Influence of Storage and Household Processing on the Agaritine Content of the Cultivated Agaricus Mushroom

Food Additives and Contaminants, 2002, Vol. 19, No. 9, 853±862 In¯uence of storage and household processing on the agaritine content of the cultivated Agaricus mushroom V. SchulzovaÂ , J. HajsÏ lovaÂ *, R. Peroutka , J. (around 10% left after 2 h). Dry baking of the y y y Gry‡ and H. C. Andersson} cultivated mushroom, a process similar to pizza baking, Institute of Chemical Technology, Department of Food Chemistry y reduced the agaritine content by approximately 25%, and Analysis, TechnickaÂ 3, 166 28 Prague 6, Czech Republic; whereas frying in oil or butter or deep frying resulted in ‡ Danish Veterinary and Food Administration, Mùrkhùj Bygade 19, DK-2860 Sùborg, Denmark; } National Food Administration, a more marked reduction (35±70%). Microwave pro- Box 622, SE-751 26 Uppsala, Sweden. cessing of the cultivated mushrooms reduced the agaritine content to one-third of the original level. Thus, the exposure to agaritine was substantially less when (Received 20 June 2001; revised 3 May 2002; accepted 19 consuming processed Agaricus mushrooms as com- May 2002) pared with consuming the raw mushrooms. However, it is not yet known to what extent agaritine and other Agaritine (N-(g-l(+)-glutamyl)-4-hydroxymethyl- phenylhydrazin e derivatives occurring in the cultivated phenylhydrazine ) was identi®ed and quanti®ed by mushroom are degraded into other biologically active high-pressure liquid chromatography and used as a compounds during the cooking procedure. marker for the occurrence of phenylhydrazine derivatives in the cultivated Agaricus bitorquis and A. garicus hortensis mushrooms. Although relatively high Keywords : agaritine, Agaricus, Agaricus bitorquis, levels of agaritine (around 700 mg kg±1) could be found Agaricus hortensis, storage, household processing, in freshly harvested A. bitorquis from early ¯ushes, cooking samples from supermarkets contained less agaritine. The content of 28 samples varied between 165 and ±1 ±1 Introduction 457 mg kg , on average being 272 69 mg kg . The § highest amounts of agaritine were found in the skin of the cap and in the gills, the lowest being in the stem. The cultivated mushroom Agaricus bisporus contains There was no signi®cant di erence in agaritine content agaritine (N-®-l(+)-glutamyl)-4-hydroxymethylphe- of the two mushroom species in our study. Pronounced nylhydrazine), two other phenylhydrazine derivatives reduction in agaritine content was observed during (4-(carboxy)phenylhydrazin e and -N-®-l(+)-glu- storage of mushrooms in the refrigerator or freezer, tamyl)-4-carboxyphenylhydrazine ) presumed to be as well as during drying of the mushrooms. The degree precursors to agaritine, and the 4-(hydroxymethyl) of reduction was dependent on the length and condition benzenediazonium ion (Andersson and Gry 2002). of storage and was usually in the region 20±75%. No The latter compound is believed to be formed when reduction in agaritine content was observed during agaritine is metabolized (LaRue 1977). Whereas agar- freeze-drying. Depending on the cooking procedure, itine is found in high quantities, usually between 200 household processing of cultivated Agaricus mush- and 450 mg kg±1 fresh weight, the other three com- rooms reduced the agaritine content to various degrees. pounds mentioned occur at lower levelsÐone-tenth Boiling extracted around 50% of the agaritine content of the amount of agaritine, or less (Andersson and into the cooking broth within 5 min and degraded 20± Gry 2002). 25% of the original agaritine content of the mushrooms. Prolonged boiling, as when preparing a sauce, Increasingly more data are accumulating from studies reduced the content in the solid mushroom further on experimental animals and from in vitro studies that the cultivated mushroom per se and the phenylhydrazine derivatives in the mushroom have adverse e ects * To whom correspondence should be addressed. e-mail: jana.hajslova on health after chronic oral exposure. Toth and co- @vscht.cz workers have demonstrated that life-time feeding of Food Additives and Contaminants ISSN 0265±203X print/ISSN 1464±5122 online # 2002 Taylor & Francis Ltd http://www.tandf.co.uk/journals DOI: 10.1080/0265203021015634 0 854 SchulzovaÂ et al. Swiss albino mice with raw, dry baked and freeze- cessing used fruiting bodies with a diameter of 4± dried A. bisporus, as well as with three of the four 6 cm. Analytical samples were prepared from at least phenylhydrazine derivative constituents of the mush- ®ve fruiting bodies, representing the size distribution room, induce tumours in the animals (Toth 2000, of the studied mushroom sample. Water used for Andersson and Gry 2002). Agaritine was the only preparation of solutions was distilled and further phenylhydrazine constituent of A. bisporus being puri®ed using the Milli G RG puri®cation system negative in such studies (Toth et al. 1981). However, (Millipore, Germany). Methanol used for extraction it was recently observed that agaritine is unstable in was supplied by Penta (Chrudim, Czech Republic), oxygenated aqueous solutions (Hajslova et al. 2002). and methanol used in mobile phase was gradient Since Toth et al. (1981) dissolved agaritine in the grade purchased from Merck (Darmstadt, drinking water given to the animals, it seems possible Germany). The agaritine standard was synthesized that the compound was successively degraded and the by Dr Henrik Frandsen, Danish Veterinary and Food animals exposed to lower concentrations of agaritine Administration, according to the method of Wallcave than expected. et al. (1979), but with some important modi®cations being introduced (Frandsen 1998). The synthesized Until it has been established whether consumption of agaritine was > 85% pure as shown at wavelength biologically relevant amounts of the cultivated mush- 200, 237 and 280 nm by high-pressure liquid chroma- room constitutes an acceptable risk for adverse e ects tography (HPLC). The purity factor of the agaritine on health or not, it seems advisable to abstain from peak reported by the Hewlett Packard ChemStation exaggerated exposures to phenylhydrazines. We have software was 999.9 (Wallbron, Germany). The main analysed the agaritine content of purchased cultivated impurity in the standard (13.6%, coming from the mushrooms and determined the distribution of agar- agaritine synthesis) did not change during the experi- itine in the fruit body. To investigate whether storage ments reported here (HajslovaÂ et al. 2002). The and household processing of the cultivated mush- standard was protected from light and stored in the room may in¯uence the level of phenylhydrazines in freezer ( 18°C . the mushroom, we have also performed a series of ¡ † experiments using various storage conditions and cooking procedures. The stored or ready to consume products were subsequently analysed for their agar- Sample preparation itine content. The agaritine content was used as a marker for all phenylhydrazine derivatives in the Agaritine standard for identi®cation. A stock solu- cultivated mushroom, assuming all of them to be ±1 in¯uenced by the processing in a similar way tion of agaritine in methanol (0.25 mg ml ) was (Gannett and Toth 1991, Toth et al. 1997). Our data prepared. From this, 100 ml were transferred into show that the agaritine content of the cultivated vials and evaporated to dryness by a stream of mushroom is reduced to various degrees by boiling, oxygen-free nitrogen. The vials were stored under dry baking, frying and microwave heating of fresh nitrogen and sealed in the freezer. The agaritine mushrooms, as well as by household drying and standard was found stable during 1 year of storage at 18°C (the amount of agaritine was reduced by storage in the freezer followed by thawing. ¡ < 1%). Before analysis, the agaritine standard was dissolved in 1 ml Milli Q water. The stability of this solution was tested. Less than 5% of the agaritine standard was broken down within 12 h. Fresh Materials and methods agaritine standard solutions were prepared for every analytical occasion, and used within 6 h after preparation. Chemicals and samples Fresh mushrooms and mushrooms stored in the For various reasons, A. bisporus is not cultivated in refrigerator. Twenty grams of fresh mushrooms the Czech Republic. Instead, two other species are were mixed with 100 ml methanol and homogenized grown, A. bitorquis and A. hortensis. Fresh cultivated for 10 min in an Ultra Turax (Janke a Kunkel, IKA- mushrooms of these species were purchased at the Werk). The homogenate was shaken for 30 min and open market in Prague. Studies on household pro- a crude extract prepared by ®ltration. The volume of Storage and household processing on the agaritine content of the cultivated Agaricus mushroom 855 the ®ltrate was adjusted to 200 ml with methanol, matographic apparatus. Separation was carried out 10 ml of the extract was evaporated to dryness and on column (250 4 mm), LiChrospher 100 RP-18 £ the residue dissolved in 2 ml Milli Q water. This (5 m), with a precolumn (4 4 mm), Li Chrospher m £ solution was ®ltered through a micro®lter (25 mm 100 RP-18 (5 mm) (all Merck). The mobile phase was Filter Unit, 5.0 mm PTFE, PP, ThermoQuest) into a methanol±water; the mobile phase gradient 10% vial and a 20-ml aliquot injected onto the HPLC methanol held for 5 min, changing to 80% over 15 column. It is important to handle the methanolic min, held at 80% methanol for 5 min. The total run extract within 48 h after it has been prepared and to time was 25 min; post-time was 5 min. The ¯ow rate inject the aqueous solution into the HPLC system as was 1 ml min±1; the column temperature 35°C. For soon as possible (not later than 6 h after the identi®cation of agaritine, the retention time, DAD preparation of the solution). spectra and peak purity software function were used. Quanti®cation of agaritine was made by comparing Processed mushrooms and mushrooms stored in the the peak area with that for a known amount of pure freezer.

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