Formation of biogenic amines in fish: Dietary intakes and health risk assessment

Abstract Biogenic amines (BAs) are toxicants that are produced during the proteolytic activities of some microorganisms, or naturally during the metabolism of their precursor amino acids. The objective of this study was to estimate the formed BAs in six types of fish retailed in Egypt including tilapia, mullet, mackerel, sardine, herring, and tuna. In addition, total mesophilic (TMC) and total psychrophilic (TPsC) bacterial counts were investigated. Furthermore, the estimated daily intakes (EDI) of BAs via the ingestion of various types of fish in Egypt were calculated, and their potential health risks were discussed. The achieved results indicated the formation of histamine (HIS), tyramine (TYR), cadaverine (CAD), putrescine, spermine, and spermidine at different concentrations. Tilapia had the lowest concentration levels for the different BAs. In contrast, mackerel and tuna had the highest concentrations of BAs. Total biogenic amines (TBAs) showed significant positive correlations with TMC in the examined fish species. The recorded EDI values of the different BAs in the current study would not have adverse effects, except for mackerel and tuna. Excessive consumption of fish contaminated with BA might have serious health hazards such as symptoms of histamine poisoning, including rashes, flushing, palpitations, and asthma. Therefore, the adoption of strict hygienic measures during the processing, storage, and distribution of fish is highly recommended to reduce the formation of BAs in fish.


| INTRODUC TI ON
Fish is considered as a major source for high biological values protein with low cholesterol, rich in trace elements and vitamins. In addition, fish is regarded as essential for a healthy life because of their content of n-3 and n-6 series of polyunsaturated fatty acids (PUFA), especially docosahexaenoic and eicosapentaenoic fatty acids (Brito et al., 2019;Lira et al., 2019Lira et al., , 2020. The demand for fish consumption is increasing worldwide to compensate the shortage of red meat. Fish is highly perishable protein source, and due to the improper hygienic and storage conditions during transportation, handling, and processing, microbial contamination and fish deterioration might occur leading to the formation of a vast array of chemicals (Huss, 1995).
Biogenic amines (BAs) are natural toxicants produced by a vast array of the microorganisms, either pathogenic or none, and via the metabolism of some amino acids. BAs are classified into 1) monoamines such as histamine (HIS) and tyramine (TYM) that are produced by a one-step decarboxylation reaction from their precursor amino acid histidine and tyrosine, respectively (Marcobal et al., 2012). 2) Diamines such as cadaverine (CAD) which is produced by a decarboxylation reaction from lysine, and putrescine (PUT). The latter is a diamine that can be produced either by a single-step decarboxylation from ornithine and agmatine or indirectly after hydrolysis of arginine ( Wunderlichová et al., 2014). 3) Polyamines such as spermine (SPM) and spermidine (SPD) are produced by various pathways that include the hydrolysis of arginine into ornithine and agmatine (Shah & Swiatlo, 2008). Biogenic amines at their physiological concentrations play essential roles in several processes in the cell, including gene expression, cell growth, and tissue repair (Benkerroum, 2016;Galgano et al., 2012;Ma et al., 2020). Unlikely, ingestion of high levels of such BAs may lead to severe symptoms such as anaphylaxis, hypertensive symptoms, nervous manifestation, and death (Medina et al., 2003). In addition, some BAs such as PUT and CAD were associated with the onset of gastric cancer as it may be converted into carcinogenic N-nitroso compounds by microorganisms in the digestive tract ( Koutsoumanis et al., 2010). Ingestion of fish containing elevated levels of BAs was associated with cases of intoxication in Europe (EFSA Panel on Biological Hazards (BIOHAZ) (2011)). Screening of the occurrence of BAs in fish is considered also as a good index for the fish quality and hygiene (Koutsoumanis et al., 2010;Visciano et al., 2012). There is a clear lack of information about the levels of BAs in different kinds of fish marketed in Egypt and their contribution to the estimated daily intakes of BAs among the Egyptian population. Furthermore, the correlation analysis between the microbial counts and the formation of BAs is less informed. Therefore, the objectives of the current study were firstly to estimate the formed BAs in six species commonly consumed in Egypt, namely, Tilapia, Mullet, Mackerel, Herrings, Sardine, and Tuna. Estimated daily intakes of BAs due to consumption of such fish kinds were additionally calculated among the Egyptian population.
Furthermore, microbial counts including total plate counts (TPC) and total psychrophilic counts (TPsC) were investigated in the fish samples and their correlations with the total biogenic amines (TBAs) were analyzed.

| Fish samples
One hundred and twenty fish samples, including Nile Tilapia (Oreochromis niloticus), Mullet (Mugil cephalus), Mackerel (Scomber scombrus Linnaeus, 1758), Herrings (Clupea harengus Linnaeus, 1758), Sardine (Sardinella aurita), and Tuna (Thunnus Albacures) (n = 20 of each fish spp.) were collected randomly from local fish markets in Egypt. Fish samples were purchased on a daily basis in the morning and within 2 hr from the arrival of the fish into the market. Fish samples were intact, with a fresh smell, and reddish gills. Fish is usually preserved in ice to keep their internal temperature as low as possible. However, the fish sellers frequently move the fish with their uncleaned bared hands. The fish market is usually in the open air, and the fish is kept in wooden boxes, which are not frequently cleaned, and the chance of cross-contamination is high. The collected samples were transferred cooled directly to the laboratory for the microbial examination and estimation of their BAs contents.

| Content of biogenic amines
Ten grams from each sample were firstly homogenized with 100 ml of 10% trichloroacetic acid for 3 min at 17,608 g. Then, the homogenates were extracted in dark for one hour with shacking at 4ºC, followed by centrifugation for 20 min at 1,956 g, 4ºC. The supernatants were filtered through a Whatman filter No. 1 and the filtrates were kept at 4ºC until analysis. An amino acid analyzer (L-8900, HITACHI, Japan) was used for quantitative estimation of BAs according to Kononiuk and Karwowska (2019). The amino acid analyzer was equipped with an Ostion LG AAA8 ion-exchange column (3,6 × 100 8 µm). Gradient elution using Na+/K+ citric buffers was employed for separation. Colorimetric detection was done at 570 nm, after postcolumn derivatization of ninhydrin. The Contents of the biogenic amines including HIS, TYR, PUT, CAD, SPM, and SPD were determined with a reference to the amine standards (Merck KGaA, Darmstadt, Germany). The biogenic amine concentrations were reported as mg/kg fish.

| Daily intake of BAs
The estimated human daily intake (EDI) for the quantified and total biogenic amines via consumption of fish in Egypt was calculated for Egyptian adults and children according to the following equation: Where Ci is the concentration of individual or total BAs in the fish, and IR is the ingestion rate of fish in Egypt. F IR is the fish ingestion rate in Egypt, which was estimated at 48.57 g/day (Food and Agricultural Organization (FAO), 2003); BW is the bodyweight, which was estimated at 70 kg for adults and 30 kg for children.

| Determination of total microbial counts
The total plate (TPC) and total psychrophilic (TPsC) counts were screened according to the methods of American Public Health Association (APHA) (2001). In short, twenty-five grams from each fish sample was homogenized in 225 ml of sterile buffered peptone water 0.1% at 2,500 rpm for 2 min. These homogenates represent the dilution of 10 -1 , and then decimal dilutions have proceeded. From each dilution, one ml was transferred into two sterile Petri dishes to which 15 ml of nutrient agar (Oxoid) were added. After thorough mixing and solidification, the inoculated plates were incubated at 37°C for 24 hr | 3125 EL-GHAREEB Et AL.
for TPC or incubation at 7ºC for ten days for TPsC. The agar plates having 30-300 pinpoint colonies were calculated and recorded as follow: TPC or TPsC/g. = average No. of colonies ×reciprocal of the dilution.
Counted colonies expressed as log 10 cfu/g.

| Statistical analysis
Measurements were carried out in duplicates and all values were expressed as means ± SE. Statistical analysis was evaluated using Tukey-Kramer HSD test. Pearson correlation analysis was done using the JMP statistical package, SAS Institute Inc. In all analyses, p < .05 was used to indicate statistical significance using the JMP statistical package, SAS Institute Inc.

| RE SULTS AND D ISCUSS I ON
In the present study, six BAs were quantified in six kinds of fish marketed in Egypt. The formed individual and total BAs varied significantly based on the kind of the fish and their microbial status.

| Histamine
The obtained results revealed formation of HIS in all examined fish samples. All fish species contained levels of HIS lower than the maxi-

| Tyramine
TYR is detected in all examined samples in the present study with levels below the recommended MPL (100 mg/kg) (Silla Santos, 1996)

| Putrescine
Regarding PUT, the achieved results indicated the formation of PUT in all examined fish samples. Similar to CAD, Tuna had significantly the highest PUT residues. PUT levels ranged between 0.700-4.100 mg/ TA B L E 1 Biogenic amines content (mg/kg) among the examined fish species

| Spermidine
SPD is the other-tested polyamine in the present study.

| Dietary intakes and health-risk assessment of BAs
The EDI values (mg/kg BW/day) of HIS via consumption of different fish species in Egypt ranged between 0.012 in adults and 0.029 in children for Tilapia, and 0.151 in adults and 0.353 in children for Tuna (Table 2) (Benkerroum, 2016). EDI values of PUT (mg/kg BW/day) ranged from 0.001 in adults and 0.002 in children for Mullet to 0.118 in adults and 0.274 in children for Tuna (Table 2). There is less information available about NOAEL or the risk assessment of PUT due to their least-toxic effects ( SPD is a precursor for the most potent carcinogenic N-nitrosamines. Furthermore, SPD is associated with ischemia and cystic fibrosis and facilitates tumor progression (Benkerroum, 2016). Mackerel is probably due to their high content of the respective-free amino acids (Biji et al., 2016). In addition, Mackerel and Tuna are imported fish species in Egypt. Frequent freezing and thawing of the imported fish during storage, transportation, and handling with the increase in the surrounding temperature give rise to the microbial decomposition and rapid spoilage of the fish ( Visciano et al., 2012).

| Microbial counts and their correlation with total BAs in fish
Therefore, we extended this study to investigate the microbial status of the examined fish species and the correlation analysis between the microbial counts (TPC and TPsC) and the total BAs. Interestingly, Tuna and Mackerel had the highest TPC (5.756 ± 0.086 and 5.757 ± 0.005 log 10 cfu/g, respectively), followed by Herrings (5.425 ± 0.005 log 10 cfu/g), Sardine (5.381 ± 0.013 log 10 cfu/g), Mullet (4.866 ± 0.016 log 10 cfu/g), and Tilapia (4.677 ± 0.012 log 10 cfu/g). TPsC was comparable to that of the TPC (Table 3). Nearly similar TPC counts were reported in Tilapia ( Kulawik et al., 2013), and Tuna ( Jääskeläinen et al., 2019). Interestingly, significant positive correlations were observed between TBAs and TPC in the different fish species, particularly in the Tilapia, Herrings, and Sardine (Table 3)

| CONCLUSION
The obtained results in the present study indicated detection of dif- serious health hazards, particularly scombroid poisoning. Therefore, efficient hygienic measures should be adopted during processing, storage, distribution, and marketing of fish in Egypt. Such hygienic measures include thorough washing and disinfection of the wooden boxes used during fish handling and storage, adequate personal hygiene, cleaning and disinfection of the floors, walls, and other surfaces of the fishery shops, proper storage, and preservation of fish.
Furthermore, continuous monitoring for BAs formation in the other fish and shellfish species marketed in Egypt is highly recommended from both the food safety and public health point of view.

| Ethical approval
This study does not involve any human or animal testing. This study was conducted according to the guidelines of King Faisal University, Saudi Arabia.

| Consent to participate
All authors approved to participate in this research work and in the manuscript.

| Consent to publish
All authors approved this manuscript to be published.

ACK N OWLED G M ENTS
The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project number IFT20087.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R CO NTR I B UTI O N S
All authors contributed to the study conception and design. Material

DATA AVA I L A B I L I T Y S TAT E M E N T
All data and materials will be available upon request.