Introduction

Biogenic amines (BAs) are low-molecular weight organic nitrogenous compounds produced during fermentative processes through the microbial decarboxylation of amino acids (resulting in the formation of corresponding amine and carbon dioxide) or amination and transamination of ketones and aldehydes (Benkerroum, 2016). Nitrogenous organic compounds are categorized based on their chemical structures into the following three main groups: aliphatic (such as putrescine, cadaverine, spermine, and spermidine), aromatic (such as tyramine and phenylethylamine), and heterocyclic (such as histamine and tryptamine) compounds (Dabadé et al., 2021; Natrella et al., 2024). BAs are naturally found in various foods, especially in fermented food items, such as cheese, wine, beer, sausages, and fish products (Burdychova and Komprda, 2007). Low concentrations of BAs in food do not adversely affect human health. BAs, which are involved in various biological functions, are well tolerated and efficiently metabolized, and detoxified by intestinal amine oxidase enzymes. However, high concentrations of BAs can exceed the detoxification capacity of these enzymes, eliciting minor allergic responses and even inducing severe health complications, such as respiratory distress, heart palpitations, and hypertension or hypotension (Turna et al., 2024). Among BAs, histamine and tyramine exert the most severe adverse effects. Histamine and tyramine are associated with ‘fish poisoning’ symptoms and the ‘cheese reaction’ syndrome, respectively (Montanari et al., 2023).

Enterococcus spp. are ubiquitous Gram-positive and facultative anaerobic bacteria that naturally inhabit the intestinal tract of humans and other mammals. These microorganisms, which are categorized as lactic acid bacteria (LAB), inhabit diverse environments. Enterococcus spp. can grow in high-salt (6.5% NaCl), wide pH (4.4–9.6), and wide temperature (10–40°C) conditions. Furthermore, they can hydrolyze esculin even in the presence of high amounts of bile salts (40%) (Geraldes et al., 2022). Enterococci are natural inhabitants of the human gastrointestinal tract (GIT) and warm-blooded animals. Additionally, enterococci have been isolated from plants, water, and soil because of their exposure to fecal sources and certain food products (Ferchichi et al., 2021). Enterococci have applications in the dairy industry as they confer sensory characteristics during the ripening of various cheese samples. This can be attributed to their role in proteolysis, lipolysis, and citrate metabolism, which collectively enhance the distinctive taste and flavor profiles of the cheese (Benkerroum, 2016). However, the safety of the presence of enterococci is controversial. Some enterococci are reported to be probiotic and can generate active bacteriocins against pathogens. However, the genus Enterococcus is associated with antibiotic resistance and exhibits virulence phenotypes, such as cytolysin expression, adherence to host tissue, invasion, abscess formation, modulation of host inflammatory responses, secretion of toxic products, and aggregation substances (Geraldes et al., 2022; Ghazvinian et al., 2024; Gök et al., 2020).

Amino acid decarboxylases, which facilitate the formation of BAs, are widely expressed in spoilage microorganisms as well as in microorganisms involved in food fermentation processes. Microorganisms that produce BA via amino acid decarboxylation include species belonging to the genera Lactobacillus, Leuconostoc, Lactococcus, Enterobacter, Escherichia, Enterococcus, Pediococcus, Pseudomonas, Streptococcus, Staphylococcus, Shigella, Salmonella, and Bacillus (Turna et al., 2024). In particular, Lactobacillus spp. are primarily responsible for the production of histamine, tyramine, and putrescine, while members of Enterobacteriaceae and Enterococcus contribute to the production of putrescine, cadaverine, and tyramine in foods (Vesković-Moračanin et al., 2022). These microorganisms with decarboxylase activity can either be intentionally introduced as starter cultures or serve as contaminants (Natrella et al., 2024). Enterococci are also reported to produce BAs (Montanari et al., 2023; Zdolec et al., 2022). BAs are generated throughout the ripening and storage phases of cheese production. During the ripening process, proteolysis breaks down proteins into amino acids. These amino acids, when present in sufficient amounts, can contribute to the production of toxic BAs. Consequently, the progression of cheese maturation is often accompanied by increased BA production (Sungur and Jobasi, 2022). The formation of BAs in cheese is influenced by multiple interrelated factors, including physicochemical parameters, such as pH, salt concentration, water activity, and proteolysis degree; technological aspects related to manufacturing, fermentation, and ripening processes; microbial factors, such as the presence and activity of decarboxylase--positive bacteria and their interactions; as well as environmental and storage conditions, such as temperature, humidity, and duration of ripening (Dabadé et al., 2021; Sungur and Jobasi, 2022; Zdolec et al., 2022).

Previous studies (Bogdanovi´c et al., 2020; Dabadé et al., 2021; Kandasamy et al., 2021; Ma et al., 2020; Sungur and Jobasi, 2022; Zdolec et al., 2022) have investigated BA content in various cheese samples consumed across different countries. However, limited studies have examined BAs produced by Enterococcus spp. in cheese samples. Therefore, this study aimed to comprehensively investigate Enterococcus strains isolated from traditional cheese samples produced without starter cultures and obtained from various supermarkets, open bazaars, and local producers in Ankara and neighboring provinces in Türkiye. The specific objectives of the study were as follows: (i) to isolate and preliminarily identify potential Enterococcus spp. from traditional cheese samples through biochemical analyses; (ii) to perform molecular characterization of the identified Enterococcus strains using 16S rDNA sequencing; (iii) to determine the production of major BAs, such as histamine, cadaverine, putrescine, and tyramine, by Enterococcus isolates; (iv) to quantify the levels of these BAs produced by Enterococcus strains using high-performance liquid chromatography (HPLC); and (v) to identify the genes responsible for BA formation in Enterococcus spp. through molecular detection methods.

Materials and Methods

Materials

Bacterial strains and culturing

The enterococcal strains isolated in this study and the reference strains were cultured in tryptic soy broth (TSB) (Merck^TM, Germany) and brain hearth infusion (BHI) broth (Merck^TM), respectively, at 37°C for 24 h. The initial isolates were stored at –80°C in 30% (v/v) aqueous glycerol (Merck^TM). Enterococcus faecalis ATCC 29212 , Enterococcus faecalis DMG 2708, Enterococcus faecium ATCC 19434, Escherichia coli LMG 3083 (ETEC), and Staphylococcus aureus ATCC 6538, which were used as reference strains for the identification of Enterococcus spp., were obtained from the culture collection of the Food Microbiology Laboratory, Department of Food Engineering, Faculty of Engineering, Ankara University, Türkiye.

Sampling

In this study, 186 traditional cheese samples were randomly purchased from various supermarkets, open bazaars, and producers in Ankara, Türkiye. This study analyzed hard, soft, and semi-soft ripened cheese varieties, such as Manyas, Sepet, Mihalic, Tulum, Civil, Orgu, Lor, Urfa, Ezine, Van Otlu, and Turkish white cheese. The collected samples were stored at 4°C during sale. Additionally, samples collected from all places were packed and non-frozen without disclosing any information about the store of origin. All cheese samples were checked for expiry dates, placed in a portable insulated cold box (<4°C), and processed immediately on the sampling day.

Methods

Isolation and biochemical characterization of Enterococcus spp.

To isolate enterococci, 25 g of each sample was briefly mixed with 225 mL of peptone water (Merck^TM) (0.1 % (w/v), and homogenized in a stomacher (Seward 400, USA) for 5 min. The mixture was incubated at 37°C for 15 min to ensure thorough homogenization. After preparing the serial dilutions of homogenates (up to 10⁻⁷ dilutions) in 0.85% (w/v) NaCl, 100 µL of each dilution was spread onto kanamycin aesculin azide (KAA) agar (Merck^TM) and incubated at 37°C for 18–24 h. Five typical colonies exhibiting a black appearance on KAA agar were randomly selected for further analysis. All Enterococcus isolates were phenotypically characterized using standard biochemical tests (Gram staining, catalase production, growth in TSB with 6.5% NaCl, growth at pH 9.6, esculin hydrolysis on bile esculin azide agar [Merck^TM], and growth at temperatures ranging from 10°C to 45°C).

Genotypic characterization

Enterococcus spp. were identified by amplifying and sequencing the 16S rDNA gene. Genomic DNA was extracted from overnight TSB cultures of enterococcal and control strains using genomic DNA purification kit, strictly following the manufacturer’s instructions (Brand: Gene All, Catalog No.: 106-101). To ensure the validity of PCR results, each assay included negative control (DNA-free) and positive control strains, with details of the reference strains provided in the Bacterial Strains and Culturing section. Cells were pelleted by centrifugation, lysed, and the DNA was subsequently purified using the kit’s spin column-based procedure. The concentration and purity of DNA were assessed using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The DNA sample was stored at –20°C. The universal primer pairs 907R (5'-CCGTCAATTCMTTTRAGTTT-3') and 27F (5'-AGAGTTTGATCMTGGCTCAG-3') were used to amplify the 16S rDNA gene (Beasley and Saris, 2004). Polymerase chain reaction (PCR) was performed in a 50-μL reaction mixture comprising 3 μL of bacterial DNA template, 34.75 μL RNase/DNase-free water, 0.25 μL Taq DNA polymerase in reaction buffer, 1 μL of 2 mM of each dNTP, 4 μL of 25-mM MgCl₂, 1 μL of each primer (forward and reverse), and 5 μL of PCR buffer. Next, PCR amplifications were performed using a thermocycler (Techne TC-512; Staffordshire, UK) under the following conditions: an initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 5 min, and a final extension at 72°C for 7 min. The amplicons were purified using a GeneJET PCR purification kit (Thermo Scientific) and subjected to 1% agarose gel electrophoresis. The amplicon bands were stained with ethidium bromide solution and visualized under ultraviolet (UV) light. The size of the amplicons was determined with an O’Gene Ruler^TM 10000-bp DNA ladder (Thermo Scientific). The sequence analysis results were compared with 16S rDNA sequences of the National Center for Biotechnology Information database using the Basic Local Alignment Search Tool program.

Decarboxylase activity of Enterococcus spp.

The production of tyramine, putrescine, cadaverine, and histamine, which were the predominant BAs in cheese samples and commonly produced by enterococcal strains, was determined. To assess the decarboxylase activity of Enterococcus isolates, a modified decarboxylase medium was used (Maijala, 1993). The medium composition was as follows: 1-g dextrose, 5-g peptone, 0.02-g bromocresol purple, and 3-g yeast extract dissolved in 1-L distilled water. Amino acids corresponding to the BAs (L-tyrosine, L-lysine, L-ornithine, and L-histidine) were sequentially added to the medium (each at a final concentration of 0.5%). The pH of the medium was then adjusted to 6.78–6.82 using 1-N NaOH and 1-N HCL before autoclaving for 15 min at 121°C. The medium with individual amino acids was prepared in a distinct broth tube. The control medium, which did not comprise amino acids, was used for comparison.

Freshly activated bacterial cultures were inoculated into 0.1 mL of decarboxylase broth with an optical density at 600 nm (OD_{600 nm}) of 0.50 and incubated at 30°C for 4–5 days. The cultures were monitored daily to observe color alterations. In the control tube, the color of the medium was expected to remain yellow (indicating a negative result). According to the criteria established by Bover-Cid and Holzapfel (1999), the color transition of the medium from yellow to purple in the tube containing amino acid was considered a positive indication of BA production.

Determination of BA production levels in Enterococcus strains using HPLC

The contents of BAs in the TSB culture supernatants were determined using acid extraction and derivatization, following the methods used by Sang et al. (2020). The strains were incubated in the medium for 24 h at 37°C, followed by culturing in TSB containing 0.25% histidine, lysine, tyrosine, and ornithine hydrochloride at 37°C for 2 days. After mixing, 1 mL of culture was mixed with 1 mL of trichloroacetic acid (5%), and the mixture was centrifuged at 6,000 rpm at 4°C for 10 min. To perform derivatization, 1 mL of supernatant was incubated with approximately 50 μL of sodium hydroxide (2 mol/L), followed by sequential incubation with 300-μL dansyl chloride (10 mg/mL), 100-μL saturated sodium bicarbonate, and 25% ammonia (50 μL). The final mixture was incubated in the dark for 30 min at 25°C. The concentrations of histamine, cadeverine, tyramine, and putrescine were measured using an HPLC system (Shimadzu, LC-2030, Kyoto, Japan) by following the methods described by Shen et al. (2020), under the following conditions: column, C18 column (Agilent ZORBAX Eclipse XDB-C18, 4.6 × 250 nm, 5 μm); mobile phase A, ultrapure water; mobile phase B, acetonitrile; flow rate, 1 mL/min; and detection wavelength, 254 nm. The gradient elution program was as follows: 0–5 min, 65%–70% B; 5–14 min, 70%–100% B; 14–18 min, 100% B; 18–20 min, 100%–65% B; and 20–22 min, 65% B. The regression parameters of the biogenic amine compounds analyzed by HPLC are presented in Table 1, demonstrating high linearity and confirming the reliability and accuracy of the analytical method employed.

Screening for decarboxylase-encoding genes

The presence of BA-encoding genes was screened using PCR with the described primer sets (Table 2). The PCR conditions were as follows: an initial denaturation at 95°C for 6 min, followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 50°C for 30 s, extension at 75°C for 5 min, and a final extension at 72°C for 5 min.

Statistical analysis

statistical analyses were performed specifically on the BA concentrations (mg/L) obtained from HPLC measurements to evaluate differences among Enterococcus isolates. Data were represented as median and quartiles or mean and standard deviation (SD). Mean values between the two groups were compared using the Mann–Whitey U test, reporting the exact p value. Meanwhile, mean values between more than two groups were compared using one-way analysis of variance (ANOVA) for homogenous variance or the Welch ANOVA test for non-homogenous variance. The significance level was set at 5%. All statistical analyses were performed using SPSS (version 27).

Nucleotide sequence accession numbers

The nucleotide sequences of the 16S rDNA genes from 135 Enterococcus isolates of the resent study were submitted and deposited to the GenBank. Accession numbers are given in Table 3.

Results and Discussion

From 186 traditional cheese samples, 135 (73.65%) probable enterococcal isolates were identified (Table 3). These 135 isolates were subjected to morphological and culture analyses. All isolates exhibited growth under the following conditions: pH 9.6, 6.5% NaCl, and 10–45°C. Additionally, these isolates exhibited Gram-positive, catalase-negative, and esculin hydrolysis-positive phenotypes. Meanwhile, 16S rDNA sequencing analysis identified 135 isolates at the species level (Figure 1, Table 3). Of the 135 Enterococcus spp., 92 were E. faecium (68.14%) and 43 were E. faecalis (31.86%). E. faecium and E. faecalis are reported to be the most commonly isolated Enterococcus spp. in cheese (Botello-Morte et al., 2022; Combarros-Fuertes et al., 2016; Gökmen and Ektik, 2022; Sanlibaba and Senturk, 2018; Souza et al., 2023). In this study, E. faecium was the predominant species. The findings of this study were consistent with those of Hajikhani et al. (2021); Raafat et al. (2016); Yerlikaya and Akbulut (2020); and Yogurtcu and Tuncer (2013). However, some studies (Aydın and Ardıç, 2019; Aydın et al., 2020; Ghazvinian et al., 2024; Jahansepas et al., 2022; Oruc et al., 2021; Souza et al., 2023) have reported that E. faecalis was the most prevalent enterococcal strain in different traditional cheese samples. The isolation rate of Enterococcus strains in this study was high. This result was consistent with that of previous studies, which reported that the percentage of Enterococcus-positive samples in traditional cheese samples was 100% in Slovakia (Kročko et al., 2011) and 96% in Iran (Jahansepas et al., 2022). However, the detection rate in this study was lower than that reported in previous studies (Aydın and Ardıç, 2019; Sanlibaba and Senturk, 2018; Togay and Karayigit, 2022) conducted in Türkiye, which reported that the prevalence of enterococci in traditional cheese samples was in the range of 83.05–99.1%.

Figure 1. Polymerase chain reaction amplification of 16S rDNA fragments of Enterococcus strains. M: M O’Gene ruler; DNA marker; 1–20: BAA17, BAA33, BAA46, BAA60, BAA71, BAA92, BAA125, BAA147, BAA184, BAA188, BAA206, BAA236, BAA251, BAA262, BAA271, BAA280, BAA298, BAA307, BAA310, BAA323, and BAA334.

This study examined the ability of 135 Enterococcus spp. to produce BAs using biochemical analysis. Among the BAs most abundantly produced by enterococcal strains in cheeses, cadaverine, histamine, tyramine, and putrescine were selected as target BAs. Among the 25 BA-producing Enterococcus strains, 18 were E. faecium (72%) and 7 were E. faecalis (28%). Additionally, histamine, tyramine, putrescine, and cadaverine were produced by 25, 24, 21, and 13 Enterococcus spp., respectively. Among the Enterococcus strains, 4 BAs were produced by 10 strains, 3 BAs were produced by 13 strains, and 2 BAs were produced by 2 strains in vitro. PCR screening revealed that 25, 24, 21, and 13 strains harbored the amino acid decarboxylase-encoding genes for histamine, tyramine, putrescine, and cadaverine production, respectively. The corresponding amplicons of these genes were also detected on agarose gels (Figures 2–5). HPLC was performed to quantify the production of histamine, tyramine, cadaverine, and putrescine by 25 Enterococcus strains. A limitation of the current study is that the analysis of BA-producing genes was restricted to their presence or absence; the future research involving the sequencing of these genes would be valuable to elucidate the molecular basis underlying the observed variations in BA production among Enterococcus strains.

Figure 2. Agarose gel electrophoresis analysis of putrescine decarboxylase-encoding gene amplicons. M: O’Gene ruler DNA marker; 1–21: BAA17, BAA33, BAA92, BAA125, BAA147, BAA184, BAA188, BAA206, BAA236, BAA251, BAA262, BAA271, BAA280, BAA298, BAA307, BAA310, BAA323, BAA334, BAA343, BAA362, and BAA388.

Figure 3. Agarose gel electrophoresis analysis of cadaverine decarboxylase-encoding gene amplicons. M: O’Gene ruler DNA marker; 1–13: BAA17, BAA33, BAA46, BAA60, BAA125, BAA184, BAA188, BAA251, BAA280, BAA307, BAA310, BAA334, and BAA343.

Figure 4. Agarose gel electrophoresis analysis of histamine decarboxylase-encoding gene amplicons. M: O’Gene ruler DNA marker; 1–25: BAA17, BAA33, BAA46, BAA60, BAA71, BAA92, BAA125, BAA147, BAA184, BAA188, BAA206, BAA236, BAA251, BAA262, BAA271; BAA280, BAA298, BAA307, BAA310, BAA323, BAA334, BAA343, BAA362, BAA368, and BAA388.

Figure 5. Agarose gel electrophoresis analysis of tyramine decarboxylase-encoding gene amplicons. M: O’Gene ruler DNA marker; 1–24: BAA33, BAA46, BAA60, BAA71, BAA92, BAA125, BAA147, BAA184, BAA188, BAA206, BAA236, BAA251, BAA262, BAA271, BAA280, BAA298, BAA307, BAA310, BAA323, BAA334, BAA343, BAA362, BAA368, and BAA388.

The concentrations of BAs in all cheese samples were in the range of 0–97.36 mg/L. In particular, the concentrations of histamine, tyramine, putrescine, and cadaverine produced by Enterococcus strains were in the range of 14.87–26.24, 2.9–33.47, 0.91–97.36, and 1.18–57.84 mg/L, respectively (Table 4). The BA188 strain produced the highest amount of BA in this study, producing putrescine at a concentration of 97.359 mg/L. The levels of histamine, tyramine, putrescine, and cadaverine did not differ significantly among the bacterial groups (p > 0.05). The statistical analyses presented in Tables 4 and 5 compare the concentrations of each BA among 25 individual Enterococcus strains, highlighting strain-specific differences in BA production (p < 0.001), rather than differences between different amine types.

Various studies (Barbieri et al., 2019; Bogdanovi´c et al., 2020; Hu et al., 2021; Li et al., 2023; Merabti et al., 2019) have reported that histamine and tyramine are the most abundantly produced BAs by Enterococcus spp. In contrast, this study reported that BA with the highest concentration in traditional cheese samples was putrescine (97.36 mg/L). High concentrations of histamine and tyramine, which are bioactive molecules, induce severe symptoms (O’Sullivan et al., 2015; Turna et al., 2024). The doses of histamine that elicit allergic reactions vary among individuals. The currently used threshold levels for histamine are derived from a limited number of human studies performed with both healthy individuals and individuals with heightened sensitivity. In healthy individuals, exposure to histamine is not expected to exert adverse effects at levels of 25–50 mg per person per meal. However, in individuals with histamine intolerance, small amounts of histamine exposure (as low as 5–10 mg) from food can trigger severe adverse health effects (Sungur and Jobasi, 2022.; Turna et al., 2024). Tyramine intoxication, commonly referred to as the ‘cheese reaction’ or ‘cheese effect’, was first observed after the consumption of cheese contaminated with high levels of tyramine. For individuals consuming monoamine oxidase inhibitors (MAOIs), dietary exposure to tyramine exerts toxic effects with a high risk of interaction with MAOIs, leading to increased blood pressure. Currently, the data to establish a definitive toxic threshold for tyramine in humans are insufficient (Turna et al., 2024). The threshold concentrations for tyramine and histamine in foods for human consumption are 100–800 mg/kg and 100 mg/kg, respectively (Burdychova and Komprda, 2007; Dabadé et al., 2021; Turna et al., 2024). In this study, the histamine (14.87–26.24 mg/L) and tyramine (2.9–33.47 mg/L) concentrations produced by Enterococcus spp. were lower than the threshold values. However, O’Sullivan et al. (2015) reported that tyramine production by Enterococcus strains was in the range of 1485–2363 mg/L. This range obtained in this study is also inconsistent with that reported by previous studies because of reporting higher concentrations of tyramine in certain cheese samples. For example, Bogdanović et al. (2020) reported that the highest tyramine concentrations in a mold-ripened cheese and a semi-hard cheese were 762.75 mg/kg and 767.03 mg/kg, respectively. Additionally, Zdolec et al. (2022) revealed that tyramine was the most abundant BA in cheese samples (Zdolec et al., 2022), accounting for 75.4%, 41.3%, and 35% of total BAs in mold cheese, hard cheese, and semi-hard cheese samples, respectively. Histamine was detected in eight cheese samples at concentrations ranging from 8.4 mg/kg to 85.1 mg/kg (O’Sullivan et al., 2015). However, Zdolec et al. (2022) reported that only two semi-hard cheese samples and three hard cheese samples exceeded the European maximum limit of 100 mg/kg.

Various regulatory agencies, including the US Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA), have not established regulatory limits for cadaverine and putrescine (EFSA Panel on Biological Hazards [BIOHAZ], 2011). The pharmacological activities of putrescine and cadaverine are lesser than those of histamine and tyramine. However, the adverse effects of these diamines include hypotension, bradycardia, lockjaw, and paresis of the extremities. Putrescine and cadaverine in food can enhance the toxicity of other amines, especially histamine. Additionally, these diamines may react with nitrite, resulting in the formation of carcinogenic nitrosamines (EFSA Panel on Biological Hazards, 2011; Rauscher-Gabernig et al., 2012). According to the EFSA Panel on Biological Hazards (2011), the levels of cadaverine in fresh cheese and hard cheese samples are in the range of 10.70–45.00 mg/kg and 47.80–83.50 mg/kg, respectively.

In this study, the concentrations of cadaverine produced by 23 Enterococcus strains were below the levels specified by EFSA Panel on Biological Hazards (2011) but those produced by two strains (BAA17 and BAA188) were within the range. An Austrian study conducted by Rauscher-Gabernig et al. (2012) proposed tolerable levels of cadaverine and putrescine in cheese. The recommended maximum levels for putrescine and cadaverine were 140–510 mg/kg and 430–1540 mg/kg, respectively. In this study, the levels of putrescine (0.91–97.36 mg/L) and cadaverine (1.18–57.84 mg/L) were below the levels recommended by Rauscher-Gabernig et al. (2012). Previous studies have reported that cadaverine concentrations significantly vary in different types of cheese. For example, Zdolec et al. (2022) reported that cadaverine levels in the cheese core and rind were in the range of 2.57–64.22 mg/kg and 3.70–38.17 mg/kg, respectively. O’Sullivan et al. (2015) detected cadaverine in all cheese samples at a concentration of 1.2–267.4 mg/kg. These results are consistent with those of the current study. The putrescine--producing enterococci also produce tyramine (Villarreal et al., 2024). However, of the 24 tyramine-producing strains, 21 also produced putrescine.

Biogenic amines, which are heat-resistant compounds, cannot be eliminated from the environment through thermal processes, such as pasteurization or cooking (Zdolec et al., 2022). The presence of BA-producing microorganisms in food can serve as an indicator of both quality of raw materials and hygiene standards maintained throughout the food-production process (O’Sullivan et al., 2015). To mitigate the formation of BAs in cheese, the following strategies are employed: the -careful selection and control of microbial starter cultures (use of competitive dairy cultures because of their possible inhibitory effect on amine-producing bacteria); the usage of high-quality and fresh raw materials and food ingredients; the implementation of stringent sanitation protocols; the appropriate usage of additives, such as sugar, salt, and antimicrobial agents; the maintenance of proper food-handling practices during fermentation (Natrella et al., 2024).

Conclusions

This study investigated the BA production profiles of Enterococcus strains isolated from various traditional cheese samples in Türkiye. These strains produce various amounts of BAs and consequently have potential implications for food safety and public health. Given the well-documented adverse health effects associated with elevated BA levels, such as histamine and tyramine, particularly in individuals sensitive to these compounds, it is crucial to monitor and control their concentrations in food products. The identification of BA producers among cheese microorganisms, especially Enterococcus spp., underscores the need for further research into the factors influencing amine production during fermentation, ripening, and storage processes. The findings of this study highlight the importance of determining the precise levels of BAs in fermented foods and their correlation with microbial activity as well as the potential risks to consumer health. Further studies are needed to explore the genetic mechanisms underlying BA production in Enterococcus strains and evaluate the dose–response relationships for amine toxicity. The future studies should focus to develop methods to reduce or control the accumulation of harmful BAs in dairy products and identify starter cultures that limit BA production without affecting the desired sensory properties of cheese. This study revealed the critical role of BA monitoring in the dairy industry and the need for comprehensive safety standards to ensure that fermented products do not pose health risks to consumers. The future studies should elucidate BA metabolism and provide strategies to mitigate BA formation in food-production processes.