DETECTION OF HISTAMINE AND TYRAMINE IN SOME CHEESE

The biogenic amines content of various food has been widely studied because of their potential toxicity. This study aims at presenting data about histamine and tyramine content in some locally and imported soft, hard, and semihard cheese available in Alexandria markets throwing lights on its public health hazard. A total of 140 random cheese samples included a large variety of types of soft cheese (kareish, old cheese (Mish), Demietta), Semihard cheese, (Roquefort and Gouda), and hard cheese (Ras and Cheddar) as twenty samples of each were analysed for histamine and tyramine using high performance liquid chromatography (HPLC). Results showed that histamine and tyramine were detected in (35–55%) and (70-100%) of samples, respectively. All examined samples contained histamine level lower than the critical oral dose toxic to human (100 mg/100 g). Tyramine exceeded the dangerous dose for patients receiving MAOI, (6 mg) by the percentage of (30, 100, 60, 100, 50, 80, and 60%), respectively. Histamin concentration increased in the order of cheddar > Mish> Gouda>Demietta> Ras > Roquefort > Kareish cheese. Also, tyramine increased in these manner Roquefort > Cheddar > Mish > Demietta > Gouda > Kariesh. The highest level of histamine and tyramine (20.46±7.73 and 32.76±10.32 mg/100g) recorded in cheddar and Roquefort cheese, respectively but the lowest level showed in kareish cheese (4.02±1.74 and 7.12±3.75 mg/100 g) of histamine and tyramine, respectively. INTRODUCTION Biogenic amines are defined as low molecular weight organic basic, aliphatic mono or polyamines, aromatic or heterocytic amines are naturally formed as a consequence of metabolic process of human, animal, plant, and microorganism (Davidek and Davidek, (1995)(1). Decarboxylation of the protein amino acids by bacterial enzymes gives rise to formation of biogenic amines (Vale and Gloria, 1997)(2). They are commonly present in high concentration in a wide range of fermented and non-fermented products including fish, meat, and dairy 797 Bull High Inst Public Health Vol.37 No.4 [2007] products (Maiijala et al., 1995; Moret and Conte 1996)(3,4). Biogenic amines in food are formed during storage, spoilage, and ripening process through degradation of protein by proteolysis together with bacterial action resulting in formation of amino acids as precursors of biogenic amines produced through the decarboxylation process (Rice et al., 1976; Summer et al., 1990; and Silla Santos 1996)(5-7). Biogenic amines are undesirable compound in food because of their potential to affect the well being of consumers or even to cause serious health problems for susceptible persons. Cheese are among those high proteincontaining food stuffs in which microbial activity results in the formation of such products of protein decomposition as biogenic amines. Cheese represent an ideal environment for amine production by bacterial decarboxylation of appropriate amino acids. Amounts of biogenic up to 1g/kg have been found in cheese products (Silla. Santos, 1996; Chang et al., 1985; Innocente, et al., 2007:)(7-9). Tyramine histamine, putrescine, cadaverine, phenylanine, spermine, and spermidine are generally considered to be the most important biogenic amines occurring in cheese (Joosten, 1988; Stratton et al., 2000)(10-11). The most notorious food borne intoxation caused by biogenic amines are related to histamine. Histamine at levels usually exceeding 1000 mg/kg has been implicated with certain food intoxications such as scombrotoxicosis (Taylor and Speckhard 1984).(12) or the cheese syndrome (Taylor et al., 1982, Summer, et al., 1985, and Stratton, et al., 1991).(13-15) Also amines in dairy products at high levels could be of great public health significance because of their possiblity in involvement in intestinal ulcers and allergic responses (USFDA, 2001; and Periago, 2003).(16,17) Several out breaks of histamine poisining have


INTRODUCTION
Biogenic amines are defined as low molecular weight organic basic, aliphatic mono or polyamines, aromatic or heterocytic amines are naturally formed as a consequence of metabolic process of human, animal, plant, and microorganism (Davidek and Davidek, (1995) (1) .
Decarboxylation of the protein amino acids by bacterial enzymes gives rise to formation of biogenic amines (Vale and Gloria, 1997) (2) .They are commonly present in high concentration in a wide range of fermented and non-fermented products including fish, meat, and dairy Bull High Inst Public Health Vol.37 No.4 [2007]   products (Maiijala et al., 1995; Moret and Conte 1996) (3,4) .
Histamine at levels usually exceeding 1000 mg/kg has been implicated with certain food intoxications such as scombrotoxicosis (Taylor and Speckhard   1984). (12)(15) Also amines in dairy products at high levels could be of great public health significance because of their possiblity in involvement in intestinal ulcers and allergic responses (USFDA,   2001; and Periago, 2003). (16,17)  et al., (1982). (13)Tyramine may provoke hypertensive crises and even death from cerebral hemorrhage in patients treated with monoamine oxidase inhibitor drugs.
Migraine headache has been triggered by consumption of cheese with high level of tyramine.Also, those amines may form nitrosamines compounds known to be carcinogenic, mutagenic, and teratogenic.1)(22) .
Despite the fact that cheese may contain exceedingly high level of histamine and other biogenic amines (> 2000 mg/kg), tolerance has not been set so far (Osman et al., 1999). (23)The level of biogenic amines in cheese is necessary to assess the health hazard arising from consumption of these products furthermore, it could be useful as a chemical index in quality assurance of material and manufacturing conditions since these tests are rapid compared to traditional microbial analysis, less subjects individual interpretion than sensory analysis.
So this study was done to obtain quantitative information for histamine and tyramine in some locally, imported soft, hard, and semihard cheese available in Alexandria markets concerning its public health.

Sample materials
A total of 140 random cheese samples (HPLC) according to the methods described by Mitz and Karamas (1978) (24) and Vale & Gloria, (1997). (2) g of ground sample was extracted with 5% trichloroacetic acid (TCA) 3  75 ml using a warning blender.Each blended mixture was centrifuged and the clear extracts were combined.The value was adjusted to 250 ml with TCA (5%) solution.
The equivalent of 2 g of sample as the TCA extract (10 ml was made alkaline by adding 1ml 50% sodium hydroxide and then extracted with n-butanol/chloroform mixture (1:1 v/v) 3  5 ml.The combined organic phase after addition of an equal of n.
heptane (15 ml) was extracted with several potions of 0.02 (1ml each) and the aqueous extract was dried using current of air.

Derivatives formation and determination
The dansyle derivatives of biogenic amines were formed by adding saturated sodium bicarbonate solution (0.5 ml) to the residue (dry film) stoppered and carefully mixed using vortex mixer, then carefully adding 1 ml dansyle chloride solution (500 mg in 100 ml acetone) and mixed thoroughly.After standing for more than 10 Detector : UV-Vis at 254 nm.
Injection : 10 ml of standard solution (as derivative) or sample was injected into HPLC apparatus.
Although there is no regulatory limits for histamine and tyramine in cheese, our study showed that all investigated samples contained histamine below 100 mg/100 g.
) With respect to the hazard level of tyramine which was reported to be dangerous dose for patients receiving MAOI, (6 mg), Blackwell & Mabbit (1965) (47) .The percentage of (30, 100, 60, 100, 50, 80, and 60%) in Kariesh, Mish, Demietta, Roquefort, Gouda, Chedder, and Ras Cheese, respectively contained tyramine in a higher level than these detected limit (6 mg).Biogenic amines are heat stable, histamine is more commonly the result of high temperature spoilage than of long term, Jargensen, et al., (2000). (48)nerally, we concluded that biogenic amines in food can be controlled by strict use of good hygiene in both raw material and manufacturing environment with corresponding inhibition of spoilage microorganisms and finally proper storage temperature.In addition, it is of great importance to establish regulatory limits for all biogenic amines which may be found in cheese to safeguard public health.
during storage, spoilage, and ripening process through degradation of protein by proteolysis together with bacterial action resulting in formation of amino acids as precursors of biogenic amines produced through the decarboxylation process (Rice et al., 1976; Summer et al., 1990; and Silla Santos 1996) (5-7) .Biogenic amines are undesirable compound in food because of their potential to affect the well being of consumers or even to cause serious health problems for susceptible persons.Cheese are among those high proteincontaining food stuffs in which microbial activity results in the formation of such products of protein decomposition as biogenic amines.Cheese represent an ideal environment for amine production by bacterial decarboxylation of appropriate amino acids.Amounts of biogenic up to 1g/kg have been found in cheese products (Silla.Santos,   1996; Chang et al., 1985; Innocente, et al., were collected separately from different retail food markets in Alexandria, including a large variety of soft cheeses (Kareish, Old cheese, and Demietta), semihard Bull High Inst Public Health Vol.37 No.4 [2007] cheeses (Roquefort and Gouda), and hard cheeses (Ras and Cheddar) as twenty samples of each.A 100-200 g portion of each sample was get up in a blender at high speed for 2 min and stored at -18°C until analysis.Analysis of biogenic amines (Histamine and Tyramine): was used by high performance liquid chromatography hours at room temperature, the densyl amines were extracted by adding 15 ml HPLC grade water and then the mixture was extracted with several portion (5 ml each) of diethyl ether (HPLC grade).The combined ether extracts were then evaporated to dryness by the aid of current of air and water bath at 35°C.The residue was dissolved in 1 ml acetonitrile.Preparation of standard solution: Histamine Dissolving 41.40 mg of histamine dihydrochloride (Sigma Chemical Co, N. 7505) in 50 ml water HPLC grade (stock solution 0.5 mg/ml) Tyramine Dissolving 31.39 mg of tyramine (4hydroxphenyl ethylamine) hydrochloride (N, T-2879) in 50 ml water HPLC grade (stock solution 0.5 mg/ml) 200 ml of each stock standard solution was transferred to glass tube (using micropipette) then evaporated using current of air.The residue was subjected to densylation.Then the residue was dissolved in 5 ml acetonitrile (1 ml = 20 g or/oug = 0.2 g each amine as derivative).Detection HPLC was used for the quantitative estimation of biogenic amines.The conditions used were as follow: Mobile solvent: solvent A: acetonitrile 0.02 N acetic acid (1: 9 v/v).Solvent B : 0.02 N acetic acid: acetonitrile: methanol (2: 9: 9v/v/v).Program : gradient program 60% solvent B in solvent A to 100% solvent B using linear program over 30 min period and 1 ml constant flow rate.
Fig. (1) illustrated the percentage of detected positive samples and concentrations of histamine and tyramine (mg/100g) in some cheese (Kareish, Old cheese (Mish), Demietta, Requfort, Gouda, Cheddar, and Ras) collected from different retail food markets in Alexandria.It is clear from these data, that, there were wide variations in the content of both histamine and tyramine versus the type of cheese and also, between samples of the same type may be attributed to the variation