- Email: [email protected]
Monitoring of biogenic amines in cheeses manufactured at small-scale farms and in fermented dairy products in the Czech Republic
Food Chemistry 141 (2013) 548–551
Contents lists available at SciVerse ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Monitoring of biogenic amines in cheeses manufactured at small-scale farms and in fermented dairy products in the Czech Republic Leona Bunˇková a,⇑, Gabriela Adamcová b, Katerˇina Hudcová c, Helena Velichová b, Vendula Pachlová c, Eva Lorencová a, František Bunˇka c a b c
Department of Environmental Protection Engineering, Faculty of Technology, Tomas Bata University in Zlín, nám. T.G. Masaryka 275, 76272 Zlín, Czech Republic Department of Food Analysis and Chemistry, Faculty of Technology, Tomas Bata University in Zlín, nám. T.G. Masaryka 275, 76272 Zlín, Czech Republic Department of Food Technology and Microbiology, Faculty of Technology, Tomas Bata University in Zlín, nám. T.G. Masaryka 275, 76272 Zlín, Czech Republic
a r t i c l e
i n f o
Article history: Received 5 November 2012 Received in revised form 28 January 2013 Accepted 11 March 2013 Available online 20 March 2013 Keywords: Cheese Fermented dairy products Biogenic amines Health risk Small-scale farm
a b s t r a c t The aim of the study was the monitoring of six biogenic amines (histamine, tyramine, phenylethylamine, tryptamine, putrescine, and cadaverine) and two polyamines (spermidine and spermine) in 112 samples of dairy products purchased in the Czech Republic, namely in 55 cheeses made in small-scale farms and in 57 fermented dairy products. The products were tested at the end of their shelf-life period. Neither tryptamine nor phenylethylamine was detected in the monitored samples; histamine was found only in four cheese samples containing up to 25 mg/kg. The contents of spermine and spermidine were low and did not exceed the values of 35 mg/kg. Significant amounts of tyramine, putrescine, and cadaverine occurred especially in cheeses produced from ewe’s milk or in long-term ripened cheeses. In about 10% of the tested cheeses, the total concentration of all the monitored biogenic amines and polyamines exceeded the level of 200 mg/kg, which can be considered toxicologically significant. In fermented dairy products, the tested biogenic amines occurred in relatively low amounts (generally up to 30 mg/kg) that are regarded safe for the consumer’s health. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Biogenic amines (BA) are low-molecular nitrogen containing substances known for their characteristic physiological effects. Some BA act as hormones, but many BA are regarded as neurotransmitters or precursors for the synthesis of hormones, alkaloids or other metabolites (Halász, Baráth, Simon-Sarkadi, & Holzapfel, 1994; Shalaby, 1996; Silla Santos, 1996). Most commonly, biogenic amines are formed by decarboxylation of amino acids or by amination or transamination of aldehydes and ketones. Microorganisms demonstrating decarboxylase activity can get into foods spontaneously or they might be contained in starter cultures added intentionally to food (Ladero et al., 2012; Silla Santos, 1996). Common levels of BA in foodstuffs and beverages (<100 mg/kg), do not represent a serious risk for healthy consumers, because they are metabolised by detoxication enzymes in human intestines. Higher BA amounts can induce undesirable psychoactive and vasoactive effects. Histamine, tyramine, and phenylethylamine might bring about the above adverse effects directly. Putrescine and cadaverine do not show direct toxic effects; however, they can intensify toxic effects of other amines (Halász et al., 1994; Silla ⇑ Corresponding author. Tel.: +420 576 031 020; fax: +420 577 210 172. ˇ ková). E-mail address: [email protected] (L. Bun 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.03.036
Santos, 1996). If the intestinal detoxication system collapses, because of excessive amount of amines consumed in food or due to its weakening by inhibitors of detoxication enzymes, BA can get into the blood circulation system and may potentially precipitate many undesirable effects (Spano et al., 2010). Most of the products fermented by lactic acid bacteria (LAB), contain traces of histamine, tyramine, putrescine, and cadaverine. Regarding the occurrence of BA, cheeses are the most important and the most commonly monitored dairy products. The amounts of BA in milk, yoghurts, curd cheese and unripened cheeses can be expected to range between milligrammes and tens of milligrammes per kg (Linares, Martín, Ladero, Alvarez, & Fernández, 2011; Spano et al., 2010). Recently, studies where products from small-scale farms in south European countries like Italy, Portugal and Greece were characterised, have been published, however, no results of an investigation on relevant products manufactured in central Europe have been reported. Recently, in the Czech Republic, farmer markets have been gaining in popularity. Many Czechs have chosen the healthy life-style inspired by nature, which directs them to the consumption of food produced in small-scale farms. A complex study on the occurrence of BA in products manufactured in the above farms, has not been published yet. The aim of this study, was to monitor contents of eight BA and polyamines (PA) (histamine, tyramine, 2-phenylethylamine,
L. Bunˇková et al. / Food Chemistry 141 (2013) 548–551
tryptamine, putrescine, cadaverine, spermidine, and spermine) in cheese samples produced in small-scale farms and in selected fermented dairy products at the end of their shelf-life period. The above products were purchased in common Czech retail and/or, in farm markets (including purchase directly in farms).
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pasteurised cow’s milk; and (v) ripened cheeses made from pasteurised cow’s milk (Table 1). Besides the above cheeses, 55 fermented dairy products purchased in common Czech and Slovak retail network shops and two fermented dairy products manufactured at small-scale farms based in east Moravia (Czech Republic) were analysed. The following fermented products were monitored: (i) 29 yoghurts (one cream yoghurt was produced at a farm); (ii) 14 fermented creams; (iii) 4 acidified milks – milks fermented with an acidophilic culture; (iv) 4 kefir milks; (v) 2 fermented buttermilks; and (vi) 4 fermented milks – milks fermented with mesophilic cultures (one made at a farm) (Table 2). None of the monitored fermented products contained flavouring. The cheese samples and fermented milk products were kept at cold store temperatures of 6 ± 2 °C. They were analysed at the end of their shelf-life period.
2. Materials and methods 2.1. Cheese samples and fermented dairy products Altogether, 55 cheeses manufactured in small-scale farms in the central Europe region (the Czech Republic and the Slovak Republic) were purchased in 2012 (April to July). All of the goods were regional products purchased in farmer markets, in retail network shops or directly in some farms. The purchased cheeses were assorted into the following categories: (i) bryndza cheeses and cheeses made from raw ewe’s milk; (ii) cheeses and curd cheeses manufactured from pasteurised ewe’s milk; (iii) cheeses produced from goat’s milk (all except one, were manufactured from pasteurised milk); (iv) fresh cheeses and curd cheeses produced from
2.2. Determination of biogenic amines and polyamine contents If necessary, all the collected samples were homogenised, and subsequently lyophilised (Christ Alpha 1–4 device). The amines
Table 1 Content of biogenic amines in mg/kg in tested cheeses produced at small-scale farms. Cheese
a b
Number of samples
Biogenic amines (mg/kg)a Total BA
Tyramine
Histamine
Putrescine
Cadaverine
Spermine
4 3 2
73.2–222.2 25.1–177.1 NDb–40.3
4/34.6–107.4 3/8.9–38.3 ND
1/24.2 ± 1.1 NDb ND
2/22.1–60.9 3/16.2–99.9 1/20.7 ± 1.3
3/16.5–42.6 2/62.6–80.7 1/19.6 ± 1.4
1/9.7 ± 0.8 ND ND
Pasteurised ewe’s milk cheeses Unripened (fresh) cheese Pasta filata type cheese Brined cheese Flavoured cheese
3 2 2 2
ND–140.3 ND–13.2 37.2–529.8 ND–223.5
2/10.2–11.1 ND 2/23.1–174.6 1/114.7 ± 8.0
ND ND ND ND
2/55.3–118.2 ND 1/229.5 ± 20.0 1/108.8 ± 7.0
2/11.4–35.8 ND 1/125.6 ± 3.0 ND
ND 1/13.2 ± 1.1 1/14.0 ± 1.1 ND
Goat’s milk cheeses Ripened cheese (raw milk without heat treatment) Unripened (fresh) cheese (unflavoured) Unripened (fresh) cheese (flavoured) Pasta filata type cheese (plant)
1 3 6 3
356.1 ND–32.7 ND–10.7 ND–89.9
1/207.1 ± 7.5 1/11.3 ± 1.0 1/10.7 ± 0.5 1/8.5 ± 0.6
ND ND ND ND
ND ND ND 1/41.1 ± 3.3
1/149.0 ± 7.3 ND ND 1/40.3 ± 2.5
ND ND ND ND
Fresh cow’s milk cheeses Unripened (fresh) cheese (unflavoured) Unripened (fresh) cheese (flavoured)
6 4
ND–161.9 19.4–45.1
2/8.3–15.2 3/7.2–22.7
ND ND
1/111.4 ± 8.6 ND
3/8.9 ± 21.7 2/7.0–22.4
4/11.4–17.9 3/11.8–15.5
Other cow’s milk Ripened cheese Pasta filata type Pasta filata type Pasta filata type
3 5 3 3
137.9–173.4 ND–49.9 12.0–25.6 9.2–39.3
2/63.8–101.4 1/25.8 ± 1.5 ND ND
ND 1/19.3 ± 0.6 ND 2/18.3–18.8
2/48.8–70.1 1/37.3 ± 1.1 2/12.2–14.7 ND
3/11.8–72.6 ND ND 1/10.7 ± 0.9
3/11.5–97.9 2/7.1–12.7 3/10.9–12.0 2/9.2–9.8
Raw ewe’s milk cheeses Bryndza Smoked cheese Fresh cheese
cheeses cheese (unflavoured) cheese (flavoured) cheese (unflavoured, smoked)
Individual biogenic amines – number of BA positive samples/range of detected BA amount. ND – biogenic amines not detected.
Table 2 Content of biogenic amines in mg/kg in fermented dairy products.
a b
Product
Number of samples
Yoghurt (fat content 3–10%) Yoghurt (fat content 10–11%) Yoghurt (fat content 2–3%) Yoghurt (fat content 0.1%) Fermented cream Acidified milk Fermented milk Fermented buttermilk Kefir milk
17 4 3 5 14 4 4 2 4
Biogenic amines (mg/kg)a Total BA
Tyramine
Putrescine
Cadaverine
0.5–29.4 NDb–26.6 1.1–13.3 1.5–10.5 5.5–15.4 2.9–7.5 3.2–6.2 4.6–5.0 3.4–15.3
17/0.3–6.3 3/0.5–2.7 3/1.1–1.7 4/0.3–2.0 12/5.5–15.4 3/2.6–7.5 3/3.2–6.2 2/4.6–5.0 3/0.8–4.0
8/1.9–25.1 2/3.2–26.1 1/1.4 ± 0.2 3/5.0–10.5 2/8.6–8.8 1/4.1 ± 0.3 ND ND 3/3.4–14.3
NDb ND 1/4.3 ± 0.3 ND ND ND ND ND ND
Individual biogenic amines – number of BA positive samples/range of detected BA amount. ND – biogenic amines not detected.
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were extracted with a perchloric acid solution (0.6 M) in triplicate. The content of eight amines was determined by liquid chromatography (LabAlliance and Agilent Technologies devices) after previous derivatisation with dansylchloride. Derivatisation, chromatographic separation (column used: ZORBAX Eclipse XDBC18, 150 4.6 mm, 3.5 lm, Agilent Technologies) and spectrophotometric detection (k = 254 nm) were performed according to Dadáková, Krˇízˇek, and Pelikánová (2009). Each sample was extracted in triplicate, each extract was derivatised in duplicate and each derivatised mixture was applied on the column in triplicate (n = 18). In individual amines, the detection limits ranged between 0.24 and 1.39 mg/kg. The results were related to the sample weight before lyophilisation. For a comparison purpose, the Kruskall–Wallis and Wilcoxon tests were applied (Unistat 5.5 statistical software; Unistat Ltd., London, UK).
3. Results 3.1. Biogenic amines in cheeses produced at farms BAs and/or PAs were detected in 40 cheeses produced at farms. No phenylethylamine or tryptamine was detected in the tested cheeses. Histamine occurrence was also insignificant and it was detected in four cheeses: in one bryndza manufactured from unpasteurised milk and in three ripened and pasteurised cow’s cheeses (18.3–24.2 mg/kg; Table 1). In the cheeses analysed, tyramine was found to be the most commonly detected BA. Twenty-four cheese samples contained between 7.2 and 207 mg/kg of tyramine. Six cheeses contained more than 100 mg/kg. Tyramine was detected in most of the cheeses produced from either pasteurised or unpasteurised ewe’s milk. Twenty products contained between 7.0 and 149.0 mg/kg of cadaverine. Cadaverine occurred mainly in ewe’s milk products, primarily in unpasteurised cheeses (P < 0.05). In cow’s milk cheeses, the fresh products showed higher contents of cadaverine. Putrescine was found in 17 cheeses at the levels of 12.2– 230 mg/kg. Four cheeses included over 100 mg/kg (Table 1). Putrescine was detected in more than half of the ewe’s milk cheeses and in one brine cheese (230 mg/kg). Putrescine was found in one quarter of cow’s milk products, primarily in ripened cheeses (P < 0.05). Nineteen cheeses contained spermine (Table 1). Spermine was detected in two thirds of cheeses made from cow’s milk. Low spermine levels were determined in ewe’s milk cheeses (P < 0.05). Most of the BA or PA containing cheeses demonstrated the presence of more than one amine. In 20% of the cheeses tested, the total amount of BA and PA did not exceed 20 mg/kg; 9% of the cheeses studied contained more than 200 mg/kg. The highest BA and PA amount (530 mg/kg) was found in a brined cheese made from pasteurised ewe’s milk. Ewe’s milk cheeses made from both pasteurised and unpasteurised milk contained generally more BA than those produced from goat’s or cow’s milk. Altogether, eight ewe’s milk cheese samples included more than 100 mg/kg of total BA; however, only one unpasteurised goat’s milk cheese and only four pasteurised cow’s milk cheeses (where only three were ripened cheeses) contained that high amounts of BA. 3.2. Biogenic amines in fermented dairy products Except for one sample, all the tested non-flavoured fermented dairy products contained BA within the range of 0.5–29.4 mg/kg
(Table 2). These included four biogenic amines – tyramine, putrescine, cadaverine, and spermine. Tyramine was the most common BA found in fermented dairy products; its presence was proved in 50 tested samples. Higher amounts of BA were solely found in fermented creams. Putrescine was quite a commonly detected BA found in 20 tested samples (Table 2). Half of the putrescine-positive samples contained up to 10 mg/kg, two yoghurt samples were found to contain slightly more than 25 mg/kg. The total amount of BA in fermented dairy products was lower than 30 mg/kg (Table 2). Ten of the tested products showed BA levels ranging between 10 and 20 mg/kg and only three yoghurts included more than 20 mg/kg. 4. Discussion Approximately 80% of the dairy products tested showed the presence of BA. These are normally present in the product ingredients; namely, spermine and spermidine are found in fresh milk (Linares et al., 2011; Spano et al., 2010). Biogenic amines can also be produced by both starter and non-starter LAB, that are used in the manufacture of these products or that can get into products during their processing, respectively. The ability of many LAB repˇ ková resentatives to produce BA has been widely reported (Bun et al., 2009; Ladero et al., 2012; Linares et al., 2011). Biogenic amines can also be produced in contaminated microflora, especially by the Enterobacteriaceae and other Gram-negative bacteria (Coton et al., 2012). In comparison, ripened cheeses showed higher BA and PA levels than the fresh ones, which can be explained by the fact that their microflora did not have enough time to produce higher amounts of BA and PA. In ripened cheeses, the concentration of BA and PA ˇ ková et al., 2010). On the usually rises gradually with time (Bun other hand, some of the shorter-time ripening cheeses produced in farms contained over 200 mg/kg of BA and PA. Higher amounts of BA could result from the activity of non-starter LAB or contaminated microflora. The levels of BA were also monitored in cheeses made from either pasteurised or unpasteurised milk. No significant differences in the amounts of BA in ewe’s milk cheeses, manufactured from raw or pasteurised milk were found. More significant differences were detected in cheeses produced from goat’s milk; cheeses produced from raw milk contained higher concentrations of BA (P < 0.05). Similar results were reported by Novella-Rodríguez, Veciana-Nogués, Roig-Sagués, Trujillo-Mesa, and Vidal-Carou (2004), who accounted the above finding, to the higher amount of microorganisms in cheeses made from raw milk. The fermented dairy products were expected to contain lower amounts of BA than cheeses. Unlike cheeses, fermented dairy products are not exposed to an environment suitable for growth of microflora, especially of Gram-negative bacteria that could produce BA (e.g. Enterobacteria, Pseudomonas prefer a higher pH than the pH of fermented dairy products). Concerning fermented products, some researchers reported low amounts of BA usually not exceeding the levels of 15 mg/kg (Chaves-López et al., 2011; Linares et al., 2011). The total amount of BA in some samples reached the amount of 29.4 mg/kg. Corresponding contents of BA were also detected by Özdestan and Üren (2010) in kefir and by Magwamba, Matsheka, Mpuchane, and Gashe (2010) in fermented milk. Regarding the impact of BA on human health, histamine and tyramine show the most adverse effects (Shalaby, 1996). Histamine was not detected in any of the studied 57 samples of fermented dairy products, but it was found in four cheeses, with
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amounts lower than 25 mg/kg. Similar levels of histamine were detected in ewe’s milk cheeses produced at farms in Portugal by Pintado et al. (2008) and in Italy by Schirone, Tofalo, Mazzone, Corsetti, and Suzzi (2011). On the contrary, Ladero, Linares, Fernández, and Alvarez (2008) reported substantially higher amounts of histamine (up to 1040 mg/kg) in cheeses produced from raw and pasteurised cow’s, goat’s, and ewe’s milk. Andiç, Gençcelep, and Köse (2010) found 680 mg/kg of histamine in cheeses which contained herbs. Ripened cheeses produced from ewe’s milk revealed substantially higher concentrations of tyramine (up to1200 mg/kg) (Andiç et al. 2010; Schirone et al., 2011). Comparing the content of BA, the tested fermented dairy products contained substantially lower amounts of BA (practically < 30 mg/kg) than the other dairy products such as ripened cheeses (Linares et al., 2011; Schirone et al., 2011). This difference is probably caused by various conditions in the manufacturing process or storage and ripening. The fermented dairy products are processed at temperatures suitable for bacteria propagation and BA formation takes only a couple of hours. Moreover, their shelf-life and thereby also period of BA generation is relatively short and it usually does not exceed the span of 3–4 weeks. Determination of the toxic level of total BA in foods is difficult due to many various factors acting in its specification. Some amines (e.g. putrescine and cadaverine) can potentiate toxic effects of other BA (especially histamine and tyramine). Researchers report values within quite a wide range of 200–800 mg of BA per one kilogramme of food (Halász et al., 1994; Silla Santos, 1996; Spano et al., 2010). Considering the lower limit of 200 mg/kg, about 10% of cheeses produced at small-scale farms contained BA amounts that can lead to health damage even in healthy consumers. Evaluating the risks of foodstuffs to consumer’s health, the safe daily BA intake should be regarded as complex. Some substances such as alcohol, can decrease the activity of enzymes that participate in degradation of BA in human intestines (Shalaby, 1996). Since cheeses are often served with alcoholic drinks such as beer or wine, even low concentrations of BA in cheeses can cause adverse effects. Beer and wine, often contain high amounts of BA, which might intensify the negative impact of BA in cheeses on human health (Ancín-Azpilicueta, González-Marco, & Jiménezˇ ka et al., 2012). Moreno, 2008; Bun The results of this study, show that BA and PA occur in cheeses quite commonly, and this might represent a serious risk to our health. As the safe levels of BA and PA in foods and beverages have not been specified by any regulation, these secondary metabolites have not been either monitored or evaluated by any control body, which might threaten the consumer’s health. Acknowledgements This work was supported by The National Agency for Agriculture Research, Project No. QJ1210300, The Complex Sustainable Systems Programme and from the Internal Grant of Tomas Bata University in Zlín (No. IGA/FT/2013/013).
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References Ancín-Azpilicueta, C., González-Marco, A., & Jiménez-Moreno, N. (2008). Current knowledge about the presence of amines in wine. Critical Reviews in Food Science and Nutrition, 48, 257–275. Andiç, S., Gençcelep, H., & Köse, Sß. (2010). Determination of biogenic amines in herby cheese. International Journal of Food Properties, 13, 1300–1314. ˇ echová, M., Drienovsky´, V., Pachlová, V., Matoulková, D., ˇ ka, F., Budinsky´, P., C Bun ˇ , V., & Bun ˇ ková, L. (2012). Content of biogenic amines and polyamines in Kubán beers from the Czech Republic. Journal of the Institute of Brewing, 118, 213–216. ˇ ková, L., Bun ˇ ka, F., Hlobilová, M., Vanˇátková, Z., Nováková, D., & Dráb, V. (2009). Bun Tyramine production of technological important strains of Lactobacillus, Lactococcus and Streptococcus. European Food Research and Technology, 229, 533–538. ˇ ablová, A., Sedlácˇek, I., Švec, P., Pachlová, V., & ˇ ková, L., Bun ˇ ka, F., Mantlová, G., C Bun Krácˇmar, S. (2010). The effect of ripening and storage conditions on the distribution of tyramine, putrescine and cadaverine in Edam-cheese. Food Microbiology, 27, 880–888. Chaves-López, C., Serio, A., Martuscelli, M., Paparella, A., Osorio-Cadavid, E., & Suzzi, G. (2011). Microbiological characteristics of kumis, a traditional fermented Colombian milk, with particular emphasis on enterococci population. Food Microbiology, 28, 1041–1047. Coton, M., Delbés-Paus, C., Irlinger, F., Desmasures, N., Le Fleche, A., Stahl, V., Montel, M.-C., & Coton, E. (2012). Diversity and assessment of potential risk factors of Gram-negative isolates associated with French cheeses. Food Microbiology, 29, 88–98. Dadáková, E., Krˇízˇek, P., & Pelikánová, T. (2009). Determination of biogenic amines in foods using ultra-performance liquid chromatography (UPLC). Food Chemistry, 116, 365–370. Halász, A., Baráth, Á., Simon-Sarkadi, L., & Holzapfel, W. (1994). Biogenic amines and their production by microorganisms in food. Trends in Food Science and Technology, 5, 42–49. Ladero, V., Cañedo, E., Pérez, M., Martín, M. C., Fernández, M., & Alvarez, M. A. (2012). Multiplex qPCR for the detection and quantification of putrescineproducing lactic acid bacteria in dairy products. Food Control, 27, 307–313. Ladero, V., Linares, D. M., Fernández, M., & Alvarez, M. A. (2008). Real time quantitative PCR detection of histamine-producing lactic acid bacteria in cheese: Relation with histamine content. Food Research International, 41, 1015–1019. Linares, D. M., Martín, M. C., Ladero, V., Alvarez, M. A., & Fernández, M. (2011). Biogenic amines in dairy products. Critical Reviews in Food Science and Nutrition, 51, 691–703. Magwamba, C., Matsheka, M. I., Mpuchane, S., & Gashe, B. A. (2010). Detection and quantification of biogenic amines in fermented food products sold in Botswana. Journal of Food Protection, 73, 1703–1708. Novella-Rodríguez, S., Veciana-Nogués, M. T., Roig-Sagués, A. X., Trujillo-Mesa, A. J., & Vidal-Carou, M. C. (2004). Evaluation of biogenic amines and microbial counts throughout the ripening of goat cheeses from pasteurized and raw milk. Journal of Dairy Research, 71, 245–252. Özdestan, Ö., & Üren, A. (2010). Biogenic amine content in kefir: A fermented dairy product. European Food Research and Technology, 231, 101–107. Pintado, A. I. E., Pinho, O., Ferreira, I. M. L. V. O., Pintado, M. M. D., Gomes, A. M. P., & Malcata, F. X. (2008). Microbiological, biochemical and biogenic amine profiles of Terrincho cheese manufactured in several dairy farms. International Dairy Journal, 18, 631–640. Schirone, M., Tofalo, R., Mazzone, G., Corsetti, A., & Suzzi, G. (2011). Biogenic amine content and microbiological profile of Pecorino di Farindola cheese. Food Microbiology, 28, 128–136. Shalaby, A. R. (1996). Significance of biogenic amines to food safety and human health. Food Research International, 29, 675–690. Silla Santos, M. H. (1996). Biogenic amines: Their importance in food. International Journal of Food Microbiology, 29, 213–231. Spano, G., Russso, P., Lonvaud-Funel, A., Lucas, P., Alexandre, H., Grandvalet, C., Coton, E., Coton, M., Barnavon, L., Bach, B., Rattray, F., Bunte, A., Magni, C., Ladero, V., Alvarez, M., Fernández, M., Lopez, P., De Palencia, P. F., Corbi, A., Trip, H., & Lolkema, J. S. (2010). Biogenic amines in fermented foods. European Journal of Clinical Nutrition, 64, S95–S100.
Contents lists available at SciVerse ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Monitoring of biogenic amines in cheeses manufactured at small-scale farms and in fermented dairy products in the Czech Republic Leona Bunˇková a,⇑, Gabriela Adamcová b, Katerˇina Hudcová c, Helena Velichová b, Vendula Pachlová c, Eva Lorencová a, František Bunˇka c a b c
Department of Environmental Protection Engineering, Faculty of Technology, Tomas Bata University in Zlín, nám. T.G. Masaryka 275, 76272 Zlín, Czech Republic Department of Food Analysis and Chemistry, Faculty of Technology, Tomas Bata University in Zlín, nám. T.G. Masaryka 275, 76272 Zlín, Czech Republic Department of Food Technology and Microbiology, Faculty of Technology, Tomas Bata University in Zlín, nám. T.G. Masaryka 275, 76272 Zlín, Czech Republic
a r t i c l e
i n f o
Article history: Received 5 November 2012 Received in revised form 28 January 2013 Accepted 11 March 2013 Available online 20 March 2013 Keywords: Cheese Fermented dairy products Biogenic amines Health risk Small-scale farm
a b s t r a c t The aim of the study was the monitoring of six biogenic amines (histamine, tyramine, phenylethylamine, tryptamine, putrescine, and cadaverine) and two polyamines (spermidine and spermine) in 112 samples of dairy products purchased in the Czech Republic, namely in 55 cheeses made in small-scale farms and in 57 fermented dairy products. The products were tested at the end of their shelf-life period. Neither tryptamine nor phenylethylamine was detected in the monitored samples; histamine was found only in four cheese samples containing up to 25 mg/kg. The contents of spermine and spermidine were low and did not exceed the values of 35 mg/kg. Significant amounts of tyramine, putrescine, and cadaverine occurred especially in cheeses produced from ewe’s milk or in long-term ripened cheeses. In about 10% of the tested cheeses, the total concentration of all the monitored biogenic amines and polyamines exceeded the level of 200 mg/kg, which can be considered toxicologically significant. In fermented dairy products, the tested biogenic amines occurred in relatively low amounts (generally up to 30 mg/kg) that are regarded safe for the consumer’s health. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Biogenic amines (BA) are low-molecular nitrogen containing substances known for their characteristic physiological effects. Some BA act as hormones, but many BA are regarded as neurotransmitters or precursors for the synthesis of hormones, alkaloids or other metabolites (Halász, Baráth, Simon-Sarkadi, & Holzapfel, 1994; Shalaby, 1996; Silla Santos, 1996). Most commonly, biogenic amines are formed by decarboxylation of amino acids or by amination or transamination of aldehydes and ketones. Microorganisms demonstrating decarboxylase activity can get into foods spontaneously or they might be contained in starter cultures added intentionally to food (Ladero et al., 2012; Silla Santos, 1996). Common levels of BA in foodstuffs and beverages (<100 mg/kg), do not represent a serious risk for healthy consumers, because they are metabolised by detoxication enzymes in human intestines. Higher BA amounts can induce undesirable psychoactive and vasoactive effects. Histamine, tyramine, and phenylethylamine might bring about the above adverse effects directly. Putrescine and cadaverine do not show direct toxic effects; however, they can intensify toxic effects of other amines (Halász et al., 1994; Silla ⇑ Corresponding author. Tel.: +420 576 031 020; fax: +420 577 210 172. ˇ ková). E-mail address: [email protected] (L. Bun 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.03.036
Santos, 1996). If the intestinal detoxication system collapses, because of excessive amount of amines consumed in food or due to its weakening by inhibitors of detoxication enzymes, BA can get into the blood circulation system and may potentially precipitate many undesirable effects (Spano et al., 2010). Most of the products fermented by lactic acid bacteria (LAB), contain traces of histamine, tyramine, putrescine, and cadaverine. Regarding the occurrence of BA, cheeses are the most important and the most commonly monitored dairy products. The amounts of BA in milk, yoghurts, curd cheese and unripened cheeses can be expected to range between milligrammes and tens of milligrammes per kg (Linares, Martín, Ladero, Alvarez, & Fernández, 2011; Spano et al., 2010). Recently, studies where products from small-scale farms in south European countries like Italy, Portugal and Greece were characterised, have been published, however, no results of an investigation on relevant products manufactured in central Europe have been reported. Recently, in the Czech Republic, farmer markets have been gaining in popularity. Many Czechs have chosen the healthy life-style inspired by nature, which directs them to the consumption of food produced in small-scale farms. A complex study on the occurrence of BA in products manufactured in the above farms, has not been published yet. The aim of this study, was to monitor contents of eight BA and polyamines (PA) (histamine, tyramine, 2-phenylethylamine,
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tryptamine, putrescine, cadaverine, spermidine, and spermine) in cheese samples produced in small-scale farms and in selected fermented dairy products at the end of their shelf-life period. The above products were purchased in common Czech retail and/or, in farm markets (including purchase directly in farms).
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pasteurised cow’s milk; and (v) ripened cheeses made from pasteurised cow’s milk (Table 1). Besides the above cheeses, 55 fermented dairy products purchased in common Czech and Slovak retail network shops and two fermented dairy products manufactured at small-scale farms based in east Moravia (Czech Republic) were analysed. The following fermented products were monitored: (i) 29 yoghurts (one cream yoghurt was produced at a farm); (ii) 14 fermented creams; (iii) 4 acidified milks – milks fermented with an acidophilic culture; (iv) 4 kefir milks; (v) 2 fermented buttermilks; and (vi) 4 fermented milks – milks fermented with mesophilic cultures (one made at a farm) (Table 2). None of the monitored fermented products contained flavouring. The cheese samples and fermented milk products were kept at cold store temperatures of 6 ± 2 °C. They were analysed at the end of their shelf-life period.
2. Materials and methods 2.1. Cheese samples and fermented dairy products Altogether, 55 cheeses manufactured in small-scale farms in the central Europe region (the Czech Republic and the Slovak Republic) were purchased in 2012 (April to July). All of the goods were regional products purchased in farmer markets, in retail network shops or directly in some farms. The purchased cheeses were assorted into the following categories: (i) bryndza cheeses and cheeses made from raw ewe’s milk; (ii) cheeses and curd cheeses manufactured from pasteurised ewe’s milk; (iii) cheeses produced from goat’s milk (all except one, were manufactured from pasteurised milk); (iv) fresh cheeses and curd cheeses produced from
2.2. Determination of biogenic amines and polyamine contents If necessary, all the collected samples were homogenised, and subsequently lyophilised (Christ Alpha 1–4 device). The amines
Table 1 Content of biogenic amines in mg/kg in tested cheeses produced at small-scale farms. Cheese
a b
Number of samples
Biogenic amines (mg/kg)a Total BA
Tyramine
Histamine
Putrescine
Cadaverine
Spermine
4 3 2
73.2–222.2 25.1–177.1 NDb–40.3
4/34.6–107.4 3/8.9–38.3 ND
1/24.2 ± 1.1 NDb ND
2/22.1–60.9 3/16.2–99.9 1/20.7 ± 1.3
3/16.5–42.6 2/62.6–80.7 1/19.6 ± 1.4
1/9.7 ± 0.8 ND ND
Pasteurised ewe’s milk cheeses Unripened (fresh) cheese Pasta filata type cheese Brined cheese Flavoured cheese
3 2 2 2
ND–140.3 ND–13.2 37.2–529.8 ND–223.5
2/10.2–11.1 ND 2/23.1–174.6 1/114.7 ± 8.0
ND ND ND ND
2/55.3–118.2 ND 1/229.5 ± 20.0 1/108.8 ± 7.0
2/11.4–35.8 ND 1/125.6 ± 3.0 ND
ND 1/13.2 ± 1.1 1/14.0 ± 1.1 ND
Goat’s milk cheeses Ripened cheese (raw milk without heat treatment) Unripened (fresh) cheese (unflavoured) Unripened (fresh) cheese (flavoured) Pasta filata type cheese (plant)
1 3 6 3
356.1 ND–32.7 ND–10.7 ND–89.9
1/207.1 ± 7.5 1/11.3 ± 1.0 1/10.7 ± 0.5 1/8.5 ± 0.6
ND ND ND ND
ND ND ND 1/41.1 ± 3.3
1/149.0 ± 7.3 ND ND 1/40.3 ± 2.5
ND ND ND ND
Fresh cow’s milk cheeses Unripened (fresh) cheese (unflavoured) Unripened (fresh) cheese (flavoured)
6 4
ND–161.9 19.4–45.1
2/8.3–15.2 3/7.2–22.7
ND ND
1/111.4 ± 8.6 ND
3/8.9 ± 21.7 2/7.0–22.4
4/11.4–17.9 3/11.8–15.5
Other cow’s milk Ripened cheese Pasta filata type Pasta filata type Pasta filata type
3 5 3 3
137.9–173.4 ND–49.9 12.0–25.6 9.2–39.3
2/63.8–101.4 1/25.8 ± 1.5 ND ND
ND 1/19.3 ± 0.6 ND 2/18.3–18.8
2/48.8–70.1 1/37.3 ± 1.1 2/12.2–14.7 ND
3/11.8–72.6 ND ND 1/10.7 ± 0.9
3/11.5–97.9 2/7.1–12.7 3/10.9–12.0 2/9.2–9.8
Raw ewe’s milk cheeses Bryndza Smoked cheese Fresh cheese
cheeses cheese (unflavoured) cheese (flavoured) cheese (unflavoured, smoked)
Individual biogenic amines – number of BA positive samples/range of detected BA amount. ND – biogenic amines not detected.
Table 2 Content of biogenic amines in mg/kg in fermented dairy products.
a b
Product
Number of samples
Yoghurt (fat content 3–10%) Yoghurt (fat content 10–11%) Yoghurt (fat content 2–3%) Yoghurt (fat content 0.1%) Fermented cream Acidified milk Fermented milk Fermented buttermilk Kefir milk
17 4 3 5 14 4 4 2 4
Biogenic amines (mg/kg)a Total BA
Tyramine
Putrescine
Cadaverine
0.5–29.4 NDb–26.6 1.1–13.3 1.5–10.5 5.5–15.4 2.9–7.5 3.2–6.2 4.6–5.0 3.4–15.3
17/0.3–6.3 3/0.5–2.7 3/1.1–1.7 4/0.3–2.0 12/5.5–15.4 3/2.6–7.5 3/3.2–6.2 2/4.6–5.0 3/0.8–4.0
8/1.9–25.1 2/3.2–26.1 1/1.4 ± 0.2 3/5.0–10.5 2/8.6–8.8 1/4.1 ± 0.3 ND ND 3/3.4–14.3
NDb ND 1/4.3 ± 0.3 ND ND ND ND ND ND
Individual biogenic amines – number of BA positive samples/range of detected BA amount. ND – biogenic amines not detected.
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were extracted with a perchloric acid solution (0.6 M) in triplicate. The content of eight amines was determined by liquid chromatography (LabAlliance and Agilent Technologies devices) after previous derivatisation with dansylchloride. Derivatisation, chromatographic separation (column used: ZORBAX Eclipse XDBC18, 150 4.6 mm, 3.5 lm, Agilent Technologies) and spectrophotometric detection (k = 254 nm) were performed according to Dadáková, Krˇízˇek, and Pelikánová (2009). Each sample was extracted in triplicate, each extract was derivatised in duplicate and each derivatised mixture was applied on the column in triplicate (n = 18). In individual amines, the detection limits ranged between 0.24 and 1.39 mg/kg. The results were related to the sample weight before lyophilisation. For a comparison purpose, the Kruskall–Wallis and Wilcoxon tests were applied (Unistat 5.5 statistical software; Unistat Ltd., London, UK).
3. Results 3.1. Biogenic amines in cheeses produced at farms BAs and/or PAs were detected in 40 cheeses produced at farms. No phenylethylamine or tryptamine was detected in the tested cheeses. Histamine occurrence was also insignificant and it was detected in four cheeses: in one bryndza manufactured from unpasteurised milk and in three ripened and pasteurised cow’s cheeses (18.3–24.2 mg/kg; Table 1). In the cheeses analysed, tyramine was found to be the most commonly detected BA. Twenty-four cheese samples contained between 7.2 and 207 mg/kg of tyramine. Six cheeses contained more than 100 mg/kg. Tyramine was detected in most of the cheeses produced from either pasteurised or unpasteurised ewe’s milk. Twenty products contained between 7.0 and 149.0 mg/kg of cadaverine. Cadaverine occurred mainly in ewe’s milk products, primarily in unpasteurised cheeses (P < 0.05). In cow’s milk cheeses, the fresh products showed higher contents of cadaverine. Putrescine was found in 17 cheeses at the levels of 12.2– 230 mg/kg. Four cheeses included over 100 mg/kg (Table 1). Putrescine was detected in more than half of the ewe’s milk cheeses and in one brine cheese (230 mg/kg). Putrescine was found in one quarter of cow’s milk products, primarily in ripened cheeses (P < 0.05). Nineteen cheeses contained spermine (Table 1). Spermine was detected in two thirds of cheeses made from cow’s milk. Low spermine levels were determined in ewe’s milk cheeses (P < 0.05). Most of the BA or PA containing cheeses demonstrated the presence of more than one amine. In 20% of the cheeses tested, the total amount of BA and PA did not exceed 20 mg/kg; 9% of the cheeses studied contained more than 200 mg/kg. The highest BA and PA amount (530 mg/kg) was found in a brined cheese made from pasteurised ewe’s milk. Ewe’s milk cheeses made from both pasteurised and unpasteurised milk contained generally more BA than those produced from goat’s or cow’s milk. Altogether, eight ewe’s milk cheese samples included more than 100 mg/kg of total BA; however, only one unpasteurised goat’s milk cheese and only four pasteurised cow’s milk cheeses (where only three were ripened cheeses) contained that high amounts of BA. 3.2. Biogenic amines in fermented dairy products Except for one sample, all the tested non-flavoured fermented dairy products contained BA within the range of 0.5–29.4 mg/kg
(Table 2). These included four biogenic amines – tyramine, putrescine, cadaverine, and spermine. Tyramine was the most common BA found in fermented dairy products; its presence was proved in 50 tested samples. Higher amounts of BA were solely found in fermented creams. Putrescine was quite a commonly detected BA found in 20 tested samples (Table 2). Half of the putrescine-positive samples contained up to 10 mg/kg, two yoghurt samples were found to contain slightly more than 25 mg/kg. The total amount of BA in fermented dairy products was lower than 30 mg/kg (Table 2). Ten of the tested products showed BA levels ranging between 10 and 20 mg/kg and only three yoghurts included more than 20 mg/kg. 4. Discussion Approximately 80% of the dairy products tested showed the presence of BA. These are normally present in the product ingredients; namely, spermine and spermidine are found in fresh milk (Linares et al., 2011; Spano et al., 2010). Biogenic amines can also be produced by both starter and non-starter LAB, that are used in the manufacture of these products or that can get into products during their processing, respectively. The ability of many LAB repˇ ková resentatives to produce BA has been widely reported (Bun et al., 2009; Ladero et al., 2012; Linares et al., 2011). Biogenic amines can also be produced in contaminated microflora, especially by the Enterobacteriaceae and other Gram-negative bacteria (Coton et al., 2012). In comparison, ripened cheeses showed higher BA and PA levels than the fresh ones, which can be explained by the fact that their microflora did not have enough time to produce higher amounts of BA and PA. In ripened cheeses, the concentration of BA and PA ˇ ková et al., 2010). On the usually rises gradually with time (Bun other hand, some of the shorter-time ripening cheeses produced in farms contained over 200 mg/kg of BA and PA. Higher amounts of BA could result from the activity of non-starter LAB or contaminated microflora. The levels of BA were also monitored in cheeses made from either pasteurised or unpasteurised milk. No significant differences in the amounts of BA in ewe’s milk cheeses, manufactured from raw or pasteurised milk were found. More significant differences were detected in cheeses produced from goat’s milk; cheeses produced from raw milk contained higher concentrations of BA (P < 0.05). Similar results were reported by Novella-Rodríguez, Veciana-Nogués, Roig-Sagués, Trujillo-Mesa, and Vidal-Carou (2004), who accounted the above finding, to the higher amount of microorganisms in cheeses made from raw milk. The fermented dairy products were expected to contain lower amounts of BA than cheeses. Unlike cheeses, fermented dairy products are not exposed to an environment suitable for growth of microflora, especially of Gram-negative bacteria that could produce BA (e.g. Enterobacteria, Pseudomonas prefer a higher pH than the pH of fermented dairy products). Concerning fermented products, some researchers reported low amounts of BA usually not exceeding the levels of 15 mg/kg (Chaves-López et al., 2011; Linares et al., 2011). The total amount of BA in some samples reached the amount of 29.4 mg/kg. Corresponding contents of BA were also detected by Özdestan and Üren (2010) in kefir and by Magwamba, Matsheka, Mpuchane, and Gashe (2010) in fermented milk. Regarding the impact of BA on human health, histamine and tyramine show the most adverse effects (Shalaby, 1996). Histamine was not detected in any of the studied 57 samples of fermented dairy products, but it was found in four cheeses, with
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amounts lower than 25 mg/kg. Similar levels of histamine were detected in ewe’s milk cheeses produced at farms in Portugal by Pintado et al. (2008) and in Italy by Schirone, Tofalo, Mazzone, Corsetti, and Suzzi (2011). On the contrary, Ladero, Linares, Fernández, and Alvarez (2008) reported substantially higher amounts of histamine (up to 1040 mg/kg) in cheeses produced from raw and pasteurised cow’s, goat’s, and ewe’s milk. Andiç, Gençcelep, and Köse (2010) found 680 mg/kg of histamine in cheeses which contained herbs. Ripened cheeses produced from ewe’s milk revealed substantially higher concentrations of tyramine (up to1200 mg/kg) (Andiç et al. 2010; Schirone et al., 2011). Comparing the content of BA, the tested fermented dairy products contained substantially lower amounts of BA (practically < 30 mg/kg) than the other dairy products such as ripened cheeses (Linares et al., 2011; Schirone et al., 2011). This difference is probably caused by various conditions in the manufacturing process or storage and ripening. The fermented dairy products are processed at temperatures suitable for bacteria propagation and BA formation takes only a couple of hours. Moreover, their shelf-life and thereby also period of BA generation is relatively short and it usually does not exceed the span of 3–4 weeks. Determination of the toxic level of total BA in foods is difficult due to many various factors acting in its specification. Some amines (e.g. putrescine and cadaverine) can potentiate toxic effects of other BA (especially histamine and tyramine). Researchers report values within quite a wide range of 200–800 mg of BA per one kilogramme of food (Halász et al., 1994; Silla Santos, 1996; Spano et al., 2010). Considering the lower limit of 200 mg/kg, about 10% of cheeses produced at small-scale farms contained BA amounts that can lead to health damage even in healthy consumers. Evaluating the risks of foodstuffs to consumer’s health, the safe daily BA intake should be regarded as complex. Some substances such as alcohol, can decrease the activity of enzymes that participate in degradation of BA in human intestines (Shalaby, 1996). Since cheeses are often served with alcoholic drinks such as beer or wine, even low concentrations of BA in cheeses can cause adverse effects. Beer and wine, often contain high amounts of BA, which might intensify the negative impact of BA in cheeses on human health (Ancín-Azpilicueta, González-Marco, & Jiménezˇ ka et al., 2012). Moreno, 2008; Bun The results of this study, show that BA and PA occur in cheeses quite commonly, and this might represent a serious risk to our health. As the safe levels of BA and PA in foods and beverages have not been specified by any regulation, these secondary metabolites have not been either monitored or evaluated by any control body, which might threaten the consumer’s health. Acknowledgements This work was supported by The National Agency for Agriculture Research, Project No. QJ1210300, The Complex Sustainable Systems Programme and from the Internal Grant of Tomas Bata University in Zlín (No. IGA/FT/2013/013).
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References Ancín-Azpilicueta, C., González-Marco, A., & Jiménez-Moreno, N. (2008). Current knowledge about the presence of amines in wine. Critical Reviews in Food Science and Nutrition, 48, 257–275. Andiç, S., Gençcelep, H., & Köse, Sß. (2010). Determination of biogenic amines in herby cheese. International Journal of Food Properties, 13, 1300–1314. ˇ echová, M., Drienovsky´, V., Pachlová, V., Matoulková, D., ˇ ka, F., Budinsky´, P., C Bun ˇ , V., & Bun ˇ ková, L. (2012). Content of biogenic amines and polyamines in Kubán beers from the Czech Republic. Journal of the Institute of Brewing, 118, 213–216. ˇ ková, L., Bun ˇ ka, F., Hlobilová, M., Vanˇátková, Z., Nováková, D., & Dráb, V. (2009). Bun Tyramine production of technological important strains of Lactobacillus, Lactococcus and Streptococcus. European Food Research and Technology, 229, 533–538. ˇ ablová, A., Sedlácˇek, I., Švec, P., Pachlová, V., & ˇ ková, L., Bun ˇ ka, F., Mantlová, G., C Bun Krácˇmar, S. (2010). The effect of ripening and storage conditions on the distribution of tyramine, putrescine and cadaverine in Edam-cheese. Food Microbiology, 27, 880–888. Chaves-López, C., Serio, A., Martuscelli, M., Paparella, A., Osorio-Cadavid, E., & Suzzi, G. (2011). Microbiological characteristics of kumis, a traditional fermented Colombian milk, with particular emphasis on enterococci population. Food Microbiology, 28, 1041–1047. Coton, M., Delbés-Paus, C., Irlinger, F., Desmasures, N., Le Fleche, A., Stahl, V., Montel, M.-C., & Coton, E. (2012). Diversity and assessment of potential risk factors of Gram-negative isolates associated with French cheeses. Food Microbiology, 29, 88–98. Dadáková, E., Krˇízˇek, P., & Pelikánová, T. (2009). Determination of biogenic amines in foods using ultra-performance liquid chromatography (UPLC). Food Chemistry, 116, 365–370. Halász, A., Baráth, Á., Simon-Sarkadi, L., & Holzapfel, W. (1994). Biogenic amines and their production by microorganisms in food. Trends in Food Science and Technology, 5, 42–49. Ladero, V., Cañedo, E., Pérez, M., Martín, M. C., Fernández, M., & Alvarez, M. A. (2012). Multiplex qPCR for the detection and quantification of putrescineproducing lactic acid bacteria in dairy products. Food Control, 27, 307–313. Ladero, V., Linares, D. M., Fernández, M., & Alvarez, M. A. (2008). Real time quantitative PCR detection of histamine-producing lactic acid bacteria in cheese: Relation with histamine content. Food Research International, 41, 1015–1019. Linares, D. M., Martín, M. C., Ladero, V., Alvarez, M. A., & Fernández, M. (2011). Biogenic amines in dairy products. Critical Reviews in Food Science and Nutrition, 51, 691–703. Magwamba, C., Matsheka, M. I., Mpuchane, S., & Gashe, B. A. (2010). Detection and quantification of biogenic amines in fermented food products sold in Botswana. Journal of Food Protection, 73, 1703–1708. Novella-Rodríguez, S., Veciana-Nogués, M. T., Roig-Sagués, A. X., Trujillo-Mesa, A. J., & Vidal-Carou, M. C. (2004). Evaluation of biogenic amines and microbial counts throughout the ripening of goat cheeses from pasteurized and raw milk. Journal of Dairy Research, 71, 245–252. Özdestan, Ö., & Üren, A. (2010). Biogenic amine content in kefir: A fermented dairy product. European Food Research and Technology, 231, 101–107. Pintado, A. I. E., Pinho, O., Ferreira, I. M. L. V. O., Pintado, M. M. D., Gomes, A. M. P., & Malcata, F. X. (2008). Microbiological, biochemical and biogenic amine profiles of Terrincho cheese manufactured in several dairy farms. International Dairy Journal, 18, 631–640. Schirone, M., Tofalo, R., Mazzone, G., Corsetti, A., & Suzzi, G. (2011). Biogenic amine content and microbiological profile of Pecorino di Farindola cheese. Food Microbiology, 28, 128–136. Shalaby, A. R. (1996). Significance of biogenic amines to food safety and human health. Food Research International, 29, 675–690. Silla Santos, M. H. (1996). Biogenic amines: Their importance in food. International Journal of Food Microbiology, 29, 213–231. Spano, G., Russso, P., Lonvaud-Funel, A., Lucas, P., Alexandre, H., Grandvalet, C., Coton, E., Coton, M., Barnavon, L., Bach, B., Rattray, F., Bunte, A., Magni, C., Ladero, V., Alvarez, M., Fernández, M., Lopez, P., De Palencia, P. F., Corbi, A., Trip, H., & Lolkema, J. S. (2010). Biogenic amines in fermented foods. European Journal of Clinical Nutrition, 64, S95–S100.