Biogenic Amines in Portuguese Traditional Foods and Wines

2293 Journal of Food Protection, Vol. 69, No. 9, 2006, Pages 2293 2303 Copyright, International Association for Food Protection Review Biogenic Amines in Portuguese Traditional Foods and Wines ISABEL M. P. L. V. O. FERREIRA 1 * A OLÍVIA PINHO 1,2 1 REQUIMTE, Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto, Rua Anibal Cunha 164, 4099-030 Porto, Portugal; and 2 Faculdade de Ciências da Nutrição e Alimentação, Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal MS 05-604: Received 30 November 2005/Accepted 25 March 2006 ABSTRACT The presence of biogenic amines in foodstuffs is an important food safety problem because of the implication of these compounds in food intolerance and intoxication. The separation and quantification of biogenic amines in foods is normally performed by chromatographic techniques. This review contains descriptions of the quantification of biogenic amines in Portuguese traditional fermented and/or ripened foods and wines, including Protected Denomination of Origin cheeses, drycured sausages, and Portuguese wines (including Port wines), using different analytical methods based on high-pressure liquid chromatography (UV or diode array and/or fluorometric detectors) and gas chromatography (with a mass spectrometry detector). The evolution of biogenic amines during fermentation, ripening, aging, or storage of those products was also evaluated. Biogenic amine concentrations ranged widely within individual food items, and storage, transport, and handling conditions can influence to some extent the biogenic amines present and their concentrations. Traditional foods are an important part of the Portuguese diet, and a high intake of harmful amounts of biogenic amines from traditional Portuguese fermented foods is possible. However, extensive research is needed to extend the current limited database. Biogenic amines are naturally occurring compounds, ubiquitous in animals and plants. These low-molecularweight bases can be aliphatic (e.g., putrescine, cadaverine, spermidine, and spermine), heterocyclic (e.g., histamine and tryptamine), or aromatic (e.g., tyramine and phenylethylamine). These active substances play roles in normal mammalian physiology; however, they also can have toxic effects when consumed in large amounts, especially if their metabolism is blocked or genetically altered. The polyamines putrescine, spermidine, and spermine are intimately involved in cell proliferation and differentiation (2 4, 46, 53). Whereas histamine, tyramine, tryptamine, serotonine, and phenylethylamine play a variety of physiological roles, resulting in toxic effects when dietary levels are too high. According to Taylor et al. (112), the threshold of danger is 1,000 mg of total amines per kg when ingestion is coupled with such potentiating cofactors as amine oxidase inhibiting drugs or alcohol or when there are preexisting gastrointestinal diseases (9). Thus, the presence of biogenic amines in food can be an important food safety problem because of the implication of these amines in cases of food intolerance and intoxication. Amines and amine-related compounds are present in food either naturally or as a result of processing or storage. The concentrations of biogenic amines usually increase during spoilage of fish and meat, which is associated with increases in microbial concentrations and deterioration of sensorial quality. In this sense, the presence of amines in * Author for correspondence. Tel: 351222078929; Fax: 351222003977; E-mail: isabel.ferreira@ff.up.pt. food may be regarded as a useful indicator of the quality or hygienic history of the raw material used in various food products (43, 44, 99, 2, 124). However, amines also can be produced by the decarboxylase activity of certain fermentative microorganisms (96, 9). The major factors that constrain the qualitative and quantitative profiles of biogenic amines in foods are the feedstock, i.e., the availability of amino acids produced as an outcome of proteolysis or fermentation, and the process, i.e., the viability of microorganisms capable of decarboxylation of amino acids. Several extrinsic processing factors also may play an important role, e.g., ph, salt concentration, water availability, and redox potential (87). Various microorganisms can produce amines because of their amino acid decarboxylase systems, e.g., species of Bacillus, Pseudomonas, Escherichia, Enterobacter, Salmonella, Shigella, Staphylococcus, Streptococcus, Lactobacillus, Enterococcus, Lactococcus, and Leuconostoc (1). Lactobacilli appear to be very active in the production (and consequent accumulation) of histamine, tyramine, and putrescine. Enterococci are notorious tyramine formers, and members of the Enterobacteriaceae can cause cadaverine and putrescine buildup, even at low biomass densities (9). Because biogenic amines have both beneficial and deleterious effects on humans, it is important to minimize our exposure to harmful amines and ensure a sufficient intake of the favorable ones (30). In highly perishable foods such as meat and fish, it is possible to establish a biogenic amine index that can express a correlation between the product and the quality and hygiene procedures used during handling (13). However,

2294 FERREIRA A PINHO J. Food Prot., Vol. 69, No. 9 this type of correlation may not be valid for fermented or ripened foods, i.e., cheese, meat products, and fermented beverages because the microorganisms used in the production of these products promote the production of biogenic amines, the most common of which are histamine, putrescine, cadaverine, tyramine, tryptamine, -phenylethylamine, spermine, and spermidine. Traditional ripened or fermented foods are especially notable for containing high concentrations of biogenic amines. Biogenic amines are difficult to analyze as a group because of their structural diversity and lack of pronounced UV visible light absorption characteristics. The usual approach, therefore, has been the derivatization of free amines with an easily detectable group to increase the sensitivity of the method. This derivatization step usually reduces the basicity of the nitrogen atom, and the use of reversed phases is usually considered the most suitable technique for this step. Several methods for separation, identification, and determination of amines have been published during the last two decades. All methods involve three main steps: extraction of the amines, purification of the extract, and derivatization. The current trend followed by most of the authors favors the use of high-pressure liquid chromatography (HPLC) methods for the simultaneous determination of various amines (55, 58, 71, 8, 118, 120). Generally, these methods include a pre- or postcolumn derivatization procedure and fluorometric detection of the corresponding derivatives. The derivatizing reagent most commonly used is o-phthaldialdehyde, usually in the presence of 2-mercaptoethanol (16, 18, 21, 59, 6), although a number of other reagents also have been used such as 9-fluorenylmethyl chloroformate (6), 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (15), 2-naphthyloxycarbonyl chloride (54), p-nitrobenzyloxycarbonyl chloride (56), and fluorescamine (4-phenylspiro[furan-2(3H)-1 -phthalan]-3,3 -dione) (20). Among the better described reagents for this derivatization are dansyl chloride (17, 27, 45, 70, 125) and dabsyl chloride (7, 8, 58), which have the advantage of producing derivatives with both primary and secondary amines that are stable enough for subsequent spectroscopic analysis. However, other chromatographic methods are used, such as thinlayer chromatography (72, 1, 3) and gas chromatography (GC) (33, 34, 84, 85, 90). The appropriate method must be chosen based on the food matrix composition and biogenic amine content. This review provides data on biogenic amine formation and concentrations in some Portuguese traditional fermented and/or ripened foods and wines. These data were collected with HPLC and GC methodologies and extraction procedures validated for separation and quantification of these compounds in Protected Denomination of Origin (PDO) cheeses, traditional cured meat products, and Portuguese wines. The evolution of biogenic amines during fermentation, ripening, aging, or storage of these products was also evaluated. BIOGENIC AMINE CONTENT OF SOME PORTUGUESE PDO CHEESES Cheese, like other fermented foods, usually contains a high concentration of biogenic amines, which result from the amino acid decarboxylase activity of microorganisms (61, 62). These amines have been studied for their potential human health risk because tyramine and histamine can cause cheese syndrome and histamine intoxication, respectively. The adverse effects of these biogenic amines include emesis, respiratory distress, heart palpitations, headache, hyper- or hypotension, and hypertensive crisis due to interactions with monoamine oxidase inhibitor drugs (38, 44). These reactions can be potentiated by other biogenic amines, such as putrescine, cadaverine, spermine, and spermidine (9). Diamines (putrescine and cadaverine) also can react with nitrites to form carcinogenic nitrosamines (98). The production of biogenic amines in cheese often has been linked to nonstarter lactic acid bacteria and members of the Enterobacteriaceae (50, 83). Several authors (50, 83, 91, 92) have reported the decarboxylase activity of lactobacilli, which produce histamine, tyramine, putrescine, and cadaverine. Many enterococci may decarboxylate amino acids to their corresponding amines, especially tyrosine to tyramine (50, 62). Many coliform bacteria can form putrescine and cadaverine by the decarboxylation of ornithine and lysine, respectively (83, 5, 9). Under normal conditions, however (50), these bacteria grow slowly and die quickly. Therefore, most cases in which large concentrations of amines have been produced in cheese have been attributed to lactic acid bacteria with decarboxylating activity. From a toxicological point of view, it is useful to produce cheeses with low concentrations of amines to avoid potential adverse reactions (51). However, the presence of free amino acids and biogenic amines in cheeses is important to achieve characteristic texture and aroma. Thus, every type of cheese has its own free amino acid and biogenic amine profile resulting from its specific degradation, interconversion, and synthesis. Many studies of cheese have been undertaken to determine the biogenic amines and their precursors. A variety of amines such as tyramine, histamine, putrescine, cadaverine, tryptamine, and -phenylethylamine have been found in many types of cheeses (19, 25, 31, 39, 47 49, 68, 77, 93), and their presence is correlated with previous treatment of the milk, microflora composition, type of rennet, and manufacturing and ripening conditions (35, 36, 67, 78, 82, 93, 99, 5, 113, 114, 121). Histamine and tyramine concentrations vary extensively both between and within different cheese varieties (9). Portugal has a strong tradition of making cheese from ovine milk, and most of these cheeses are protected by the PDO designation (37). However, studies on the biogenic amine concentrations of these PDO cheeses are scarce. A method of simultaneous extraction and separation of the free amino acids and biogenic amines developed to optimize derivatization with dabsyl chloride and chromatographic separation was validated in ovine cheese samples (87). Several advantages of the dabsyl and dansyl methods over methods involving other derivatizing reagents have been reported (58). Extraction of biogenic amines from the cheese matrix is a critical step for obtaining adequate re-

J. Food Prot., Vol. 69, No. 9 BIOGENIC AMINES IN PORTUGUESE FOODS 2295 FIGURE 1. Changes in concentrations of cadaverine, putrescine, and tyramine in Terrincho cheese throughout ripening. (Adapted from Pinho (86).) FIGURE 2. Changes in concentrations of ethylamine, dimethylamine, tryptamine, phenylethylamine, histamine, and cystamine in Terrincho cheese throughout ripening. (Adapted from Pinho (86).) covery of each amine. An acid medium was beneficial for the analysis of the cheeses (71, 87). The evolution of biogenic amines and the native microflora (i.e., viable numbers of lactobacilli, enterococci, enterobacteria, staphylococci, and yeasts), ph, acidity, water activity, and free amino acids were evaluated during the 60-day ripening period for Terrincho ovine cheese. This cheese is an uncooked pressed product made from raw whole milk from Churra da Terra Quente sheep in northern Portugal. It is produced by slowly draining the curds obtained after coagulation by the action of animal rennet. No starter cultures are added. This cheese is cylindrical (dish type) with a flat rind, it weighs between 0.8 and 1.2 kg, and it has a semisoft consistency. The concentrations of biogenic amines in Terrincho PDO cheese change significantly during ripening, except for histamine, which was consistently at very low concentrations. Putrescine, cadaverine, and tyramine were the dominant biogenic amines and increased significantly during ripening (Fig. 1). Ethylamine, tryptamine, phenylethylamine, and cystamine reached maxima by 30 days of ripening and decreased thereafter (Fig. 2). During this period, water activity ranged from 0.97 to 0.93. The amines and their corresponding amino acid precursors and microbial potential producers were correlated (88). Cheeses from five dairy farms located throughout the Terrincho cheese PDO region were evaluated after 30 days of ripening (86). Differences were observed in terms of microbiological qualitative and quantitative profiles and types of biogenic amines. The differences in biogenic amines were associated with the different values obtained for the ripening extension and depth indices; biogenic amines were more concentrated when more proteolysis had occurred. The higher ph and the low salt content of some cheeses accelerated amino acid accumulation and hence stimulated amine production. However, during fermentation and ripening, the environmental factors that affect the activity of the decarboxylating enzymes were more important than the availability of the precursor (86). For example, despite the high concentrations of the precursor amino acid tyrosine in some cheeses, tyramine is not found among the biogenic amines. The factor most likely to negatively influence tyramine production in these cheeses was low ph. Tyramine production by Clostridium divergens was lower at ph 4.9 than at ph 5.3, an observation related to reduced cell yield (14). Putrescine was the major biogenic amine in Terrincho cheeses from two dairies, whereas phenylethylamine, tyramine, and cadaverine were the major biogenic amines in cheeses from the other three dairies. High concentrations of putrescine and cadaverine also have been described for other types of cheeses (1). The concentrations of tyramine and especially histamine in Terrincho cheese were lower than those in other traditional cheeses (25, 47, 93, 118). Terrincho cheeses with high concentrations of lactic acid bacteria had high concentrations of tyramine, and Terrincho cheeses with high concentrations of enterococci had high concentrations of phenylethylamine, as found by other authors (9, 49). Terrincho cheeses with high concentrations of enterobacteria and Pseudomonas contain high concentrations of tryptamine, cystamine, putrescine, and cadaverine. Durlu-Özkaya et al. (26) also reported that several enterobacteria were able to produce high concentrations of putrescine and cadaverine in vitro. Thus, biogenic amine concentrations in Terrincho cheeses were closely correlated with the presence of viable microbes in both qualitative and quantitative terms (86). Another Portuguese cheese that has the PDO designation is Azeitão cheese. This cheese is made from raw ovine milk using an extract of cardoon flower (Cynara cardunculus L.) as rennet; it ripens for a minimum of 20 days. The major biogenic amines in Azeitão cheese at 30 days of ripening were tyramine (122 mg/kg of dry matter), cadaverine (181 mg/kg of dry matter), histamine (458 mg/kg of dry matter), and spermine (61 mg/kg of dry matter), a biogenic amine profile different from that of Terrincho cheese (Fig. 3). Storage at room temperature (25 C) promoted a significant increase in the concentration of tyramine and putrescine; these two biogenic amines may serve as indicators of temperature changes in ripened cheese (87). These results support the need for implementation of a well-designed refrigeration scheme throughout transportation, commercial distribution, purchase, and consumer storage of

2296 FERREIRA A PINHO J. Food Prot., Vol. 69, No. 9 TABLE 1. Mean biogenic amine concentration in raw meat from cattle and chickens a Amine concn (mg/kg) Meat origin Tryptamine Putrescine Cadaverine Tyramine Spermidine Spermine Cattle Chickens 18 22 15 35 6 21 3 6 36 15 a Compiled from Vinci and Antonelli (124). Raw meat was maintained for 5 days at 4 C., not detected. FIGURE 3. Comparison of mean concentrations of biogenic amines in Terrincho and Azeitão cheeses after 30 days of ripening. (Compiled from Pinho et al. (87) and Pinho (86).) cheese to guarantee low concentrations of biogenic amines (and therefore decreased risk of health hazards) at the time of consumption. BIOGENIC AMINE CONTENT OF LINGUIÇA, A PORTUGUESE CURED SAUSAGE The fermentation process of sausage manufacturing provides both the microorganisms and the free amino acids required for amine formation and environmental conditions (ph and temperature) favoring bacterial growth and decarboxylase activity. To avoid the formation of hazardous levels of biogenic amines during ripening of dry-cured sausages, the critical factors for biogenic amine formation must be known (1, 11, 12, 14, 26, 29, 44, 69, 91, 92, 95, 116). A review of this topic was recently published by Ruiz- Capillas and Jiménez-Colmenero (94). The type of meat influences the initial concentration of biogenic amines. Table 1 presents concentrations of biogenic amines found in raw chicken and bovine meat that had been stored for 5 days at 40 C (124). The microbial quality of raw materials appears to be one factor affecting amine formation in dry sausage (57, 64, 117). Contaminant lactic acid bacteria have been associated especially with formation of histamine and tyramine in dry-cured sausages (28, 65). Another important way to prevent amine accumulation is control of natural fermentation by addition of amine-negative starter cultures (66, 112). However, other authors reported that the addition of starter culture did not affect formation of biogenic amines (5). Concentrations of biogenic amines in processed meat products differ according to product origin. In general, tyramine is the most abundant biogenic amine, with concentrations of to 560 mg/kg of dry matter, followed by putrescine, histamine, and cadaverine ( to 190 mg/kg of dry matter) (Table 2) (13, 24, 40, 43, 0, 7, 119). Portuguese traditional cured dry meat products are very popular in the Portuguese market. These products include chouriço, linguiça, salpição, and paio, and their production of this regulated by different rules (73 76). However, little information is available on the biogenic amine content of these meat products. Some studies have been performed on linguiça, a traditional cured dry processed meat product made with meat and animal fat (90.3%) plus red wine (7.6%), salt (1.3%), and garlic (0.8%) (76). During manufacture, which involves mixing of the ingredients, fermentation, and ripening to obtain the final product, the proteins and other nitrogenated compounds are transformed, causing the development of desirable sensorial characteristics but also undesirable compounds, such as some biogenic amines. Changes in biogenic amine composition also occur during product storage (115). An HPLC-diode array and fluorescence detection procedure with dansyl derivatization was validated for following biogenic amine formation during the dry-curing process of traditional Portuguese sausage and during storage. The influence of additives (acidifying and antioxidant) on biogenic amine production also was evaluated (52). Biogenic amines were extracted from 2.5 g of cured dry sausage by solid-liquid extraction with 5% tricloroacetic acid and by liquid-liquid extraction with bis-2-ethylhexylphosphate. Derivatization was performed with dansyl chloride in acetone (7.5 mg/ml) for 15 min at 60 C followed by addition of proline (0 mg/ml) for neutralization of derivatizing excess. Derivatives were extracted with toluene. After being dried, samples were dissolved in acetonitrile and then used for HPLC injection (52). The total concentration of amines increased during the manufacturing of traditional linguiça (from 0 to 440 mg/ kg of dry matter) and then decreased slightly during storage (380 mg/kg of dry matter). The decrease during storage also was observed by Bover-Cid et al. () in dry-cured meat products during 20 days of storage at 4 C. During product manufacture, water activity ranged from 0.97 to 0.92. The addition of additives with acidifying and/or antioxidant properties was not advantageous; the samples that contained these additives had high concentrations of amines (up to 460 mg/kg of dry matter) during manufacture, which decreased (to 400 mg/kg of dry matter) during storage (52). The dominant amines were the same as those found in the Terrincho PDO cheeses, i.e., cadaverine, putrescine, and tyramine, and their concentrations increased during manufacture to 29.2, 146.6, and 165.7 mg/kg of dry matter, respectively, in the final product (52) (Table 3). Similar concentrations have been reported for other types of cured sausages (29, 4). Histamine concentrations were also high (95 mg/kg of dry matter). Paulson and Bauer (81) found similar concentrations of histamine in sausages, but in gen-

J. Food Prot., Vol. 69, No. 9 BIOGENIC AMINES IN PORTUGUESE FOODS 2297 TABLE 2. Mean biogenic amine concentration in dry-cured meat products from different countries Amine concn (mg/kg) Product origin Tryptamine -Phenylethylamine Putrescine Cadaverine Histamine Tyramine Spermidine Spermine Reference(s) Belgium France Spain Chourizo Salchichón The Netherlands Norway Czech Republic Egypt a 150 190 20 170 40 140 370 560 119 119 16 9 1 7 60 3 20 12 18 7 282 280 4 5 26 15 43 43 5 13 50 1 39 60 30 19 2 30 8 1 0 14 3 5 6 20 30 3 13, 24 40 7 0 a, not detected. eral lower concentrations have been reported. A different trend was observed for spermidine and spermine, which decreased during fermentation from 1.7 to 1.5 mg/kg of dry matter and from 25 to 18.69 mg/kg of dry matter, respectively. TABLE 3. Biogenic amine concentrations in Portuguese traditional linguiça a Biogenic amine Tryptamine -Phenylethylamine Putrescine Cadaverine Histamine Tyramine Spermidine Spermine a From Judas (52). Concn (mg/kg of dry matter) 14.5 5.69 146.6 29.23 94.59 165.7 1.50 18.69 BIOGENIC AMINE CONTENT OF PORTUGUESE WINES The first reference to the presence of biogenic amines in wines was by Tarantola in 1954 (111). Since then, the problem has become a major concern for many researchers and international wine organizations, as reflected in the many published studies (15 18, 20 23, 79, 80, 89, 97, 6, 122, 123, 126). The amounts and composition of biogenic amines depend on the type and degree of ripeness of the grapes, the climate and soil of the viticultural area, and the vinification techniques. The biogenic amines usually found in wines are agmatine, spermine, spermidine, putrescine, cadaverine, histamine, and tyramine. Recently, several studies on biogenic amines in Portuguese wines have been conducted (Table 4). The biogenic amine concentrations of 30 Portuguese wines, including fortified wines Porto, Madeira, and Moscatel de Setúbal, red wine Dão, and white wine Vinho Verde were evaluated (63). The o-phthaldialdehyde fluorescent derivatives of amines (histamine, tyramine, phenylethylamine, tryptamine, putrescine, cadaverine, ethylamine, methylamine, isoamylamine, and ethanolamine) were separated by reverse-phase HPLC. The amines suspected to have toxicological effects (histamine, tyramine, and -phenylethylamine) are not of concern because their concentrations did not exceed 5 mg/liter. The concentrations of amines associated with deficient sanitary conditions (putrescine and cadaverine) were low, between 0.2 and 0.6 mg/liter in the analyzed samples. Tryptamine was not detected. Another HPLC method using derivatives that fluoresced with o-phthaldialdehyde 3-mercaptopropionic acid 9-fluorenylmethyl chloroformate chloride was developed and validated to study free amino acids and biogenic amines in musts and wines (42). Thirty-three wine and 39 must samples were analyzed. Each sample was analyzed in 138 min, and each analysis involved quantification of 23 amino acids and amines. In wines, ethanolamine, histamine, ethylamine, and putrescine together represented 93% of the amine content. The same procedure was applied to characterize free amino acids and biogenic amines in musts and wines from southern Portugal (Alentejo) (42). Characterization of 200 samples of musts and wines from Alentejo subregions, varieties, and vintages was performed (41). The generation of volatile and biogenic amines also was studied throughout microvinifications. During alcoholic or malolactic fermentation (which occurred in all red wines), no significant increase was noticed in the concentrations of total volatile amines. The concentration of histamine did not vary greatly during alcoholic fermentation, staying close to that initially found in musts (average 1.2 mg/liter for red musts). Although higher histamine concentrations were found only during the storage period, an increase in tyramine concentration was verified immediately after malolactic fermentation. In the red wines, malolactic fermentation seems to be the main source of tyramine and putrescine. Grape variety, region of production, and vintage can affect free amino acid and amine contents in both musts and wines, although alcoholic and malolactic fermentation can overcome that influence. The varieties with the highest final concentrations of biogenic amines also had the highest free amino acid concentrations (41).

2298 FERREIRA A PINHO J. Food Prot., Vol. 69, No. 9 TABLE 4. Evaluation of biogenic amines in Portuguese wines Type of wine Region of origin Method, derivatization, detector a Biogenic amines Reference Port wine Madeira wine Red and white wine Red wine White wine Monovarietal musts at different stages of fermentation Monovarietal wines Port wines Grade juices Port wines Grape juices Monovarietal musts Port wines recently prepared Tawny and vintage port wines Douro Madeira Island Dão, Vinho verde Douro, Bairrada, Dão, Beira, Estremadura, Ribatejo, Alentejo Alentejo (Evora, Portalegre, Borba, Reguengos, Redondo, Vidigueira) HPLC, OPA, fluorometric detector HPLC, OPA and mercaptoethanol, fluorometric detector HPLC, OPA and FMOC, fluorometric detector Douro GC, heptafluorobutyric anhydride, mass detector Douro GC, pentafluorobenzyl bromide, mass detector Douro GC, isobutyl chloroformate, mass detector Histamine, tyramine, -phenylethylamine, trypatamine, putrescine, cadaverine, ethylamine, methylamine, isoamylamine, ethanolamine Mafra et al. (63) Putrescine, tyramine, cadaverine, histamine Leitão et al. (60) Ethanolamine, methylamine, ethylamine, histamine, tyramine, -phenylethylamine, tryptamine, isoamylamine, cadaverine, putrescine -Phenylethylamine, tyramine, 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine Herbert et al. (41) and Herbert et al. (42) Fernandes and Ferreira (33) and Fernandes (32) Histamine Fernandes and Ferreira (34) and Fernandes (32) Methylamine, dimethylamine, ethylamine, isopropylamine, diethylamine, propylamine, butylamine, 2-methylbutylamine, isoamylamine, pirrolidine, morfoline, amylamine, piperidine, hexylamine, -phenylethylamine, diaminopropane, putrescine, cadaverine, 1-6 diaminohexane a OPA, o-phthaldialdehyde; FMOC, 9-fluorenylmethyl-chloroformate.

J. Food Prot., Vol. 69, No. 9 BIOGENIC AMINES IN PORTUGUESE FOODS 2299 The putrescine, tyramine, cadaverine and histamine composition of about 300 commercial Portuguese wines originating from different regions from north to south during a -year period was evaluated (60) using HPLC with fluorometric detection by derivatives that fluoresced with o- phthaldialdehyde 2-mercaptoethanol. The concentrations of biogenic amines were higher in red wines than in white wines, and this difference can be explained by the differences in the vinification techniques. Most commercial Portuguese wines had low concentrations of biogenic amines. Only 28% of the wines analyzed had concentrations higher than 8 mg/liter, and 25% had concentrations lower than 2.5 mg/liter. The concentrations of biogenic amines in white wines never exceeded 17 mg/liter, although in red wines concentrations could reach 28 mg/liter. In the last few years, putrescine, tyramine, and histamine concentrations increased slightly in red wines (60). Most of the commercial Portuguese white wines appear to be safe (6); only 6.5% of the wines analyzed had concentrations of tyramine and histamine that were higher than 8 mg/liter. For red wines, 45% had concentrations of tyramine and histamine higher than 8 mg/liter, which means that according to this work some of these wines could be dangerous to human health. This study included more samples than did the previous studies. Biogenic amines are also present in Port wine, a fortified dessert wine of great economical significance for Portugal, in relatively low quantities compared with other food products. Biogenic amine quantification by GC mass spectrometry (MS) is specific and sensitive. Different methods based on ion-pair extraction and using derivatization reagents such as heptafluorobutyric anhydride, pentafluorobenzyl bromide, and isobutyl chloroformate were applied to study the origin and evolution of these compounds in Port wines. Three different analytical methods were therefore developed and improved, based on the use of the GC- MS technique, which enabled correct quantification of the biogenic amines of interest in musts and Port wines (32). The first of those methods consists of using a previous ion-pair extraction of the amines, followed by derivatization of compounds with heptafluorobutyric anhydride. This method was used for determining aromatic amines, diamines, and polyamines, all of them characterized by their lower volatility. The amines were extracted from previously dealcoholized and buffered samples by ion-pairing reagent bis-2-ethylhexylphosphate dissolved in chloroform, followed by a back-extraction with 0.1 M HCl, and then converted to the corresponding heptafluorobutyric derivatives. GC electron impact MS in selected ion-monitoring mode was the last step in the analysis (33). The second method is based on the same ion-pair extraction procedure, followed by a derivatization with pentafluorobenzyl bromide for the determination of histamine (34). This method includes isolation of histamine and its deuterated analogue [,,, - 2 H 4 ]histamine, used as the labeled internal standard, by ion-pair extraction with bis-2- ethylhexylphosphate dissolved in chloroform, and the corresponding volatile derivatives are formed via reaction with pentafluorobenzyl bromide. The resulting derivative trispentafluorobenzyl-histamine was used for capillary GC separation and measurement of the abundance ratio of the (M- 181) ions from labeled and unlabeled derivatives (34). The third method consists of a two-phase derivatization procedure with isobutyl chloroformate directly applied to the samples. It was used in the determination of all studied amines, both volatile and nonvolatile, except for histamine and the polyamines (32). In all cases, an accurate selection of the best internal standards for several classes of amines was based on the use of deuterated isotopic analogues as much as possible. Quantification was achieved in the selected ion-monitoring mode to take advantage of the improved sensitivity provided by the analytical equipment. The three developed methods were applied in the study of the biogenic amine contents of samples of monovarietal musts, young Port wines, and aged tawny and vintage ports. A total of 17 amines were quantified. The results obtained allowed better characterization of musts and Port wines of the five most representative varieties, Touriga Nacional, Touriga Francesa, Tinta Roriz, Tinta Barroca, and Tinto Cão, which have lower concentrations of amines than do other types of wines (32). A comparison of biogenic amines for wines recently prepared and of changes in amine composition during Port wine aging, both in the bottle (reducing environment) and in wood (oxidant environment), was also performed. Total biogenic amine concentration for musts of the five most representative varieties ranged between 4.505 and 19.061 mg/liter, with a mean value of 8.676 mg/liter. Putrescine (mean, 2.909 mg/liter), spermidine (2.338 mg/liter), and ethylamine (1.453 mg/liter) were the most abundant biogenic amines in musts. These three amines accounted for 77.2% of the total biogenic amine concentration. Spermine mean concentration in musts was 0.527 mg/liter and cadaverine mean concentration was 0.197 mg/liter (32). Total biogenic amine concentration for samples of the five types of monovarietal Port wines (from 1995 to 1998) ranged between 3.976 and 17.470 mg/liter, with a mean value of.472 mg/liter. Spermine and spermidine were not detected in Port wine samples because these amines are used as a nitrogen source by microorganisms during fermentation. Ethylamine, isoamylamine, pirrolidine, and phenylethylamine increased in the wines, and putrescine, cadaverine, tyramine, and histamine increased slightly. However, histamine and tyramine concentrations were less than 0.5 mg/liter. During Port wine aging, a decrease in biogenic amine concentrations was observed, especially for methylamine, ethylamine, putrescine, and cadaverine. Pirrolidine increased during aging; this amine results from decarboxylation of proline and can be used to determine Port wine age (32). DISCUSSION The intake of harmful amounts of biogenic amines from traditional Portuguese fermented foods is possible. PDO cheeses and traditional cured dry meat products and red wines can contribute significantly to daily intake of

2300 FERREIRA A PINHO J. Food Prot., Vol. 69, No. 9 these compounds. Thus, in addition to basic foodstuffs generally consumed everywhere, typical Portuguese foods and drinks should be taken into account during calculation of Portuguese consumer intake of biogenic amines, because these products are commonly consumed. Little information is available concerning the exposure of the Portuguese consumer to biogenic amines and, consequently, the real risk associated with the Portuguese diet. This information is essential for proposing modifications in the food chain production system that will lead to a reduction of the concentration of biogenic amines. The conditions that support growth and activity of certain groups of microorganisms directly involved in proteolytic degradation and/or biogenic amine synthesis are associated with variations in the hygienic quality of raw materials and with different handling and manufacturing practices. Reliable analytical procedures based on HPLC and GC methods have been validated and applied to determine biogenic amine concentrations in different food matrices. In the same PDO cheeses, dairy-to-dairy differences can be observed, mainly in terms of microbiological profiles and/ or profiles of proteolysis products and, consequently, biogenic amines. Such differences could be related to differences in the hygienic quality of raw materials and in handling and cheese-making practices. Little information is available concerning biogenic amines in Portuguese cured dry meat products. In general, low levels of biogenic amines were found in Portuguese wines. However, because alcohol increases the permeability of the mucosa, biogenic amines from alcoholic beverages or from food consumed simultaneously with alcohol can be transferred into the blood stream without being metabolized. More studies are needed on the composition of biogenic amines in Portuguese traditional foods and beverages. 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