamine on the accumulation of histamine, tyramine, putrescine, cadaverine, tryptamine, phenylethylamine, spermine, and spermidine in fish muscle. This aims to provide direct scientific evidence and technical guidance for the application of protamine in the low-temperature preservation of Esox lucius. Furthermore, given the continuous expansion of the local cold-water fish industry, this technology demonstrates excellent process transferability and can be extended to broader cold-water fish processing sectors, offering a common technical solution for addressing storage and preservation challenges in bulk cold-water fish. In terms of commercial value, protamine, as a naturally sourced biological preservative, aligns with current consumer demands for clean labels and green processing. Compared to traditional chemical preservatives, it offers significant advantages in high safety and environmental friendliness. By optimizing the effective concentration and application method of protamine, this study is expected to effectively slow the deterioration process of Esox lucius during distribution without altering existing cold chain processes, thereby enhancing economic benefits and resource utilization efficiency. More importantly, by effectively inhibiting biogenic amine formation during storage, this technology reduces the risk of harmful substance intake—such as histamine—providing technical safeguards for seafood consumption safety. This approach offers new insights for developing efficient, safe green preservation technologies for fish products.

2. Materials and Methods

2.1. Sample Preparation

Live Esox lucius purchased from a local supermarket (body weight 1000 ± 50 g), totaling 15 fish, were randomly divided into 5 groups of 3 each and transported to the laboratory in oxygenated water bags. After stunning and euthanizing, the head, tail, scales, skin, viscera, and visible intermuscular bones were sequentially removed. The fish surface and muscle tissue were thoroughly rinsed with sterile physiological saline to remove residual blood and tissue debris. Within a clean bench, the dorsal muscle was dissected along muscle fiber orientation into 2 cm^3^ fish chunks for later use. Pre-chilled (4 °C) sterile physiological saline served as the solvent for preparing working solutions of protamine at mass-to-volume ratios (w/v) of 0% (negative control), 0.1%, 0.5%, and 1.0%. Completely submerge the fish pieces in the corresponding concentration working solution and maintain at 4 °C for 2 min. After treatment, promptly remove and drain the surface solution under sterile conditions for 30 s. Establish an additional group of untreated fish pieces as a blank control (CK). All treated and control fish samples were individually placed in sterile polyethylene bags, sealed, and stored at 4 °C, −3 °C, and −18 °C. On days 1, 4, 7, 10, and 13 of storage, samples were randomly selected from each storage condition for subsequent analysis.

2.2. Determination of Total Colony Count

Refer to GB 4789.2-2016 [16] National Food Safety Standard, Methods for Microbiological Examination of Foods, Determination of Total Colony Count, with minor adjustments based on experimental conditions. The specific procedure is as follows: Place the Esox lucius sample into a sterile sampling bag, cut it into small pieces, and accurately weigh 5.0 g. Transfer the sample to a conical flask containing 45 mL of sterile physiological saline. Thoroughly mix the sample, then take an appropriate amount of the homogenized solution for serial dilutions and pour plates. Incubate all plates at 30 °C in a constant-temperature incubator for 72 h before colony counting.

2.3. Methods for Analyzing Microbial Community Structure

Following the method of Tao et al. [17] with minor modifications, total genomic DNA from microbial communities was extracted from fish muscle samples in both the control and treatment groups. DNA integrity was assessed by 1% agarose gel electrophoresis (JY600C, Beijing Junyi, Beijing, China), and concentration and purity were determined using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Using the extracted DNA as a template, Polymerase Chain Reaction (PCR) (T100 Thermal Cycler, BIO-RAD Laboratories, Hercules, CA, USA) amplification was performed targeting the V3-V4 region of the 16S rRNA gene. Amplification products were separated by 2% agarose gel electrophoresis, purified using a DNA gel recovery and purification kit (PCR Clean-Up Kit, Yuhua, China), and quantified using Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Purified amplicons were used to construct libraries with the NEXTFLEX Rapid DNA-Seq Kit. Subsequently, paired-end sequencing was performed on an Illumina PE250 platform (Shanghai Meiji, Shanghai, China). Fast Length Adjustment of Short reads (version 1.2.11) was used to assemble paired-end sequences into complete tags. Sequences were clustered into operational taxonomic units (OTU) at a 97% similarity threshold after quality-controlled assembly. Species annotation was performed using the Ribosomal Database Project classifier (version 2.11) to align OTUs, with a confidence threshold set at 70%. This enabled the statistical analysis of microbial community composition at different phylum and genus levels across samples. Finally, the abundance matrix of OTUs obtained through clustering at a 97% similarity threshold was used to calculate α-diversity indices for each sample group using the summary.single core analysis command in the mothur [18] software (v1.48.0). To mitigate the impact of sequencing depth variation across samples on diversity estimates, rarefaction analysis was performed on all samples. The following metrics were calculated and output: Abundance-based Coverage Estimator (Ace), Chao1 richness estimator (Chao 1), Shannon–Wiener index (Shannon), Observed species richness (Sobs), and Good’s coverage estimator (Coverage).

2.4. Extraction of Biogenic Amines and Method Development

To optimize the extraction process for biogenic amines, a single-factor experimental system was first employed to investigate the effects of perchloric acid concentration, ultrasonic power, ultrasonic duration, ultrasonic temperature, and extraction frequency on biogenic amine extraction efficiency. The levels of each factor were set as follows: perchloric acid concentration at 0.4, 0.6, and 0.8 mol/L; ultrasonic power at 80, 100, 120, and 140 W; ultrasonic duration at 5, 10, 15, and 20 min; ultrasonic temperature at 30, 40, 50, and 60 °C; and number of extractions at 1, 2, 3, and 4 times. Based on the single-factor trial results, three significantly influential factors were selected to design an orthogonal experiment to determine the optimal process combination for biogenic amine extraction from Esox lucius. Weigh 5.0 g of fish muscle sample, add 10 mL of perchloric acid solution, and after ultrasonic treatment, centrifuge at 8000× g for 10 min (Multifuge ×1R, Thermo Fisher Scientific, USA). Take the supernatant and make up to 25 mL with perchloric acid solution.

2.5. Detection of Biogenic Amines and Method Development

This study employed high-performance liquid chromatography (HPLC) to determine the biogenic amine content in Esox lucius muscle. Key conditions for the derivatization reaction were optimized by investigating the effects of Dansyl chloride (Dns-Cl) concentration (2, 4, 6, 8, 10 mg/mL in acetone solvent), derivatization time (15, 30, 45, 60, 70 min), and reaction temperature (30, 40, 50, 60, 70 °C) on derivatization efficiency. Take 1.0 mL of sample extract, sequentially add 200 μL of 2 mol/L sodium hydroxide solution for alkalization, followed by 300 μL of saturated NaHCO_3_ solution as the buffer system. Add 2.0 mL of Dns-Cl acetone solution to the mixture and incubate in the dark at the appropriate temperature for the specified duration. After the reaction, add 100 μL of ammonia solution to terminate the derivatization reaction and eliminate interference from excess derivatization reagents. Finally, diluted to 5.0 mL with acetonitrile. Filter the derivatized solution through a 0.22 μm organic-phase microporous membrane, transfer it to a chromatographic injection vial, store it at 4 °C in the dark, and perform instrument analysis as soon as possible.

The primary biogenic amines affecting food quality include histamine, tyramine, putrescine, cadaverine, tryptamine, 2-phenylethylamine, spermine, and spermidine [19]. Accurately weigh 50.0 mg of each of the eight biogenic amines (accurate to 0.1 mg), dissolve them separately in perchloric acid solution, and transfer them to a single standard stock solution diluted to 1.0 mg/mL. Based on the expected concentration range of each biogenic amine in the sample, prepare a single-standard stock solution and a mixed standard working solution using a stepwise dilution method. Store at 4 °C in the dark for later use. Derivatization of the mixed standard working solution and sample solutions was performed using the optimized Dns-Cl derivatization conditions described above. Analysis was conducted using a high-performance liquid chromatograph (LC-2010A HT, Shimadzu, Japan). Chromatographic conditions were as follows: Column: Inertsil ODS-C_18_ (4.6 mm × 250 mm, 5 μm); Column temperature: 30 °C; injection volume 20 μL; detection wavelength 254 nm; flow rate 0.8 mL/min. Mobile phase A was ultrapure water, and B was acetonitrile. A gradient elution program was employed for sequential injection and analysis of the mixed standard solution and samples.

2.6. Data Analysis

Each parameter was assessed using three independent biological replicates, with three technical replicates performed for each experimental group. Results are expressed as mean ± standard deviation. A p < 0.05 indicates statistically significant differences. Data were graphed using OriginPro 2025 software (v10.2.5).

3. Results and Analysis

3.1. The Effect of Protamine on Microorganisms of Esox lucius During Storage

3.1.1. Changes in Total Colony Count

Total colony count is a key indicator of fish freshness and shelf life, while microorganisms also play a significant role in the accumulation of biogenic amines in aquatic products [20]. Research findings (Figure 1) reveal marked differences in the dynamic changes in total colony count in Esox lucius flesh across various storage temperatures. At 4 °C, total colony count steadily increased with storage duration, exhibiting typical refrigerated spoilage characteristics. At −3 °C, total colony counts also exhibited an upward trend, but their growth rate was markedly lower than that observed in the 4 °C storage group. In stark contrast, under −18 °C frozen storage conditions, total colony counts remained essentially stable throughout the storage period without a significant increase. These differences primarily stem from the multi-level effects of temperature on microbial physiological activities. At −18 °C, all enzymatic reaction rates in microorganisms decrease drastically, nearly halting metabolism. Furthermore, the majority of free water forms ice crystals, causing the system’s water activity to plummet below the critical threshold for microbial growth. This induces severe cellular osmotic dehydration. Concurrently, mechanical action by ice crystals can cause physical damage to cellular structures [21]. Under the combined effects of these factors, microbial cellular metabolism tends toward stagnation, and growth and reproduction are inhibited, thereby maintaining stable total colony counts. In contrast, while −3 °C is below the freezing point, it causes only partial water freezing, leaving the system with some liquid water. Although this condition imposes both cold and osmotic stress on microorganisms, it allows some cold-tolerant microorganisms to maintain low-level metabolic activity and grow. Under refrigeration conditions below 4 °C, the inhibitory effect of temperature on most spoilage microorganisms is weaker, and their intracellular enzyme activity remains relatively high, resulting in the most rapid increase in total colony count. This phenomenon aligns with the findings of Li et al. [22], who concluded that low-temperature conditions effectively inhibit microbial growth during fish storage.

Further analysis revealed that total colony counts began to rise significantly by the 7th day of storage at 4 °C. By the 10th day, total colony counts in the CK group, 0%, and 0.1% protamine-treated group reached 7.67, 7.70, and 7.65 lg CFU/g, respectively, exceeding the acceptable upper limit for total bacterial counts in fish (7.0 lg CFU/g) set by the International Commission on Microbiological Standards for Foods (ICMSF). This indicates that the samples had undergone significant spoilage by this point. Notably, protamine exhibited concentration-dependent inhibition of microbial growth: the 0.5% treatment group reached the spoilage threshold only by day 13, while the 1% treatment group maintained total colony counts below 7.0 lg CFU/g throughout storage. Under −3 °C storage conditions, the dynamic trend of total colony counts resembled that of the 4 °C group, yet the antibacterial effect of protamine remained significant: Compared to the CK group, 0.1%, 0.5%, and 1% protamine treatments all inhibited microbial growth to varying degrees. The 0.5% and 1% treatments demonstrated the most pronounced effects, calculations based on the total colony counts at Day 0 and Day 13 in Figure 1 indicate that by the end of storage, the increases in total colony counts for these treatments were only 46.93% and 61.81%, respectively—significantly lower than other treatment groups. Under −18 °C frozen storage, although no significant changes in total colony counts were observed in any treatment group within 13 days, the protamine-treated groups still exhibited a slight antibacterial trend. This may stem from its further action on damaged cells during freeze–thaw cycles. However, its primary preservation effect likely lies not in inhibiting growth but in potentially delaying the secondary growth of revived microorganisms after thawing. These results indicate that protamine effectively inhibits microbial growth in Esox lucius muscle during low-temperature storage, with the inhibitory effect concentration-dependent. This phenomenon aligns with Chen et al.’s findings that the inhibitory effect of protamine on Escherichia coli increases with concentration [23]. From a mechanistic perspective, as a cationic polypeptide rich in arginine residues, protamine carries a dense surface charge. It targets and binds to negatively charged components on the microbial cell membrane via electrostatic interactions, thereby disrupting membrane structural integrity [10,11]. Other studies indicate that protamine can penetrate into the cell interior, interfering with microbial nucleic acid replication, transcription, and the function of key metabolic enzymes [12]. At lower concentrations (e.g., 0.1%), protamine molecules cannot fully coat bacterial surfaces, causing only minor membrane disruption and metabolic interference. This fails to induce lethal membrane damage, resulting in limited antibacterial efficacy. When concentrations reach 0.5% or 1%, protamine forms a dense coating on bacterial membranes, effectively compromising membrane integrity and leading to cell death or severe damage, thereby significantly enhancing antibacterial activity [24].

3.1.2. Analysis of Microbial Community α-Diversity

Esox lucius, as a cold-water commercial fish species, exhibits quality deterioration during low-temperature storage that is closely linked to microbe-mediated biogenic amine accumulation. Microbial-secreted decarboxylases catalyze the conversion of free amino acids in fish muscle into biogenic amines such as putrescine and cadaverine, while the dynamic evolution of microbial community abundance and structure directly regulates the accumulation rate and composition of these biogenic amines [25]. As a natural cationic antimicrobial peptide, protamine has been demonstrated to inhibit food spoilage microorganisms through multiple mechanisms [26]. However, the targeted selection effect on microbial communities during cold storage of Esox lucius, and its synergistic interaction with cold stress, remain unclear. Therefore, this study quantifies α-diversity and analyzes community structure to reveal succession patterns of microbial communities in Esox lucius during low-temperature storage (4 °C and −3 °C) under protamine treatment. This provides theoretical support for targeted interventions to modulate biogenic amine formation and enhance the safety of stored aquatic products.

As shown in Table 1, both the Ace index (an estimate of community richness) and the Chao1 index (species abundance prediction) exhibited non-monotonic changes across treatment groups, initially increasing and then decreasing. This pattern reflects the dynamic adaptive strategy of microbial communities in response to protamine treatment and low-temperature stress. Zhong et al. also observed that the Chao1 and Ace indices exhibited a consistent decline over storage time in yellowtail fillets stored at −1 °C, 4 °C, and 20 °C [27]. During the initial storage phase, dominant bacteria in fish muscle were suppressed to varying degrees by both protamine and low temperature, releasing nutritional niches. Concurrently, the 4 °C refrigeration and −3 °C partial freezing conditions may have stimulated metabolic activity in certain psychrophilic microorganisms [28]. These relatively scarce microbial species rapidly proliferated within the released niches, leading to a significant increase in community species richness. As storage time extends, the persistent dual selective pressures of protamine and low temperature allow only a few highly tolerant strains to survive and dominate. These dominant strains further inhibit other microorganisms by rapidly consuming nutrients and accumulating metabolic byproducts, ultimately leading to a decline in community species numbers and structural simplification. Additionally, the accumulation of substances such as ammonia and organic acids produced by microbial metabolism during storage causes fluctuations in the pH of fish muscle. This further accelerates the elimination of intolerant species and speeds up community simplification, as evidenced by decreases in the Ace and Chao1 indices. Notably, the Sobs index—reflecting observed species richness—exhibited distinct patterns at 4 °C and −3 °C. At 4 °C, it showed an overall decline, though the 1% protamine-treatment group exhibited an initial rise followed by a decrease. Conversely, at −3 °C, all treatment groups followed an initial increase, followed by a decrease. This discrepancy may stem from the synergistic interaction between the intensity of temperature stress and the antibacterial effect of protamine. At −3 °C, partial freezing triggers ice crystal formation and osmotic pressure changes, causing severe physical damage to mesophilic bacteria. Concurrently, protamine’s antibacterial action accelerates the demise of sensitive bacteria, temporarily freeing up ecological niches and nutrients for cold-tolerant species—manifesting as a brief increase in species count. As storage prolonged, cumulative ice crystal damage combined with the sustained antibacterial effect of protamine, allowing only a few extremely tolerant strains to survive and monopolize the ecological niche, causing the Sobs index to decline. In contrast, 4 °C represents conventional refrigeration temperatures where sensitive bacteria may not be immediately suppressed by temperature alone. Here, psychrophilic and cold-tolerant bacteria become the dominant adapters. At this stage, low concentrations (0.1% or 0.5%) of protamine are insufficient to achieve broad-spectrum bactericidal effects but may impose additional stress on certain sensitive bacteria. Under dual stress from low temperature and protamine, non-adaptive species gradually die off or decline below detection limits. Simultaneously, a few dominant spoilage bacteria adapted to low temperatures and possessing some protamine tolerance begin rapid proliferation. Leveraging their growth advantage, they expand rapidly, eliminating opportunities for other species to recover or grow. The community evolves toward reduced species diversity but increased abundance of specific species. The high concentration of protamine in the 1% treatment group strongly inhibited a broad spectrum of microorganisms, including many potentially dominant spoilage bacteria, disrupting the original community balance. Previously suppressed rare cold-tolerant or peptide-resistant microorganisms gained growth opportunities, leading to a temporary increase in species richness. As storage time extended and nutrients gradually depleted, stress-tolerant microorganisms began competing for limited resources. Dominant strains gradually gained supremacy, causing species richness to decline once more.

The Shannon index reflects both species richness and species evenness. During cold storage, the Shannon index declined. This indicates that, under combined stress from protamine treatment and low temperatures, fish muscle sustained extensive damage to microbial cells. Due to significant variations in repair and tolerance capacities among different species, intense selection occurred. Only groups possessing specific tolerance phenotypes survived and became dominant. Notably, even in the control group without protamine and the CK group, low temperature itself serves as a potent directional selective pressure, driving the community toward a structure dominated by cold-tolerant bacteria [29,30]. The addition of protamine accelerates and intensifies this selection process. It likely eliminates initial competitors more rapidly, paving the way for the rise in dominant spoilage bacteria. Duan et al. [31] also demonstrated that protamine treatment inhibited the growth of putrefactive bacteria and affected the progression of changes in the microbial community structure. Simultaneously, different protamine concentrations may select for slightly distinct dominant species, yet all outcomes lead to community simplification and reduced evenness. By comparing with existing studies, we found that the results of this study are highly consistent with Zhang et al.’s conclusion that protamine regulates microbial community structure without altering the final dominant microbial communities [32]. Consequently, the α-diversity analysis above did not reveal completely opposite trends between the CK and 0% treatment groups. This precisely indicates that, throughout storage, the community’s species composition may undergo reorganization and replacement. However, the core process of rapid rise and monopolization by specific dominant bacteria is universal and irreversible [33].

3.1.3. Analysis of Microbial Community Structure

Analysis at the phylum level based on 16S rRNA gene sequencing (Figure 2a) revealed that the dominant phyla in all treatment groups during the early storage phase were Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and Cyanobacteria. This composition aligns with the typical microbial community observed during the initial stages of low-temperature storage in freshwater fish [34,35]. By the end of storage, Proteobacteria became the dominant phylum, followed by Firmicutes. The dominance of Proteobacteria stems from its broad environmental adaptability; strains within this phylum are predominantly facultative anaerobes or aerobes capable of rapidly proliferating by utilizing nutrients such as proteins and amino acids in fish muscle. Furthermore, most species possess functional traits, such as cold tolerance and resistance to fluctuations in osmotic pressure [36], which confer a competitive advantage under combined stress conditions. This finding aligns with the results reported by Nimnoi et al. [37], who found that Proteobacteria was the dominant phylum in short mackerel (Rastrelliger brachysoma) across various storage conditions, accounting for 40.17–97.40% of the microbial community. Cui et al. [38] observed a significant reduction in bacterial species richness and diversity during chilled salmon storage, with Pseudomonas, a Proteobacteria, ultimately becoming the dominant group. Similar conclusions were reached by Sheng et al. [39] in pufferfish (Takifugu obscurus) and Wang et al. [40] in tilapia during storage. Genus-level analysis (Figure 2b) revealed a complex microbial community composition in fish muscle during early storage, primarily comprising Pseudomonas, Delftia, Acinetobacter, Stenotrophomonas, Rhodoferax, Aquabacterium, Lactobacillus, Pelomonas, Brochothrix, and Shewanella. During this stage, fish muscle is rich in nutrients, with relatively mild physicochemical conditions such as pH, osmotic pressure, and metabolic waste accumulation. No strong selective pressure has yet emerged, allowing multiple microorganisms to survive temporarily and resulting in high species richness. By the end of storage, the community structure significantly simplified, with Pseudomonas and Delftia becoming dominant genera, both belonging to the Proteobacteria phylum. High-throughput sequencing results indicated that the dominant microbial communities in the Fresh group were Delftia (47.61%) and Pseudomonas (23.71%); After 13 days of storage at −3 °C and 4 °C, Pseudomonas became the dominant genus in the CK group, accounting for 97.62% and 98.67%, respectively; in the 0% treatment group, Pseudomonas was also the dominant genus, representing 47.29% and 73.75%, respectively; In the 0.1% treatment group, Delftia was the dominant genus at 51.13% after 13 days of storage at −3 °C. At 4 °C, Pseudomonas and Acinetobacter were the dominant genera at 66.70% and 11.29%, respectively. In the 0.5% treatment group, Pseudomonas was the dominant genus at 99.73% and 84.47%; in the 1.0% treatment group, Pseudomonas was the dominant genus at 95.96% and 98.95%. These findings align with previous studies. Hu [41] and Tian et al. [42] observed that Pseudomonas and Shewanella dominated grass carp at the end of 4 °C storage. Jia et al. identified Pseudomonas, Aeromonas, Acinetobacter, and Shewanella as dominant bacteria in silver carp (Hypophthalmichthys molitrix) [43]. Liu et al. [44] identified specific spoilage bacteria in late-stage spoiled bighead carp (Aristichthys nobilis) as Aeromonas, Pseudomonas, Shewanella, and Acinetobacter. These findings corroborate α-diversity analyses, indicating that under combined stress, the community tends toward simplification and succession toward dominant species. From a functional perspective, Pseudomonas (e.g., Pseudomonas aeruginosa) and Delftia (e.g., Delftia acidovorans) represent typical psychrophilic or cold-tolerant spoilage bacteria capable of maintaining high metabolic activity at low temperatures. They secrete key enzymes, such as lysine decarboxylase and ornithine decarboxylase, which catalyze the conversion of free amino acids into biogenic amines, such as histamine and putrescine [36,45]. Notably, while protamine treatment influenced community succession rates, it did not alter the dominance patterns of Pseudomonas and Delftia species. This may be closely related to their tolerance to protamine and cold adaptation. Pseudomonas species can form biofilms by synthesizing extracellular polysaccharides, reducing protamine binding efficiency to cell membranes [46,47]. Delftia also exhibits strong organic nitrogen utilization capabilities, surpassing other initial microbial communities in nutrient uptake efficiency under stress conditions. During low-temperature storage, protamine shapes microbial community structure through selective pressure, promoting the accumulation of cold-tolerant and protamine-resistant strains, which may subsequently influence biogenic amine formation dynamics.

As shown in Figure 3a, a total of 816 OTUs were detected in the fresh Esox lucius muscle samples (Fresh group). This initial microbial community primarily originated from environmental bacteria during the fishing and processing stages, as well as the fish’s own gut microbiota. At this stage, niche differentiation within the community was incomplete, dominant microbial groups had not yet formed, and competitive relationships between species were loose. The community exhibited high sensitivity to environmental disturbances such as cold stress and changes in nutrient substrates, making it prone to dramatic succession driven by subsequent environmental changes [48]. By day 7 of cold storage, sample OTU numbers surged to 1376, a 68.6% increase over the Fresh group—indicating rising microbial diversity. These shared 507 OTUs (62.1% of the initial OTUs) with the Fresh group. This shared microbiota represents initial communities that successfully survive and proliferate by adapting to low temperatures and utilizing early nutrients. Research by Weedon et al. indicates that the direct effect of temperature on OTU abundance stems from the temperature adaptability of the community [49]. The newly added OTUs predominantly belonged to opportunistic microbial groups. These communities colonize through ecological niche complementarity, utilizing protein hydrolysis products or tolerating early metabolic byproducts to occupy new ecological niches within the community [50]. By the late stage of low-temperature storage (13 days), OTU numbers sharply decreased by 65.1% to 480. Notably, samples from this stage shared 403 OTUs with the 7-day samples, accounting for 83.9% of the OTUs present at this stage. Figure 3b shows the PCoA analysis of microbial communities at the OTU level. At the end of storage (13 days), samples from each group exhibit highly clustered positions and are closely grouped, indicating a highly similar microbial community structure. Zhong et al. also found that bacterial community structure differences decreased among samples of greater amberjack (Seriola dumerili) fillets at different storage temperature endpoints [27]. As storage time increased, the microenvironment of fish muscle underwent systematic changes. Further pH shifts, accumulation of microbial secondary metabolites, and nutrient depletion intensified environmental stress. These harsh conditions selected for highly specialized stress-tolerant microbial communities through competitive exclusion, ultimately forming a stable community structure centered on specific spoilage bacteria [29,51]. This dynamic process reveals the structural transformation patterns of spoilage microbial communities in Esox lucius muscle during cold storage, providing theoretical foundations for identifying core spoilage bacteria and for developing strategies to regulate biogenic amine production.

3.2. Effect of Protamine on Biogenic Amines During Storage of Esox lucius

3.2.1. Establishment of Biogenic Amine Detection Method

To achieve accurate quantitative detection of biogenic amines in Esox lucius flesh, this study established an efficient and stable HPLC detection method by optimizing the derivatization conditions of Dns-Cl and the sample pretreatment process. Since most biogenic amines lack intrinsic UV-absorbing or fluorescent groups, they cannot be directly detected with high sensitivity using conventional HPLC. Dns-Cl reacts with primary and secondary amines in biogenic amines to form derivatives that exhibit strong UV absorption and fluorescence, significantly enhancing detection sensitivity and chromatographic separation efficiency [52]. Pataca also employed pre-column derivatization with Dns-Cl during the biogenic amine analysis of tuna samples [53]. As shown in Figure 4, using the chromatographic response value of the bioamine derivatives as the evaluation criterion, and comprehensively considering the adequacy of the derivatization reaction and the stability of the derivative products, the optimal derivatization conditions were identified as: Dns-Cl concentration of 10 mg/mL, derivatization time of 45 min, and derivatization temperature of 50 °C.

Meanwhile, traditional biogenic amine extraction processes rely on mechanical agitation or static extraction, which can lead to incomplete extraction of target compounds due to uneven mass transfer. Ultrasound-assisted extraction utilizes high-frequency microjets generated by cavitation effects and localized high-temperature/high-pressure environments to thoroughly disrupt cellular structures. This facilitates the rapid release of biogenic amines from complex fish muscle matrices into the solvent system. The process significantly shortens extraction time while reducing amine oxidation or degradation that may occur during prolonged extraction, thereby more accurately reflecting the initial sample content. Therefore, based on the optimized derivatization system described above, optimal extraction parameters were selected through single-factor experiments using bioamine extraction rate as the evaluation criterion (Figure 5): perchloric acid concentration 0.4% (v/v), ultrasonic power 140 W, ultrasonic duration 10 min, ultrasonic temperature 40 °C, and extraction frequency 2 times. Further optimization was conducted using an orthogonal design, selecting ultrasonic power (A), ultrasonic temperature (B), and ultrasonic duration (C) as factors. Results (Table 2) indicated that the order of influence on extraction rate was: ultrasonic duration > ultrasonic temperature > ultrasonic power. Through comprehensive evaluation and model prediction, the optimal extraction conditions were determined as: ultrasonic power 100 W, ultrasonic temperature 40 °C, and ultrasonic duration 10 min. Chromatograms of eight biogenic amines from Esox lucius samples showed symmetrical peaks without tailing. Retention times of all components closely matched those of the corresponding standards, and response values showed excellent agreement. These results confirm that under these conditions, while ensuring a high extraction rate, the sensitivity and accuracy of HPLC detection of biogenic amines in fish muscle are significantly enhanced. This provides reliable technical support for monitoring biogenic amines in Esox lucius-specific samples and demonstrates strong potential for practical application.

3.2.2. Analysis of Biogenic Amine Changes

To investigate the effects of protamine treatment on biogenic amine formation and accumulation during low-temperature storage of fish muscle, this study systematically analyzed the dynamic changes in various biogenic amines by combining the effects of different storage temperatures (4 °C, −3 °C, −18 °C) and protamine concentrations (0.1%, 0.5%, 1%). The results are shown in Figure 6. The primary biogenic amines in fresh Esox lucius were tryptamine, phenylethylamine, histamine, and spermidine. During storage, cadaverine and putrescine were the main compounds generated. This phenomenon is consistent with Chiesa et al.’s findings in a study of 84 bluefin tuna (Thunnus thynnus) specimens, which similarly demonstrated significant accumulation of cadaverine and putrescine in certain samples [54]. Furthermore, Atmaca’s evaluation of biogenic amines in farmed rainbow trout (Oncorhynchus Mykiss) confirmed that putrescine levels increased significantly across different storage temperatures (0 °C, 2 °C, 4 °C) [55]. Research by Fan et al. also indicates that the levels of tryptamine, 2-phenylethylamine, putrescine, cadaverine, and tyramine significantly increased (p < 0.05) in black carp fillets stored at different temperatures [56]. With prolonged storage, all experimental groups showed progressive increases in tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermine, and spermidine. Storage temperature and protamine treatment significantly regulated biamine accumulation. Taking the CK group as an example, by day 13 of storage at 4 °C, putrescine and cadaverine increased to 57.5 mg/kg and 45.2 mg/kg, respectively, indicating significant spoilage. At −3 °C, their levels were 48.9 mg/kg and 39.4 mg/kg, respectively. while at −18 °C, putrescine and cadaverine accumulated to only 4.9 mg/kg and 5.7 mg/kg, respectively. Phenethylamine exhibited a similar pattern: under 4 °C storage, its content in the CK group increased from an initial 5.08 mg/kg to 30.96 mg/kg (approximately a 6-fold increase); whereas at −18 °C, its content increased only slightly and remained significantly lower than in the 4 °C storage group. Hu et al. also observed similar phenomena in their studies of fish, squid, and octopus: freezing effectively prevents biogenic amine formation, but levels of putrescine, cadaverine, histamine, and tyramine significantly increase [57]. Li et al. also confirmed that temperature influences biogenic amine formation when evaluating changes in rainbow trout (Oncorhynchus Mykiss) fillets at different temperatures (−3, 3, 9, and 15 °C), with higher storage temperatures more readily promoting histamine accumulation [58]. Concurrently, protamine treatment significantly reduced the accumulation of all biogenic amines, with the inhibitory effect positively correlated with concentration. Taking the 1% protamine-treated group as an example, at the end of storage, the tryptamine content in the 1% treatment group at 4 °C, −3 °C, and −18 °C was 53.89%, 54.24%, and 90.63% of the CK group, respectively. Phenethylamine levels were 55.74%, 62.08%, and 76.43% of the CK group, respectively. Putrescine levels were 18.3, 18.2, and 3.9 mg/kg, respectively; cadaverine levels were 13.8, 12.5, and 2.2 mg/kg, respectively, all significantly lower than the corresponding control group at the same temperature. At 4 °C, the histamine content in the 1% treatment group was only 58.43% of that in the CK group; at −3 °C and −18 °C, histamine levels were reduced by approximately 3.0 mg/kg and 1.6 mg/kg, respectively, compared to the CK group. At 4 °C and −3 °C, tyramine levels in the 1% treatment group were 38.80% and 36.24% of the CK group, respectively. After 13 days of storage at 4 °C, the CK group showed increases of 23.0 mg/kg and 17.2 mg/kg in spermidine and spermine, respectively, whereas the 1% treatment group showed increases of only 13.5 mg/kg and 4.8 mg/kg. Protamine at all concentrations inhibited biogenic amines, with the 1% treatment demonstrating the most pronounced effect.

Overall, as storage time increased, the levels of all eight biogenic amines in fish muscle across all experimental groups showed an upward trend. Storage temperature significantly regulates biogenic amine accumulation, with higher temperatures accelerating their formation rate and increasing their accumulation levels. Protamine treatment inhibited the formation of all biogenic amines, with the most pronounced suppression observed at 1%. Biogenic amines are products of amino acid decarboxylation catalyzed by decarboxylases. During postmortem storage of fish, this reaction is primarily driven by endogenous tissue enzymes and decarboxylases secreted by microorganisms [6,8]. As storage time increases, microbial proliferation and accumulation of enzyme activity become the primary pathways for biogenic amine formation. Extensive research has demonstrated that various bacteria, particularly certain Gram-negative bacteria, possess strong amino acid decarboxylase capabilities and are key factors in biogenic amine formation in food. For example, Özogul et al. confirmed Pseudomonas aeruginosa exhibits strong tyramine production capacity [59]. Margareta et al. demonstrated that despite refrigeration and strict contamination control, naturally occurring bacteria in mackerel continued to grow and produce histamine [60]. During cold storage, lower temperatures prolong microbial division cycles, slow biomass accumulation, and, correspondingly, reduce decarboxylase production. As a protein, amino acid decarboxylase exhibits highly temperature-dependent catalytic activity, with lower temperatures decreasing enzyme reaction rates. Consequently, even under identical initial microbial conditions, biogenic amine accumulation at −18 °C is significantly lower than at 4 °C. Certain strains of the Pseudomonas and Delftia genera have been reported to possess amino acid decarboxylase activity, converting amino acids into biogenic amines [6]. Thus, their subsequent dominance directly drives accelerated accumulation of biogenic amines. Meanwhile, increased protamine concentration forms an effective antimicrobial barrier on fish muscle surfaces, significantly reducing total viable bacteria counts—particularly inhibiting proliferation of dominant spoilage bacteria. This reduction in microbial biomass directly lowers total decarboxylase activity [12], thereby slowing biogenic amine production rates. The most pronounced inhibitory effect is observed at a 1% concentration. This study not only provides theoretical support for elucidating the role of protamine in regulating biogenic amines during fish preservation but also offers technical references for developing efficient and safe natural preservation strategies. It contributes to extending the shelf life of aquatic products and enhancing food safety, holding significant practical implications for advancing the quality and upgrading of the aquatic product storage and processing industry. It should be emphasized that although this study systematically investigated the effects of protamine on the microbial communities and biogenic amine metabolism of Esox lucius during refrigeration and freezing, and preliminarily confirmed its potential application as a natural preservative, research on the association between protamine regulation of microbial community succession and biogenic amine metabolism in cold-water fish remains limited. This study has not yet delved into how protamine regulates specific microbial metabolic pathways at the molecular level, particularly gene expression related to amino acid decarboxylase activity. Future research could further elucidate the intrinsic mechanisms by which protamine regulates specific biogenic amine accumulation to construct a more comprehensive regulatory mechanism map. Additionally, the refrigeration and freezing conditions employed in this study were relatively uniform. Future investigations could explore different temperature gradients and storage durations to systematically evaluate protamine’s antibacterial efficacy and its impact on biogenic amine accumulation under varying cold chain conditions, thereby advancing its practical application in seafood preservation. Of course, from a technological application and industrialization perspective, the practical promotion of protamine still faces several technical and economic challenges. First, extraction costs and yield are key factors limiting its large-scale application. Although the protamine used in this study was derived from fish processing by-products, achieving resource reuse, its industrial extraction process, purification efficiency, and batch consistency still require further optimization to reduce production costs. Second, a systematic assessment of its impact on product sensory qualities is required. While protamine effectively inhibits microorganisms, its potential negative effects on sensory attributes such as color, odor, and texture of fish muscle necessitate comprehensive evaluation considering consumer preferences. Additionally, economic viability is a critical factor for technological adoption. Future research should systematically compare the usage costs of protamine with traditional chemical preservatives, evaluating its cost–benefit ratio under various storage conditions within real-world cold chain logistics scenarios to clarify its competitive market advantages. Finally, standardized application research must be conducted for diverse aquatic products and processing scenarios to determine optimal protamine dosage, treatment methods, and complementary cold chain parameters. This will establish a precise, standardized technical application system, providing technical specifications for its targeted use in the food industry.

4. Conclusions

This study systematically evaluated the regulatory effects of protamine on microbial community succession and biogenic amine accumulation in Esox lucius under storage conditions of 4 °C, −3 °C, and −18 °C. Results indicated that protamine significantly delayed microbial growth, with its antibacterial effect exhibiting concentration dependence, most pronounced in the 1% treatment group, though its inhibitory action tended to plateau under −18 °C frozen storage conditions. Microbial community analysis revealed that microbial diversity during refrigeration and freezing generally exhibited a non-monotonic change pattern, initially increasing and then decreasing. Although protamine delayed the succession rate, it did not alter the succession pattern where Pseudomonas and Delftia ultimately became the dominant genera. This finding suggests that protamine’s antibacterial mechanism primarily regulates microbial community succession rates rather than reshaping microbial structures or altering dominant spoilage bacteria species, offering new insights into the action patterns of natural preservatives.

Regarding biogenic amine regulation, this study optimized and established a trace detection method for biogenic amines suitable for fish muscle matrices, enabling accurate quantification of eight biogenic amines. Analysis revealed that storage temperature (4 °C, −3 °C, −18 °C) significantly regulates biogenic amine accumulation: higher temperatures accelerate biogenic amine production rates and increase accumulation levels. Protamine treatment exhibited inhibitory effects on all biogenic amine production, with the 1% concentration treatment demonstrating the most pronounced suppression. In summary, this study provides scientific evidence for the precise application of protamine as a natural preservative in aquatic product storage and offers technical guidance for the resource utilization of aquatic processing by-products.