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Chemical Composition and Occurrence of Mycotoxins in Six Blue- Mould-Ripened Cheeses in Egypt El-Zahar K.1, Abdel Galeel A.A

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Chemical Composition and Occurrence of Mycotoxins in Six Blue- Mould-Ripened Cheeses in Egypt El-Zahar K.1, Abdel Galeel A.A Alex. J. Fd. Sci. & Technol. Vol. 5, No. 1, pp. 43-50, 2008 Chemical Composition and Occurrence of Mycotoxins in Six Blue- Mould-Ripened Cheeses in Egypt El-Zahar K.1, Abdel Galeel A.A. 1 & Hendawi M.Y.2 1Food Science Dept., Fac. of Agric., Zagazig Univ., Zagazig, Egypt. 2Plant Protection Dept., Fac. of Agric., Zagazig Univ., 44511, Zagazig, Egypt. ABSTRACT Chemical composition (fat, moisture, protein, salt, titratable acidity, and pH), proteolysis (free amino acids), lipolysis (total volatile fatty acids and free fatty acids), and some mycotoxins were determined in all cheese samples studied. Saint Agur was characterised by its high concentrations of total protein (22.04%), total amino acids (5.21mg/ g), fat (33.79%), and TVFA (25.3 ml NaOH/100 mg cheese) and lowest concentrations of salt (2.41%) and moisture Bleu d’Auvergne, had a pH around 6.0, and the highest concentrations of salt in moisture (8.0%), soluble .(40.20٪) nitrogen (51.98%), moisture (47.09%) and salt (3.76%) of all cheeses investigated. Bleu d’Auvergne had the lowest fat content (28.42%) and TVFA (20.1 ml NaOH/100mg cheese) of all the cheeses studied. Cheese samples were analyzed for seven mycotoxins (Roqurfortine C, Mycophenolic acid, Penicillin acid, Afla- toxin G1, Aflatoxin M1, Aflatoxin B1, Aflatoxin B2). Roqurfortine C was detected in all of the blue mould cheese samples in concentrations of 1.6-14 µg kg-1. Two blue-cheese samples (Saint Agur and Moby blue) contained also 0.35 and 0.47 µg kg-1 mycophenolic acid, respectively. Penicillic acid was detected (0.12, 0.41 and 0.53-1 µg/ kg) in Gorgonzola, Saint Agur and Moby blue, respectively. Aflatoxin G1, aflatoxin M1, aAflatoxin B1 and aflatoxin B2 were not detected in the tested cheeses, this is likely to be due to best manufacturing practises applied in the cheese production, and at the same time the storage conditions are all strictly controlled in order to avoid mould contamina- tion and aflatoxins formation. Keywords: blue cheese, chemical properties, ripening indices, aflatoxins, mycotoxins, organoleptic properties. INTRODUCTION has been used as a secondary starter culture for the ripening of blue–mould cheeses, such as Gor- Blue-type cheeses, characterised by the growth gonzola, Danabule, Roquefort, Bleu de Bresse and of Penicillium roqueforti in fissures throughout the Stilton for centuries. Because of the coevolution cheese matrix, are one of the most easily identi- with lactic acid bacteria, Penicillium roqueforti is fiable cheese types. During ripening, blue-type resistant to lactic acid, acetic acid, carbon dioxide cheeses undergo extensive proteolysis and lipolysis and several other lactic acid bacterial metabolites resulting in odour, flavour, appearance and texture (Boysen et al., 2000). development (Fox et al., 2000). The production of blue cheese with typical high quality flavour is There are many false reports on mycotoxin mainly influenced by milk type, species of moulds production from Penicillium roqueforti. Myco- used, rate of starter growth and salt content. It is toxins are toxic secondary metabolites produced well established that cheese ripening is a complex by many species of fungi. The presence of these process involving the breakdown of the curd by substances in dairy products can have two origins; proteolysis, lipolysis and other enzymes-catalyzed (a) indirect contamination, which results from lac- reactions to give the flavour and texture changes tating animals ingesting contaminated feed and (b) typical of the different cheese varieties. These reac- direct contamination, which occurs because of in- tions are brought about by enzymes present in the tentional or accidental growth of moulds on dairy milk, added rennet, starter cultures, the moulds and products (van Egmond 1989). Some of the fungal yeasts and the cheese milk micro flora (Kinsella & starters used in the cheese manufacture have been Hawng 1976). Lactic acids bacteria were reported shown to produce various mycotoxins on culture to the dominant group from salting on words, while media. P. roqueforti, widely used as a starter in moulds and yeasts were dominant during ripening the blue mould cheeses, is known to synthesise (Lopez-Diaz et al., 1996). Penicillium roqueforti Roquefortine C, PR-toxin, mycophenolic acid and 43 Vol. 5, No. 1, pp. 43-50, 2008 Alex. J. Fd. Sci. & Technol. isofumigaclavins (Samson & Frisvad, 2004). The determined according to the procedure of (BSI) occurrence of Roquefortine C and PR toxin in do- (1985). The modified Gerber method was used to mestic and imported blue cheeses in Italy, the iden- determine fat in cheese samples as described by BSI tification of the penicillia used as starter, and the (1985).The level of pH (pH 4.6-SN) was measured investigation of their capacity for producing tox- using the method of Kuchroo & Fox (1982). Free ins in culture media were investigated (Frisvad et amino acids concentrations were determined using al., 2004). Roquefortine C was always found in the the method of Fenelon et al., (2000). The concen- cheeses with levels ranging from 0.05 to 1.47 µg trations of free fatty acids (C2:0, C3:0, C4:0, C6:0) kg-1, whereas PR-toxin or penicillic acid could not were determined using the method described by find in blue cheeses (Schoch et al., 1984). Fungal Kilcawley et al. (2001). Total volatile fatty acids strains present in the domestic cheeses included of cheese samples were determined according to Penicillium glabrum, P. roqueforti and P. cyclo- the method of Kosikowki (1978). Cheese samples pium in the Gorgonzola “dolce” and P. roqueforti were analysed in duplicate, and they are stored at in the Gorgonzola “naturale”. (Erdogan et al., 4°C prior to analysis. 2003, Samson & Frisvad, 2004). Other mycrobiota besides starters may be present in mould cheeses as Determination of mycotoxins contaminants, since various fungi are encountered Samples preparation in cheese factory environments and some of these The samples were prepared with a slight modi- organisms can be potential mycotoxin producers. fication as described by the method of Rundber- Few reports indicating the occurrence of mycotox- get & Wilkins (2002). Ten grams of cheese were ins due to contaminating fungi in mould cheeses. weighed and 60 ml of acetonitrile (containing 0.1% Lafont et al., (1990) detected penicillic acid in one formic acid) and 50 ml of hexane were added. The blue cheese sample of dubious organoleptic prop- sample was homogenized by Ultra Turrax homog- erties, indicating this might be because of contami- enizer for five minutes and centrifuged (4000 rpm, nating fungi rather than actual starters. for 10 min,). The sample was filtered through an The objectives of the present study were to de- S & S 2329 filter paper and a 10ml portion of the termine the gross compositional constituents of a acetonitrile phase was evaporated to dryness under number of blue–type cheeses in Egyptian markets a stream of nitrogen at +50°C. The residue was dis- and to evaluate their nutritional values. The second solved in 0.2ml of methanol and filtered through a goal of this study was to determine the level of my- 0.2 mm syringe filter into an auto-sampler vial. cotoxins in blue cheeses samples collected in order Capillary liquid chromatography-tandem to evaluate its safety. mass spectrometric (LC-MS/MS) analysis Chromatographies were carried out on a Wa- MATERIALS AND METHODS ters 2695 separation module equipped with a Waters Collection of samples 996 photodiode array detector (Waters, Millford, MA, USA), by injecting 10 µl of the sample on a Six blue-type cheeses selected to represent a Symmetry C18 column (2.1x100 mm, 3.5 µm; Wa- rang of different blue-moulds cheeses (2-4 months ters, Milford, MA, USA). Elution was performed old), were purchased from the Egyptian super- by using a gradient (74.9% water, 25% acetonitrile markets at Cairo and Alexandria. Cheeses includ- and 0.1% acetic acid, v/v/v), which were held for 16 ed the following: Danish Blue (5 samples), Blue min, then changed to (9.9% water, 90% acetonitrile d’Auvergne (7 samples), Blue Stilton (4 samples), and 0.1% acetic acid, v/v/v) for 24 min with flow Gorgonzola (4 samples), Moby blue (6 samples) rate 0.2 ml /min. LC was coupled to a Micro-Mass and Saint Agur (6 samples). Quattro Micro triple-quadrupole mass spectrometer Chemical analysis (Micro mass Ltd, Manchester, UK) equipped with an electrospray ionization (ESI) probe operating in Gross chemical composition was analysed us- the positive mode. Multiple reactions monitoring ing standard methods; moisture contents were esti- (MRM) mode was used for identification and quan- mated by the oven drying method (AOAC, 1990). tification of the compounds. The retention times of Salt content was determined according to (AOAC, the standard mycotoxins were 3.5, 4.2, 4.7, 8.2, 8.6, 1990). Acidity content expressed as lactic acid was 11.3 and 27 min for Penicillic acid, Roquefortine 44 Alex. J. Fd. Sci. & Technol. Vol. 5, No. 1, pp. 43-50, 2008 C, Aflatoxin M1, Aflatoxin B2, Aflatoxin G1, Afla- tively), soluble nitrogen value (48.47 and 46.82%, toxin B1 and Mycophenolic acid, respectively. respectively) and high concentrations of fat in the dry matter (56.50 and 55.15%, respectively) (Ta- RESULTS AND DISCUSSION ble 1).Bleu d’Auvergne a French blue-type manu- factured using cow’s milk, had a pH of 5.9, and Chemical composition the highest concentrations of salt (3.76%), salt The chemical composition and the concentra- in moisture (8.00%), soluble nitrogen (51.98%), tions of free amino acids (FAA), free fatty acids moisture (47.09%). Bleu d’Auvergne had the low- (FFA), in six blue-mould cheeses are shown in est fat content (28.42%), TVFA (20.1 ml NaOH Tables (1, 2 and 3), respectively.
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