Patulin Production by Penicillium roqueforti Thom from grape

Patulin Production by Penicillium roqueforti Thorn from grape J. Harwig, B. J. Blanchfield and P.M. Scott Health Protection Branch, Tunney's Pasture, Ottawa, Ontario, Canada, KIA OL2

Abstract

Experimental Methods

Seven isolates of Penicillium roqueforti were isolated from moldy rapes and examined for toxin production. In 5-ml cultures, all produced ~atulin a~t~r 9 days. of gro~th at 25 C in yeast extract (2% )-sucr~se (15%) medium In amounts varyIng from 20 to 1,267 Ilg per culture. SIX cheese isolates of P. roqueforti did not produce patulin under these conditions. One grape isolate tested also produced patulin in fresh grapes. The physicochemical properties of crystalline patulin from one grape isolate corresponded with those .of a~then~ic patulin. Gent!s~l alcohol and I,4-hydroquinone were also IdentIfied In extracts of thIS Isolate.

Isolation of P. roqueforti. Information on the crushed, moldy Gamay Beaujolais grapes examined 1n this study has been presented previously (Scott et al., 1977a). For isolation of Penicillium spp., a thawed piece of grape was placed on each of 50 Petri plates of potato dextrose agar (PDA) and malt extract agar which were then incubated at 25 C. Plates were examined periodically under a stereoscopic microscope for Penicillium expansum Link and P. roqueforti. P. expansum HPB 050576 and P. roqueforti HPB 110376-8 were used as reference cultures; both cultures had earlier been isolated from grapes and had been identified by Dr. R. A. Samson, Centraalbureau voor Schimmelcultures (CBS), Baarn, the Netherlands. After single spore isolation on PDA, subcultures of isolates resembling P. roqueforti were sent to CBS for confirmation. Stock cultures were maintained by successive single spore transfers on PDA.

Resume Sept souches de Penicillium roqueforti isolees de raisins moisis ont ete examinees pour la production de mycotoxines. La patuline etait produite par chacune des souches; Ie dosage variait entre 20 et 1,267 Ilg par 5-ml de culture apres 9 jours d'incubation a 25 C dans un milieu de culture comprenant de l'extrait de levure (2%) et de la saccharose (15%). Six souches de P. roqueforti isolees de fromage n'ont pas produit de patuline dans ces memes conditions de culture. La presence de patuline a ete decelee dans des raisins ensemences avec une des souches isolees de raisins. Les proprietes physicochimiques de la patuline crystallisee provenant d'une souche isolee de raisins correspondent a celles de la patuline authentique; l'alcohol gentisique et Ie 1,4-dihydroxybenzene ont aussi ete identifies dans les extraits.

Introduction P. roqueforti is a major component of the microflora of Roquefort and blue cheese, and of moldy corn silage (Pelhate, 1974; Raper and Thorn, 1968). The mold is sometimes predominant in silage associated with mycotoxicoses of livestock (Kanota, 1970, Wei et al., 1973). Some isolates from chestnuts, pecans, rice, corn silage, and cheese are toxic to laboratory animals, tissue cultures, or brine shrimp (Durackova et al., 1976, Kurata et al., 1968, Lafont et al., 1976, Wei et al., 1973, Wells and Payne, 1975 and 1976). However, long term feeding of blue cheese and a P. roqueforti strain used commercially for blue cheese production gave no evidence of toxic or carcinogenic effects in rats (Frank et al., 1977). Known metabolites of P. roqueforti isolated from different sources include PR toxin (Wei et al., 1975) and related metabolites (Moreau et al., 1976), three partially characterized compounds (Kanota, 1970), and various alkaloids (Ohmomo et al., 1975, Scott et al., 1977b). With the exception of PR toxin which has an oral LD 50 of 115 mg/kg in weanling rats (Wei et al., 1973), the oral toxicities of these metabolites are unknown. It is not clear which compounds account for the toxicity evident in some P. roqueforti isolates. PR toxin was not present in the most toxic extracts tested by Lafont et ale (1976). The mycotoxin patulin is produced by a variety of molds (Escoula, 1975, Stott and Bullerman, 1975). To our knowledge, no published evidence is available for patulin production by P. roqueforti, but in an oral presentation Leistner and Pitt (1976) have mentioned its production by P. roqueforti isolates from meat products. This paper provides evidence for the production of patulin by P. roqueforti isolates from moldy grapes. Can. Inst. Food Sci. Technol. J. Vol. II, No.3, July 1978

Inoculation and Incubation. Single spore isolates obtained in this work and cheese isolates obtained previously (Scott et al., 1977b) were tested for patulin production on a semi-micro scale (Scott et al., 1970). They were subcultured on PDA for about 1 week at 25 C. Conidia were released from the mycelium by flooding and shaking the cultures with 0.05% aqueous Tween 80 (polyoxyethylene sorbitan mono-oleate). Conidial suspensions were used to inoculate yeast extract (2%)-sucrose (15%) medium (YES) at a concentration of 10 6 conidia/ml YES. In small-scale experiments, vials containing 5-ml YES were incubated at 25 C. To obtain patulin on a large scale, adequate for chemical identification, P. roqueforti HPB 011276-17 (10 6 conidia/ml YES) was also cultured in 800-ml Roux bottles. The bottles containing 200-~1 YES were incubated at 25 C _ for 16 days. The suitability of grapes as a substrate for patulin production by P. roqueforti was examined. Fresh Tokay grapes were damaged by applying a I-em, longitudinal, superficial cut to each grape. A loopful of conidia of P. roqueforti HPB 110376-8 was then put into the cut. Duplicate samples of inoculated and non-inoculated grapes were incubated at 25 C. Analysis and isolation of patulin. Small-scale cultures were extracted by boiling with chloroform (Scott et al., 1970) and blending three times with chloroform. Chloroform extracts were passed through Na2 S0 4 before concentration under N 2 • Residues were redissolved in 0.2-20.0 ml chloroform. Amounts of patulin were determined semiquantitatively by thin layer chromatography (TLC) with chloroform-acetone (9: 1, vlv, CA) or toluene-ethyl acetate-90% formic acid (5:4:1, v/v/v, TEF) as solvent systems and phenylhydrazine hydrochloride or 3-methyl-2benzothiazolinone acidified with formic acid as spray rea-

149

gents (Scott et al., 1977a, Scott and Somers, 1968). The estimated detection limit of patulin in the chloroform extract of a single culture was 2 fLg. For large-scale isolation of patulin, filtrates of 'P. roqueforti cultures (HPB 011276-17) were extracted with ethyl acetate. Combined extracts Jrom 16 Roux bottles were evaporated, and the residue (2.60 g) was partially dissolved in benzene-ethyl acetate (60:1, v/v) and chromatographed on 25 g of silica gel 60 (E. Merck). Column fractions (20 ml) were collected and monitored by TLC on silica gel 60 layers developed with CA, using 4% phenylhydrazine hydrochloride (Scott and Somers, 1968) or acidic anisaldehyde (Scott et al., 1970) as spray reagents. Patulin (R f 0.2) was eluted-with benzene-ethyl acetate (9: 1) in 39 fractions. Combined major fractions were crystallized separately from benzene. Inoculated and non-inoculated grape samples (50 g) were analyzed for patulin as described (Scott et al., 1977a). Determination of dry weight and pH.. Mycelial mats were weighed after overnight drying of washed mats at 70 C. The pH of culture liquids was determined electrometrically before extraction of cultures.

Results and Discussion Colonies resembling P. roqueforti and P. expansum were observed in 25 and in 16 of 100 Petri plates, respectively. Therefore, P. roqueforti occurred in this sample at least as frequently as P. expansum. Figure 1 shows that both grape isolates tested for patulin follow essentially the same pattern of production and decline. Maximum amounts of patulin in small scale cultures were present be800

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Fig. I.

150

Amounts of patulin and pH changes in 5-ml YES cultures of 2 grape isolates of P. roque/orti grown at 25 C. Vertical bars represent standard errors.

tween 7 and 9 days; at this time, the pH of the culture liq. uid had declined from 6.4 to about 5.0. Dry weights of my. celial mats did not increase significantly after 5-6 days. 1\ sharp decrease in patulin after 9 days coincided with an in. crease in the pH, presumably due to onset of autolysis Patulin may disappear by reaction with autolysis produc~ of the mold; reactions of patulin with sulihydryl com. pounds increase with-increasing-pH (Hofmann et al. 1971~

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Five other isolates from grapes and (5 isolates from cheese were also examined for patulin after 7 and 9 days of incubation (Table 1). Only the grape isolates produced patulin and lowered the pH considerably. Toxin production ranged from 20 to 1,267 fLg per culture after 9 days of incubation. The great differences between some of the iso. lates indicate that these isolates are genetically unrelated and may have originated from several sites of infection. The large scale isolation experiment confirmed the production of patulin by P. roqueforti HPB 011276-17. This experiment yielded a total of 119 mg of crystalline patulin. An additional 86 mg of patulin was estimated by TLC to be present in the mother liquors. The purest sample of patulin had m.p. 109-110 C and ultraviolet abo sorption (in ethanol) at 276 nm (e 14,925); its mass spectrum, mixed m.p., and infrared spectrum agreed with that of authentic patulin isol~ted from P. patulum. Earlier column fractions containing patulin were con· taminated by 1,4-hydroquinone (R f 0.16), which was ob· tained as chloroform-insoluble crystals (6.5 mg), m.p. 174 C, from 8 fractions collected before the elution of patulin and identified by comparison with a standard (mixed m.p. and ultraviolet, infrared and mass spectra). 1,4-hydroquinone is a hitherto unknown fungal metabolite. It was not detected in non-inoculated YES. Fifty-two mg of a second chloroform-insoluble crystalline material, C7 Hs 0 3 (from high resolution mass spectrometry), m.p. 92-94 C, R f 0.05, was eluted from the column by benzene-ethyl acetate (85: 15 to 75:25). Although not completely pure, it was structurally characterized as gentisyl alcohol from its ultraviolet spectrum (maximum 296 nm, in ethanol), infrared spectrum, mass spectrum (m/e 140, 122, 110,94,82, 81, 66, 65, 55, 39), and 100 MHz nuclear magnetic resonance spectrum in DMSO-dolo 4.41 (d, 2H, J = 5 Hz; C!:!20H), 4.88 (t, IH, J = 5 Hz; - CH 20H), 6.47 (m, 2H; Ar-H), 6.73 (d, IH; Ar-H), and 8.54 (s, 2H; Ar-OH) ]. According to Scott and Beadling (1974), gentisyl alcohol is a precursor of patulin in P. patulum. A mauve spot at R f 0.16 was detected by TLC with the anisaldehyde reagent; this unidentified metabolite started to elute from the silica gel column after most of the patulin but was only partially separated from gentisyl alcohol. It was isolated by preparative TLC and crystallized from methanol-water as colorless needles (32 mg); after 2 recrystallizations the purest sample had m.p. 139-142 C, showed no ultraviolet absorption above 205 nm (in ethanol), and had infrared absorption (in chloroform) at 3680, 3600, 3460, 2950, 2862 and 1722 cm,l; mass spectrometry (electron impact and chemical ionization) suggested a molecular weight of 236. In a previous publication (Scott et al., 1977a), it was concluded that patulin in moldy Rauschling grapes had probably been produced by P. expansum. Our present results indicate that in patulin grapes may also result from 1. Inst. Can. Sci. Technol. Aliment. Vol. II. No.3. Juillet 1978

Table 1.

Amounts of patulin and pH changes in 5-m1 YES cultures of grape and cheese isolates of P. roqueforti grown at 25 C. 7 days incubation

Isolate No.

pHa

HPB 011276-1 c HPB 011276-2 c HPB 011276-3 c HPB 011276-4 c HPB 011276-5 c HPB061175-1 d HPB 061175-2 d HPB 061175-3 d HPB 061175-4 d HPB 111275 d 596Ad

4.6 4.6 4.5 5.1 4.4 5.0 5.7 5.7 6.3 6.2 5.1

9 days incubation

/Lg patulin/culture b

pH

J-Lg patu1in/culture b

0

4.4 4.5 4.2 5.3 4.2 5.1 5.5 5.5 6.0 6.1 5.2

20±4 800±100 1,267 ±133 333 ±41 680±227 0 0 0 0 0 0

1,067±167 300±50 30±16 8±5 0 0

0 0 0 0

aInitial pH of YES was 6.4. bMean ± standard error, triplicate cultures. cGrape isolates. dCheese isolates.

growth of P. roqueforti; 4.7 and 7.3 ppm patulin were detected in inoculated grapes after 13 and 16 days of incubation, respectively. There is a need to identify more definitively the species of Penicillium known to invade sometimes grapes before harvest, in transit, and in storage (Harvey and Pentzer, 1960). Escoula (1974) linked the presence of patulin in moldy corn silage to patulin-producing strains of Byssochlamys nivea. We suggest that P. roqueforti isolates from corn silage also be examined for patulin production. None of the cheese isolates examined here produced patulin in YES (Table I). Even if they would produce patulin under the conditions provided by a cheese substrate, patulin might not be detectable. The compound re·acts with cheese components, thereby yielding reaction products with diminished antibiotic activity (Stott and Bullerman, 1976).

Conclusions P. roqueforti isolates from grape are capable of producing patulin in culture and in fresh grapes. In addition to P. expansum, P. roqueforti may therefore be one of the molds responsible for the presence of patulin in naturally moldy grapes and grape products.

Acknowledgements Weare grateful to R. A. Samson for identifying the P. roqueforti isolates, W. F. Miles, F. Benoit and R. O'Brien for recording the mass spectra, and R. R. Capoor and R. R. Fraser for the nuclear magnetic resonance spectrum.

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Can. Inst. Food Sci. Technol. J. Vol. 11, No.3, July 1978

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