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Article

Novel Insight into Metabolism Mechanism of Biogenic Amines During Fermentation of Chinese Traditional Fermented Mandarin Fish (Chouguiyu) Based on Metabolism Pathway and Correlation Network

by
Jun Li
1,
Daqiao Yang
2,3,*,
Yongqiang Zhao
2,
Di Wang
2,
Hui Huang
2 and
Chunsheng Li
2,*
1
Guangdong Provincial Key Laboratory of Lingnan Specialty Food Science and Technology, Key Laboratory of Green Processing and Intelligent Manufacturing of Lingnan Specialty Food, Ministry of Agriculture and Rural, College of Light Industry and Food, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
2
Key Laboratory of Aquatic Product Processing, Ministry of Agriculture and Rural Affairs, National R&D Center for Aquatic Product Processing, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China
3
College of Life Sciences, Linyi University, Linyi 276005, China
*
Authors to whom correspondence should be addressed.
Foods 2025, 14(16), 2863; https://doi.org/10.3390/foods14162863
Submission received: 24 June 2025 / Revised: 28 July 2025 / Accepted: 11 August 2025 / Published: 18 August 2025
(This article belongs to the Special Issue Food Microorganism Contribution to Fermented Foods)
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Abstract

A complex microbial community gives the possibility to produce biogenic amines in traditional fermented foods. In this study, the metabolism mechanisms of biogenic amines during fermentation of fermented mandarin fish Chouguiyu were revealed based on the metabolic pathways and correlation analysis. Functional genes based on KEGG orthology related to biogenic amine metabolism were selected from the metagenome and were used to construct the biogenic amine metabolic pathways in Chouguiyu. A total of 91 and 75 genera were related to the synthesis and degradation of biogenic amines, respectively. High concentrations of cadaverine and putrescine were observed, while the other biogenic amines were detected in relatively low concentrations. The metabolic mechanisms of various biogenic amines were illuminated by correlation network maps between biogenic amines and microbial synthesis/degradation enzymes. Lactococcus, Flavobacterium, Tessaracoccus, and Yoonia could only degrade and not produce biogenic amines. Acinetobacter and Enterococcus possessed more abundant enzymes for degradation than synthesis. Amine oxidase (K00276), diamine N-acetyltransferase (K00657), and gamma-glutamylputrescine synthase (K09470) were the main biogenic amine degradation enzymes in the microbial community. Lactococcus garvieae, Flavobacterium gelidilacus, Tessaracoccus antarcticus, Yoonia vestfoldensis, Acinetobacter haemolyticus, and Enterococcus ureasiticus were the main microbial species for biogenic amine degradation and could be isolated as the potential strains for biogenic amine degradation in fermented foods.

1. Introduction

Biogenic amines (BAs) in foods are mainly produced by the decarboxylation of amino acids under the action of microbial decarboxylases [1]. Excessive BAs can cause a variety of harmful reactions, such as headaches, rashes, nausea, diarrhea, flushing, and neurotoxicity [2]. BAs are easily produced in fermented foods because of the high concentrations of free amino acids and complex microbial community [3]. Many researchers have studied the BA synthesis and degradation ability of microorganisms in fermented foods after isolation and identification [4,5]. However, it is difficult to give a comprehensive understanding of the microbial metabolism mechanism of BAs in fermented foods.
With the development of microbial analysis, high-throughput sequencing technology can comprehensively analyze the complex microbial community in fermented foods [6,7]. Many studies have discovered BA-producing and BA-degrading microorganisms through correlation analysis between microbial communities and BAs [8,9]. However, correlation analysis only considers whether the abundance of the microbial community is consistent with the change in BA content and ignores the relevant enzymes for BA metabolism in these microorganisms. It cannot determine the core microorganisms that contribute to the synthesis and degradation of BAs in fermented foods. Metagenomics can predict microbial function by analysis of the whole genetic material of microbial community [10,11]. It provides an effective method to select the enzymes related to BA metabolism from microorganisms in fermented foods [12].
Chouguiyu is a traditional fermented food made from mandarin fish in China, and it has a unique flavor and taste. Most studies have focused on the study of changes in free amino acids [13], volatile flavor compounds [14], and microbial community [11,15,16] during fermentation of Chouguiyu. There are plenty of microorganisms in Chouguiyu due to the low-salt fermentation process, which can not only generate unique flavor and taste substances but also produce BAs. However, there is a lack of research on the changes in BAs during fermentation of Chouguiyu. Moreover, the synthesis and degradation mechanisms of BAs based on microbial metabolism have rarely been reported.
Therefore, in this study, metagenome sequencing was performed using an Illumina Hiseq Xten to analyze the whole genetic material of the microbial community during Chouguiyu fermentation. The key genes that were associated with the synthesis and degradation of various BAs in the microbial community were selected after KEGG orthology (KO) annotation. Combined with the results of BA concentrations, the metabolism mechanisms of different BAs during fermentation of Chouguiyu were revealed based on the BA metabolic pathways and correlation networks between microorganisms and BAs. The core microorganisms for BA degradation in Chouguiyu were also identified. This study is expected to provide a comprehensive understanding of the synthesis and degradation mechanisms of BAs in Chouguiyu, which is helpful in the isolation of functional strains for the degradation of BAs.

2. Materials and Methods

2.1. Fermentation of Chouguiyu

Chouguiyu was made by fermenting mandarin fish coming from Guangdong, China, (Siniperca chuatsi, av. 500 g, frozen and thawed before fermentation) at 15–20 °C for 8 days in Anhui, China, according to previous studies [11,15,16]. Briefly, frozen mandarin fish was thawed, gutted, and cleaned. Then, the prepared fish was soaked in brine (6% salt and 0.02% spices). Fermentation used wild microbiota (no starter cultures). Three samples of Chouguiyu were collected on Day 1 (D1), Day 2 (D2), Day 4 (D4), Day 6 (D6), and Day 8 (D8) for further study.

2.2. Metagenome Sequencing of Microbial Community in Chouguiyu

The microorganisms in Chouguiyu were obtained after centrifugation of fermented liquid in each sample. Shotgun metagenomics was adopted to analyze the microbial community, as per previous studies [11,15,16]. Briefly, total genomic DNA was extracted from microorganisms using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA). After the verification of DNA purity, the genomic DNA from microorganisms was fragmented to construct the DNA library using NEXTFLEX® Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). The DNA library was then used for paired-end sequencing through the Illumina Hiseq Xten (Illumina Inc., San Diego, CA, USA).

2.3. Gene Assembly and Functional Annotation

Sequencing data were assembled using MEGAHIT (version 1.1.2) to obtain the contigs. Open reading frames (ORFs) from each assembled contig were predicted using MetaGene. CD-HIT (version 4.6.1) was used to cluster the gene sequences (90% identity and 90% coverage). The longest gene in each class was selected as the representative sequence to construct the non-redundant gene set. The obtained genes were aligned to the KEGG and COG databases to obtain their annotation. The BA metabolic pathways in the microbial community of Chouguiyu were constructed based on the KO in the KEGG pathways. The abundance of enzymes from microbial genera was calculated based on the sum of KO abundance related to BA metabolism. Representative sequences of non-redundant gene catalogs were aligned to the NCBI NR database with an e-value cutoff of 1e-5 using Diamond (version 0.8.35).

2.4. Determination of Biogenic Amines

The quantification of tryptamine, β-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine was carried out as previously described [17] with some modifications. Briefly, a total of 5.00 g of samples was added to centrifuge tubes with 10 mL perchloric acid (0.4 mol/L) and was ground for 20 min at 40 Hz using a Multi-Tube Vortexer (EFAA-HM-01, Yalin, Shanghai, China). After centrifugation at 4000 r/min for 20 min using a high-speed tabletop refrigerated centrifuge (TDZ5-WS, Xiangyi, Changsha, Hunan, China), the supernatant (1 mL) was mixed with 100 μL sodium hydroxide (2 mol/L) and 300 μL saturated sodium bicarbonate. The derivative reaction was performed by adding dansyl chloride (1 mL) into the mixture, followed by incubation in the dark at 40 °C for 45 min. The BAs were detected using a high-performance liquid chromatograph (LC-20AD, Shimadzu, Nakagyo-ku, Kyoto, Japan) equipped with an ultraviolet detector (SPD-M20A, Shimadzu, Nakagyo-ku, Kyoto, Japan) on the WondaCract ODS-2 column (Shimadzu-GL, Nakagyo-ku, Kyoto, Japan). Solvent A and solvent B were acetonitrile and ammonium acetate, respectively. Elution program was performed as follows: 0.1 min, 55% B; 7 min, 65% B; 14 min, 70% B; 20 min, 70% B; 27 min, 90% B; 30 min, 100% B; 31 min, 100% B, 32 min, 55% B, and 37 min, 55% B with a flow rate of 0.80 mL/min. The BA contents of each sample were calculated according to the standard curves (Table S1).

2.5. Statistical Analysis

The statistical analysis was conducted using SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) with one-way analysis of variance and multiple comparison Tukey tests. The correlation network maps between microbial BA metabolism enzymes and BA contents were built according to Pearson’s correlation by Cytoscape v3.9.1. Heatmaps were produced using Excel (Microsoft Office Home and Student 2019, Redmond, WA, USA).

3. Results and Discussion

3.1. Metagenomic Analysis of Microbial Community in Chouguiyu

BAs are potential risks in Chouguiyu due to high concentrations of free amino acids and the complex microbial community. To investigate this, we analyzed the metagenome of the microbial community during Chouguiyu fermentation (Figure 1). According to the taxonomic analysis of obtained genes of the metagenome, Firmicutes and Proteobacteria were the main phyla and their total abundance accounted for over 85% during fermentation (Figure 1A). With increasing fermentation time, Firmicutes obviously increased while Proteobacteria obviously decreased, in accordance with the results in other fermented aquatic products [7,18], reflecting a successional pattern common to fermented fish. As shown in Figure 1B, there were a total of 66 microbial genera (relative abundance > 0.1%). Vagococcus, Acinetobacter, Psychrobacter, Peptostreptococcus, Enterococcus, Myroides, and Streptococcus were the dominant genera during the fermentation of Chouguiyu. Among these genera, Acinetobacter was the most abundant genus in the initial fermentation but was significantly inhibited thereafter. A similar trend was also found in Myroides. With an increase in fermentation time, the abundance of Vagococcus, Peptostreptococcus, and Enterococcus obviously increased, while Psychrobacter first increased and then decreased. Vagococcus, Peptostreptococcus, Psychrobacter, and Enterococcus became dominant at the end of fermentation.
In order to obtain gene annotations of the metagenome of the microbial community, the gene sequences were further annotated using the KEGG and COG databases. As shown in Figure 1C, the KEGG pathways mainly focused on metabolism, among which carbohydrate metabolism and amino acid metabolism had the highest abundance. As shown in Figure 1D, except for function unknown (S), most gene sequences are annotated as replication, recombination, and repair (L) and amino acid transport and metabolism (E). The high abundance of genes related to amino acid metabolism provided the basis for analyzing the metabolism process of BAs. KEGG orthology (KO) is a straight-homologous classification system that can group genes with similar sequences and functions and use the functions of known genes as the KO functions for cross-species annotation. All the gene sequences of the microbial community in Chouguiyu were totally annotated with 13,968 KO functions. The top 50 KO functions are shown in Figure 1E. K01990, K02004, K06147, and K01992 had the highest gene abundance during the fermentation of Chouguiyu. The KO functions related to BA metabolism were further identified (Figure 1F). A total of 16 KO functions were found in the metagenome of the microbial community. Among these, K00797, K01476, K01480, K01581, K01582, K01590, K01593, K10536, K12251, K13746, K13747, and K23385 were related to BA synthesis, while K00274, K00276, K00657, K00797, K09251, K09470, K12256, K13746, and K13747 contributed to BA degradation. K00797, K13746, and K13747 function in both synthesis and degradation pathways (Figure 2).

3.2. Enzymes Related to Biogenic Amine Metabolism in Chouguiyu Based on KO Functions

In order to fully understand the microbial metabolism mechanism and related enzymes of BAs in Chouguiyu, the BA metabolic pathways in Chouguiyu were constructed according to KO functions (Figure 2, Tables S2 and S3). For tryptamine metabolism, tryptamine was produced from tryptophan by L-tryptophan decarboxylase (EC 4.1.1.28 and EC 4.1.1.105) in K01593, while monoamine oxidase (EC 1.4.3.4) in K00274 could degrade tryptamine. Cadaverine was mainly produced from lysine by D-lysine decarboxylase (EC 4.1.1.116) in K23385 and lysine decarboxylase (EC 4.1.1.18) in K01582, while putrescine aminotransferase (EC 2.6.1.82) in K09251 could degrade cadaverine into 5-aminopentanal. Tyramine was obtained from tyrosine using L-tryptophan decarboxylase (EC 4.1.1.28) in K01593 and could be degraded by monoamine oxidase (EC 1.4.3.4) in K00274 and primary-amine oxidase (EC 1.4.3.21) in K00276. Histamine was generated from histidine by histidine decarboxylase (EC 4.1.1.22) in K01590, while no histamine-degrading enzymes were identified in Chouguiyu.
As one of the most abundant BAs, putrescine could be synthesized in a variety of ways. Arginine was first catalyzed by arginase (EC 3.5.3.1, K01476) into ornithine, which was further transferred into putrescine by ornithine decarboxylase (EC 4.1.1.17, K01581). This pathway was also the main putrescine-producing pathway in fermented foods [19]. Putrescine could also be synthesized through agmatine deiminase (EC 3.5.3.12) in K10536 and N-carbamoylputrescine amidase (EC 3.5.1.53) in K12251 or agmatinase (EC 3.5.3.11) in K01480 using agmatine as the substrate. Meanwhile, many enzymes contributed to the degradation of putrescine in Chouguiyu, including putrescine aminotransferase (EC 2.6.1.82) in K09251 and pyruvate transaminase (EC 2.6.1.113) in K12256 for 4-aminobutanal production, gamma-glutamylputrescine synthase (EC 6.3.1.11) in K09470 for γ-L-glutamyl-putrescine production, and diamine N-acetyltransferase (EC 2.3.1.57) in K00657 for N-acetyl-putrescine production.
In addition, putrescine was also catalyzed into spermidine by spermidine synthase (EC 2.5.1.16) in K00797 or was first degraded into carboxy-spermidine by carboxynorspermidine synthase (EC 1.5.1.43) in K13746 and transferred into spermidine by carboxynorspermidine decarboxylase (EC 4.1.1.96) in K13747. No spermidine-degrading enzyme, i.e., spermine-synthesizing enzyme, was observed in Chouguiyu.
Phenylethylamine was produced from phenylalanine by L-tryptophan decarboxylase (EC 4.1.1.28) in K01593. The enzymes for phenylethylamine degradation were the same as those for tyramine degradation, resulting in the production of phenylacetaldehyde.

3.3. Microbial Genus Related to Biogenic Amine Metabolism in Chouguiyu Based on KO

3.3.1. Microbial Genera for BA Synthesis

After analyzing the KO abundance, there were a total of 111 microbial genera related to BA metabolism (Figure 3). Among these, 91 microbial genera possessed BA synthesis ability (Figure 3A). Pseudomonas was the only microbial genus that possessed L-tryptophan decarboxylase (K01593), which was the most important enzyme for producing tryptamine, tyramine, and phenylethylamine. The gene abundance of Pseudomonas first increased and then decreased to none. Similarly, previous studies found that Pseudomonas was one of the main microorganisms to produce tryptamine [20], tyramine [5], and phenylethylamine [21].
The lysine decarboxylase in Hafnia can effectively produce cadaverine [22]. In this study, Hafnia was the most important microbial genus for the production of D-lysine decarboxylase (K23385) and lysine decarboxylase (K01582) for cadaverine synthesis. In addition, a high abundance of lysine decarboxylase (K01582) was found in Obesumbacterium, Citrobacter, Raoultella, Serratia, and Enterobacter, contributing to abundant cadaverine synthesis. Similar results were found showing that the expression of the decarboxylase gene in Serratia [23], as well as Citrobacter and Enterobacter [24,25,26], was associated with cadaverine production in foods. There were five microbial genera related to histamine production, among which Morganella and Raoultella contained the most abundant histidine decarboxylase (K01590). The histamine-producing ability of Morganella [27] and Raoultella [28] has also been reported in other studies.
Putrescine was mainly produced by a two-step reaction of agmatinase (K01476) and ornithine decarboxylase (K01581). A total of 20 microbial genera were responsible for the production of agmatinase, and most arginase-producing genera increased along with the fermentation process. Among these, Vagococcus, Kurthia, Brevibacillus, and Pelagivirga were the main microbial genera for agmatinase production. More microbial genera (27 kinds) could produce ornithine decarboxylase, and Psychrobacter, Citrobacter, Kluyvera, and Moraxella were the main microbial genera. However, there were only six microbial genera that could produce agmatinase and ornithine decarboxylase together, including Pelagivirga, Psychrobacter, Serratia, Kluyvera, Paracoccus, and Citrobacter. For putrescine synthesis from agmatine, there were 38 microbial genera secreting agmatinase (K01480), among which Myroides, Pseudoalteromonas, Citrobacter, and Vibrio played an important role in enzyme secretion. Most agmatinase-producing genera with high abundance decreased in the late fermentation.
In addition, putrescine could also be produced by a two-step reaction of agmatine deiminase (K10536) and N-carbamoylputrescine amidase (K12251) from agmatine. A total of 31 and 11 microbial genera were responsible for the production of agmatine deiminase and N-carbamoylputrescine amidase. Most genera with a high abundance of agmatine deiminase production increased with the increasing fermentation time. Acinetobacter, Psychrobacter, Lactococcus, and Vagococcus were the main microbial genera for agmatine deiminase production, while Psychrobacter, Pseudoalteromonas, Bacteroides, and Empedobacter were the main microbial genera for N-carbamoylputrescine amidase. Only Acinetobacter, Psychrobacter, Aeromonas, Chryseobacterium, and Pseudomonas produced these two enzymes together. The pathways of putrescine production are more complicated than those of other BAs and many studies have reported that putrescine can be generated from similar microorganisms, such as Enterobacter [29], Citrobacter [10], Proteus [24,30], Serratia [31], Aeromonas [32], and Lactococcus [32].
A total of 20 microbial genera possessed a high abundance of spermidine synthase (K00797) to produce spermidine, among which Pseudoalteromonas, Psychrobacter, Myroides, Serratia, Lysobacter, Raoultella, Macrococcus, and Shewanella were the main microbial genera. Spermidine was also produced by carboxynorspermidine synthase (K13746) and carboxynorspermidine decarboxylase (K13747) from putrescine. However, only Vibrio could produce carboxynorspermidine synthase, while there were nine microbial genera that possessed the ability to produce carboxynorspermidine decarboxylase, such as Psychrobacter, Bacteroides, and Vibrio. Similarly, Psychrobacter [33], Bacteroides [34], Serratia [35], and Shewanella [36] showed greater capacity to produce spermidine. Spermidine was further degraded into spermine using spermine synthase. However, there was no microbial metabolic gene for spermine in Chouguiyu.

3.3.2. Microbial Genera for BA Degradation

Besides the abundant microorganisms for the production of BAs, many microorganisms (75 microbial genera) possessed various enzymes related to the degradation of BAs in Chouguiyu (Figure 3B). In this study, a total of 73 microbial genera possessed BA degradation ability and 19 microbial genera were potentially able to produce monoamine oxidase (K00274) for tryptamine, tyramine, and phenylethylamine degradation, among which Psychrobacter, Lysobacter, Primorskyibacter, Pelagivirga, and Dietzia possessed the highest abundance and increased along with the fermentation process. Tyramine and phenylethylamine could also be degraded by primary-amine oxidase (K00276). A total of 14 microbial genera generated primary-amine oxidase. Hafnia, Acinetobacter, Kluyvera, and Raoultella were the main microbial genera to produce the enzyme. Most genera with a high abundance of primary-amine oxidase production decreased with the prolongation of fermentation time. A total of 7 microbial genera played an important role in secreting putrescine aminotransferase (K09251) for cadaverine and putrescine degradation, and Hafnia, Kluyvera, Citrobacter, Enterobacter, and Lactococcus possessed the highest abundance. Lactococcus has previously been found to possess the enzyme activities responsible for BA degradation in cheese [37]. In addition, putrescine could be degraded by pyruvate transaminase (K12256), spermidine synthase (K00797), carboxynorspermidine synthase (K13746), gamma-glutamylputrescine synthase (K09470), and diamine N-acetyltransferase (K00657). Pyruvate transaminase was produced from 13 microbial genera, such as Pelagivirga, Acinetobacter, Sulfitobacter, and Aeromonas. The microbial genera for putrescine degradation using spermidine synthase and carboxynorspermidine synthase were the same as those for spermidine synthesis. There were nine genera that produced gamma-glutamylputrescine synthase, among which Acinetobacter, Serratia, Hafnia, Citrobacter, and Raoultella were the main genera. More genera (32 kinds) had the ability to produce diamine N-acetyltransferase, and Vagococcus, Enterococcus, Myroides, and Lactococcus were the main genera with high abundance. It was reported that Enterococcus’s multicopper oxidases could degrade phenylethylamine, putrescine, histamine, and tyramine [25]. Interestingly, some microbial genera possessed various enzymes for the degradation of various BAs in Chouguiyu, such as Acinetobacter, Aeromonas, Arthrobacter, Hafnia, Lysobacter, Raoultella, and Pseudomonas. Similar results were found showing that Arthrobacter was widely used to produce various amine oxidases for phenylethylamine, tyramine, and histamine degradation [38].
Interestingly, huge differences in microbial composition were found using total genes of the metagenome (Figure 1B) and genes related to BA metabolism (Figure 3). It was more scientific to analyze the mechanism of BA production and degradation based on functional gene abundance.

3.4. Change in Biogenic Amines During Fermentation of Chouguiyu

In order to clarify the actual influence of microbial genera on BAs of Chouguiyu, the change rules of eight BAs in Chouguiyu at different fermentation time are shown in Figure 4. As the fermentation time increased, tryptamine and phenylethylamine first increased and then decreased, and all reached maximums at 2 d of 4.30 and 2.86 mg/kg, respectively. Interestingly, the gene abundance of K01593 in Pseudomonas was low in Chouguiyu (Figure 3), resulting in the relatively low content of these two BAs. Cadaverine, tyramine, histamine, and putrescine markedly increased with the increasing fermentation time, while spermidine and spermine decreased. Histamine is considered the most toxic BA in fermented foods [7]. Worldwide, the legal limit for histamine in aquatic products varies by country and product type [39]. Among these, the minimum limit is 30 mg/kg and the United States suggests that the histamine safe limit in aquatic products is 50 mg/kg [40]. In this study, the histamine content reached a maximum of 15.91 mg/kg after fermentation for 8 d, indicating the safety of Chouguiyu. And the abundance of histidine decarboxylase genes decreased in the late fermentation, which might be an important reason for the low production of histamine. Tyramine also shows high toxicity, and its excessive intake can cause headaches and high blood pressure [24]. In this study, the maximal tyramine content was 25.65 mg/kg after 8 d fermentation.
Cadaverine and putrescine, despite being non-toxic in themselves, can inhibit the metabolic enzyme activity of histamine and tyramine (such as monoamine oxidase and diamine oxidase), therefore enhancing the toxicity of histamine and tyramine [31]. In this study, cadaverine and putrescine were the highest BAs in Chouguiyu at D8, reaching their maximums of 105.73 mg/kg and 105.30 mg/kg, respectively. The smelly flavor of these two BAs might contribute to the unique flavor of Chouguiyu. Interestingly, the abundance of the cadaverine-producing genes in almost all microbial genera first increased and then decreased during fermentation of Chouguiyu. There were low contents of spermidine, spermine, tryptamine, and phenylethylamine in the late fermentation of Chouguiyu, especially spermidine, which was not even detected.

3.5. Relationship of Biogenic Amine and Metabolic Pathway in Chouguiyu

The relationship between microorganisms and their metabolites can be clearly visualized by correlation network maps [41,42]. To elucidate the microbial sources of biogenic amines (BAs), Pearson’s correlation between microbial synthesis enzymes and BAs was analyzed within each KO (Figure 5 and Figure S1 and Table S4). L-tryptophan decarboxylase (K01593) from Pseudomonas was positively correlated with tryptamine and phenylethylamine, while a negative correlation was observed with tyramine. Pseudomonas was similarly identified as an important genus for tryptamine production in correlative analyses [43]. Cadaverine levels were significantly positively correlated with D-lysine decarboxylase (K23385) from Salmonella but negatively correlated with the enzyme from Hafnia. Cadaverine was significantly positively correlated with lysine decarboxylase (K01582) from Caldisalinibacter, Gottschalkia, Anaerosphaera, and Citrobacter but was negatively correlated with that from Serratia, Hafnia, and Escherichia.
Salmonella, Caldisalinibacter, Gottschalkia, Anaerosphaera, and Citrobacter might play a major role in cadaverine synthesis. Similarly, cadaverine in Dongbei Suancai (fermented cabbage from Dongbei) was highly related to Citrobacter [9]. The change in putrescine was significantly positively correlated with arginase (K01476) from Pelagivirga, Lysobacter, Vagococcus, Paracoccus, and Brevibacillus, ornithine decarboxylase (K01581) from Ruegeria, Sedimentitalea, Paracoccus, and Pelagivirga, N-carbamoylputrescine amidase (K12251) from Lysobacter, and agmatinase (K01480) from Pseudosulfitobacter, Pelagivirga, Sulfitobacter, Andreesenia, Tissierella, and Agrococcus. The enhanced abundance of enzymes from these genera might play an important role in putrescine production.
Histamine was positively correlated with histidine decarboxylase (K01590) from Morganella and Raoultella, with Morganella—an established histamine-producing bacterium in seafood—being particularly notable [44]. Spermidine levels were significantly positively correlated with carboxynorspermidine decarboxylase (K13747) from Vibrio and Arcobacter and with spermidine synthase (K00797) from Myroides, Lelliottia, and Serratia. Vibrio, Arcobacter, Myroides, Lelliottia, and Serratia might be the major spermidine-synthesizing bacteria. Based on correlation analysis, Serratia was positively correlated with most of the BAs in soybean paste [26].
For the correlation between BAs and their degradation enzymes from microorganisms in Chouguiyu, Pearson’s correlation between microbial degradation enzymes and BAs in each KO was analyzed (Figure 6 and Figure S2 and Table S5). Tryptamine levels were significantly positively correlated with monoamine oxidase (K00274) from Kocuria, Flavobacterium, and Tessaracoccus. Cadaverine was significantly positively correlated with putrescine aminotransferase (K09251) from Lactococcus but was negatively correlated with that from Citrobacter and Lelliottia. Declining Citrobacter and Lelliottia abundances correlated with cadaverine accumulation, suggesting reduced degradation capacity, and the decrease in Lactococcus was beneficial to the control of cadaverine accumulation. Tyramine levels were significantly positively correlated with monoamine oxidase from Primorskyibacter, Lysobacter, Roseovarius, Dietzia, Paracoccus, Gillisia, Tessaracoccus, Pseudorhodobacter, and Pelagivirga and positively related with primary-amine oxidase from Brevibacterium, Agrococcus, Paracoccus, and Psychrobacter. The increasing enzymes in these genera might contribute to the inhibition of tyramine accumulation. Conversely, tyramine concentration was significantly negatively correlated with monoamine oxidase from Pseudomonas and Pseudoalteromonas and with primary-amine oxidase from Acinetobacter, Arthrobacter, Galactobacter, Kocuria, and Hafnia, indicating their role in tyramine increase.
The change in putrescine was also significantly positively correlated with putrescine aminotransferase (K09251) from Lactococcus, pyruvate transaminase (K12256) from Pelagivirga, Sulfitobacter, Roseovarius, and Hoeflea, spermidine synthase (K00797) from Lysobacter, Brevibacterium, and Dietzia, and diamine N-acetyltransferase (K00657) from Oceanobacillus, Bacillus, Enterococcus, Salinicoccus, Jeotgalibaca, Vagococcus, and Aequorivita. These increasing enzymes in the above genera were important for the inhibition of putrescine in Chouguiyu. Meanwhile, putrescine was significantly negatively correlated with putrescine aminotransferase from Citrobacter, Lelliottia, Kluyvera, and Hafnia, pyruvate transaminase from Pseudomonas, Aeromonas, Halomonas, and Shewanella, spermidine synthase from Pseudoalteromonas, Shewanella, Lelliottia, Myroides, Chryseobacterium, and Comamonas, carboxynorspermidine synthase (K13746) from Vibrio, gamma-glutamylputrescine synthase (K09470) from Enterobacter, Serratia, Acinetobacter, and diamine N-acetyltransferase from Vibrio, Aeromonas, Pseudoalteromonas, Carnobacterium, Kluyvera, Myroides, Hafnia, Serratia, and Weissella. The decreasing enzymes in these genera contributed most to the increase in putrescine during fermentation of Chouguiyu.
The change in phenylethylamine was significantly positively correlated with monoamine oxidase from Kocuria, Flavobacterium, and Tessaracoccus and correlated with primary-amine oxidase from Leclercia and Paeniglutamicibacter. These microbial genera played a crucial role in the maintenance of phenylethylamine concentration. Meanwhile, the KOs of K09251, K00657, K00274, K12256, K09470, and K00276 were the major biological amine degradation pathways (Figure 2).
Interestingly, microbial degraders of tryptamine, tyramine, and phenylethylamine outnumbered producers, explaining their low concentrations. Conversely, putrescine and cadaverine accumulated due to scarce degraders (Figure 6).

3.6. Identification of Core Microorganisms for Biogenic Amine Degradation in Chouguiyu

In order to more clearly focus on BA-degrading microbial species, the microorganisms associated with key degradation pathways are shown in Figure 7. In this study, the synthesis of cadaverine by microorganisms in Chouguiyu was higher than its degradation process, resulting from the increasing cadaverine concentration along with fermentation (Figure 7A). Salmonella, Gottschalkia, Caldisalinibacter, and Anaerosphaera contributed most to the production of cadaverine, while Enterobacter and Lactococcus were the main cadaverine-degrading microbial genera.
Among the histamine-producing genera, only Morganella showed a close relationship with histamine, and its low abundance resulted in the low content of histamine.
Pseudomonas was the only genus that was responsible for the synthesis of phenylethylamine, tryptamine, and tyramine, leading to the relatively low production of these BAs in Chouguiyu. More microbial genera were helpful in the degradation of phenylethylamine, among which Psychrobacter, Hafnia, Lysobacter, and Acinetobacter were the main degradation bacteria of phenylethylamine, while Psychrobacter and Lysobacter contributed most to tryptamine and tyramine degradation. Vagococcus and Psychrobacter, which possessed high enzyme abundance, were the main putrescine synthesis microorganisms, while Vagococcus and Enterococcus were beneficial for putrescine degradation. The high abundance of synthesis enzymes in Psychrobacter, Pseudoalteromonas, Myroides, and Bacteroides was important for spermidine synthesis, while there was no related enzyme for spermidine degradation. Interestingly, Lactococcus, Flavobacterium, Tessaracoccus, and Yoonia possessed only the BA degradation enzymes. Similarly to Korean jeotgal [45], Chouguiyu’s low-salt process permits diverse BA-producing microbes, but unlike jeotgal, it harbors unique degraders like Yoonia. In addition, Acinetobacter and Enterococcus had a much higher abundance of degradation enzymes than synthesis enzymes for BA metabolism.
The microbial species in the above genera for BA degradation were further explored (Figure 7B,C). For Lactococcus, a total of four microbial species had the BA degradation enzyme genes, among which Lactococcus garvieae possessed the most abundant putrescine degradation enzymes. Lactococcus lactis possessed both putrescine aminotransferase (K09251) and diamine N-acetyltransferase (K00657) for cadaverine and putrescine degradation, while Lactococcus raffinolactis, Lactococcus piscium, and Lactococcus garvieae only possessed diamine N-acetyltransferase for putrescine degradation. Flavobacterium gelidilacus, Tessaracoccus antarcticus, and Yoonia vestfoldensis were the only species that possessed monoamine oxidase (K00274) for tryptamine, tyrosine, and phenylalanine degradation in Flavobacterium, Tessaracoccus, and Yoonia, respectively. For Acinetobacter, a total of 19 microbial species possessed plenty of BA degradation genes, and they were observed to have a variety of putrescine, tyrosine, and phenylalanine degradation enzymes, including pyruvate transaminase (K12256), gamma-glutamylputrescine synthase (K09470), and primary-amine oxidase (K00276). Among these species, Acinetobacter haemolyticus possessed the highest enzyme activity for tyrosine and phenylalanine degradation. For Enterococcus, there were six microbial species that had putrescine degradation enzymes, i.e., diamine N-acetyltransferase. Enterococcus ureasiticus possessed more abundant enzymes than the other microbial species in Enterococcus.
It can be concluded that Lactococcus garvieae, Flavobacterium gelidilacus, Tessaracoccus antarcticus, Yoonia vestfoldensis, Acinetobacter haemolyticus, and Enterococcus ureasiticus were the main microbial species for BA degradation during the fermentation of Chouguiyu, which could be isolated as the potential functional strains for the degradation of BAs in fermented foods. According to the above results, the degradation of undesirable BAs in Chouguiyu could be reduced and isolated strains could be applied in fermented foods like fish sauce or cheese to reduce BA risks.

4. Conclusions

Based on KO analysis, there were 91 BA-synthesizing microbial genera, among which Psychrobacter, Vagococcus, Pseudoalteromonas, Hafnia, Citrobacter, and Myroides were the main genera, especially for putrescine synthesis. Seventy-five genera possessed degradation enzymes, primarily Acinetobacter, Psychrobacter, Vagococcus, Pseudoalteromonas, Enterococcus, Myroides, and Hafnia, especially for putrescine. After determination of BAs, high concentrations of cadaverine and putrescine were observed in Chouguiyu, while histamine and tyramine exhibited relatively low concentrations, and these four BAs increased with the increasing fermentation time, similar to KO analysis. The correlation networks revealed Lactococcus, Flavobacterium, Tessaracoccus, and Yoonia as non-producers but potential BA degraders. Acinetobacter and Enterococcus possessed more abundant degradation enzymes than synthesis enzymes. Amine oxidase from Acinetobacter, as well as diamine N-acetyltransferase from Enterococcus and Lactococcus, played the main role in biogenic amine degradation in Chouguiyu. Lactococcus garvieae, Flavobacterium gelidilacus, Tessaracoccus antarcticus, Yoonia vestfoldensis, Acinetobacter haemolyticus, and Enterococcus ureasiticus were the main microbial species for BA degradation and could be isolated as the potential functional strains for the degradation of BAs in fermented foods.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods14162863/s1, Table S1: Standard curves of different biogenic amine; Figure S1: Correlation heatmap between microbial synthesis enzymes and biogenic amines in each KO; Figure S2: Correlation heatmap between microbial degradation enzymes and biogenic amines in each KO. Table S2: Enzymes and relative abundance of reads involved in biogenic amine production in Chouguiyu; Table S3: Enzymes and relative abundance of reads involved in biogenic amine degradation in Chouguiyu; Table S4: Correlations and p-value between synthesis genus and biogenic amine in Chouguiyu. Table S5: Correlations and p-value between degradation genus and biogenic amine in Chouguiyu.

Author Contributions

J.L., D.Y.: formal analysis, methodology, and writing—original draft. Y.Z.: data curation and funding acquisition. D.W.: visualization. H.H.: funding acquisition. C.L.: writing—review and editing, and conceptualization. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the Earmarked fund for CARS (CARS-46), the National Natural Science Foundation of China (32272348), the Key Laboratory of Aquatic Product Processing, Ministry of Agriculture and Rural Affairs, China (NYJG202405), the Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams (2024CXTD26), and the Central Public-interest Scientific Institution Basal Research Fund, CAFS (2023TD78).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Metagenomic analysis of microbial community during fermentation of Chouguiyu. (A) Microbial taxonomic composition at the (A) phylum and (B) genus levels. Gene annotations of metagenome with the (C) KEGG and (D) COG databases. The (E) top 50 KO functions of the gene sequences and the (F) KO functions related to BA metabolism.
Figure 1. Metagenomic analysis of microbial community during fermentation of Chouguiyu. (A) Microbial taxonomic composition at the (A) phylum and (B) genus levels. Gene annotations of metagenome with the (C) KEGG and (D) COG databases. The (E) top 50 KO functions of the gene sequences and the (F) KO functions related to BA metabolism.
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Figure 2. Biogenic amine metabolic pathways of microbial community in Chouguiyu.
Figure 2. Biogenic amine metabolic pathways of microbial community in Chouguiyu.
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Figure 3. Abundance of enzymes for the (A) synthesis and (B) degradation of biogenic amines from the microbial community in Chouguiyu.
Figure 3. Abundance of enzymes for the (A) synthesis and (B) degradation of biogenic amines from the microbial community in Chouguiyu.
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Figure 4. Change in biogenic amines during fermentation of Chouguiyu. Bars with different lowercase letters differ at p < 0.05 by one-way analysis of variance and Tukey test.
Figure 4. Change in biogenic amines during fermentation of Chouguiyu. Bars with different lowercase letters differ at p < 0.05 by one-way analysis of variance and Tukey test.
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Figure 5. Correlation network map between microbial synthesis enzymes and biogenic amines. The red and blue lines, respectively, represent positive and negative correlation.
Figure 5. Correlation network map between microbial synthesis enzymes and biogenic amines. The red and blue lines, respectively, represent positive and negative correlation.
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Figure 6. Correlation network map between microbial degradation enzymes and biogenic amines. The red and blue lines, respectively, represent positive and negative correlation.
Figure 6. Correlation network map between microbial degradation enzymes and biogenic amines. The red and blue lines, respectively, represent positive and negative correlation.
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Figure 7. Core microbial genera for the degradation of biogenic amines. (A) Total abundance of functional enzymes related to biogenic amine synthesis and degradation in different microbial genera and their correlation with biogenic amines. (B) Core microbial species with the ability to degrade biogenic amines during the fermentation of Chouguiyu. (C) Relationship of core microbial species and enzymes related to biogenic amine degradation.
Figure 7. Core microbial genera for the degradation of biogenic amines. (A) Total abundance of functional enzymes related to biogenic amine synthesis and degradation in different microbial genera and their correlation with biogenic amines. (B) Core microbial species with the ability to degrade biogenic amines during the fermentation of Chouguiyu. (C) Relationship of core microbial species and enzymes related to biogenic amine degradation.
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Li, J.; Yang, D.; Zhao, Y.; Wang, D.; Huang, H.; Li, C. Novel Insight into Metabolism Mechanism of Biogenic Amines During Fermentation of Chinese Traditional Fermented Mandarin Fish (Chouguiyu) Based on Metabolism Pathway and Correlation Network. Foods 2025, 14, 2863. https://doi.org/10.3390/foods14162863

AMA Style

Li J, Yang D, Zhao Y, Wang D, Huang H, Li C. Novel Insight into Metabolism Mechanism of Biogenic Amines During Fermentation of Chinese Traditional Fermented Mandarin Fish (Chouguiyu) Based on Metabolism Pathway and Correlation Network. Foods. 2025; 14(16):2863. https://doi.org/10.3390/foods14162863

Chicago/Turabian Style

Li, Jun, Daqiao Yang, Yongqiang Zhao, Di Wang, Hui Huang, and Chunsheng Li. 2025. "Novel Insight into Metabolism Mechanism of Biogenic Amines During Fermentation of Chinese Traditional Fermented Mandarin Fish (Chouguiyu) Based on Metabolism Pathway and Correlation Network" Foods 14, no. 16: 2863. https://doi.org/10.3390/foods14162863

APA Style

Li, J., Yang, D., Zhao, Y., Wang, D., Huang, H., & Li, C. (2025). Novel Insight into Metabolism Mechanism of Biogenic Amines During Fermentation of Chinese Traditional Fermented Mandarin Fish (Chouguiyu) Based on Metabolism Pathway and Correlation Network. Foods, 14(16), 2863. https://doi.org/10.3390/foods14162863

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