Letters in Applied Microbiology 1986, 3, 123-125 MOM/W8

The effect of water activity and pH on the production of mycotoxins by fungi growing on a bread analogue

M A R G A R E T PATTERSON 8z A . P . DAMOGLOU Department of Agricultural and Food Bacteriology, Queen’s University of Belfast and Agricultural and Food Bacteriology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5 P X , Northern Ireland

Received I 1 July 1986 and accepted 17 September 1986

PATTERSON, M . & DAMOGLOU, A.P. 1986. The effect of water activity and pH on the production of mycotoxins by fungi growing on a bread analogue. Letters in Applied Microbiology 3, 123-125.

Mycotoxin production by various toxigenic fungi, growing on a bread analogue, was investigated at various water activities (a,) and pH combinations. Citrinin, ochratoxin A and sterigmatocystin could be detected at a, > 0.80, while patulin was only observed at a, = 0.95. These results show that some toxins may be produced at lower water activities than have been reported on synthetic media and suggest that, where possible, natural substrates should be used to investigate factors affect- ing mycotoxin production in foodstuffs.

Environmental conditions such as water activity (a,), pH and temperature are known to influ- ence growth of fungi and mycotoxin production (Northolt & Bullerman 1982). Northolt et al. (1978, 1979) studied the effect of a, and tem- perature on mycotoxin production by growing toxigenic fungi on semi-synthetic media and adjusting the a, with glycerol or sucrose. Roland & Beuchat (1984) studied the effect of a, and temperature on patulin production in apple juice by Byssochlarnys niuea, with the a, being altered by the addition of sucrose. The use of sucrose to adjust the a, is not entirely satisfac- tory as this may be utilized as an additional energy source. However, it has been suggested that the rate of toxin production on natural substrates may be different from that on syn- thetic media (Jarvis 1971).

Bread is a suitable substrate for the pro- duction of various toxins (Patterson 1984). It was proposed that a bread analogue could be prepared at a variety of a, and pH values and the growth of toxigenic fungi and mycotoxin production be investigated.

Materials and Methods

P R E P A R A T I O N OF T H E B R E A D A N A L O G U E

Bread dough was prepared by mixing together flour (100 g), grated margarine ( 5 g), salt (0.5 g), baking powder (0.5 g) and yeast extract (0.5 g) with the liquids to give the appropriate a, (Table 1). The pH of the dough was 5.6. The pH was adjusted by the addition of 1 ml 10 mol/l HCl in the liquid mixture to give pH 4.2 or 1 ml 10 mol/l KOH to give pH 8.2. The dough was shaped into a block, approximately 8 x 8 x 2 cm, wrapped in greaseproof paper and autoclaved at 121°C for 15 min. When cool, the substrate was sliced aseptically and the pieces placed in sterile petri dishes (sample weight 8.0 f 0.5 9). The samples were inocu- lated, as described by Damoglou et al. (1984). with 5 x lo3 spores of Penicilliurn uiridiccitvn; ATCC 26169 for the production of citrinin zinc ochratoxin A; P. expansurn CMI 39761 for the production of patulin; or Aspergillus uersicolor 44605 (supplied by H.P. van Egmond, Labor- atory for Chemical Analysis of Foodstuffs, The

124 Margaret Patterson and A . P . Damoglou Table 1. Adjustment of water activity

Liquids added (m11100 g bread mix)

a Water Glvcerol

0.95 70 0.92 60 0.87 50 0-85 47 0.80 45 0.75 35

-

10 20 23 25 35

Results

The water activity and pH of the uninoculated control samples did not vary significantly during the 10 d incubation. Growth of P. viridicatum and A. versicolor was observed at a,., = 0.80 and above, but P. expansum did not grow below a, = 0.85. The effects of a, and pH on toxin production are shown in Fig. 1.

Netherlands) for the production of sterigma- tocystin. The pH and a, of control, unin- oculated samples were measured before and after incubation for 10 d at 25°C. The pH was measured with a surface probe fitted to an E512 meter (Metrohm Herasui); the a, was measured with a dewpoint meter (Protimeter DP680). The samples were incubated in a humidity cabinet (Townson & Mercer, England) placed inside a 25°C incubator. A saturated solution with the same a, as the breads (Robinson & Stokes 1955) was placed in the cabinet and the system left to equilibrate for 3 d. The a, inside the cabinet was checked before the samples were incubated.

M Y C O T O X I N A N A L Y S I S

Triplicate samples were extracted after 7 and 10 d incubation.

Citrinin and ochratoxin A

Each sample was stomached (Colworth 400 Stomacher) with a mixture of 50 ml chloroform and 5 ml 0.1 mol/l H,PO, for 30 s. Citrinin and ochratoxin A were extracted and quantified as described by Damoglou et al. (1984).

Patulin

The samples were stomached for 30 s with 50 ml ethyl acetate and extracted and quantified as described by Damoglou et al. (1985).

Sterigmatocystin

The samples were stomached for 30 s with a mixture of 50 ml acetonitrite and 5 ml KCl con- taining 40 g/1 KC1 and the toxin extracted and quantified using the method of Stack & Rod- ricks (1971), omitting the clean-up procedure.

Discussion

The results indicate that ochratoxin A, citrinin and sterigmatocystin can be produced over a wide range of a, and pH values at 2 5 T , the optimum temperature of the producing fungi. The toxins were detected in low concentrations at a, = 0.80 with greatest production after 10 d at a, = 0.92 and pH 5.6. Northolt et al. (1979) grew P. viridicatum on a Czapek maize agar with the addition of sucrose or glycerol to adjust the a, and found the minimum for ochra- toxin A production to be 0.83-0.86, depending on the strain of organism. They also reported that optimum toxin production was achieved at a, 0-95-0.99 compared with an optimal a, of 0.92 on the bread substrate. Citrinin produced simultaneously with ochratoxin A showed similar values for minimal and optimal toxin production, but was generally produced in much greater quantities than ochratoxin A.

Sterigmatocystin production was found to be optimal at a, 0.92 and at a minimum at 0.80 in contrast with the results of Northolt & van Egmond (1981) who reported that sterigmatocy- stin production on agar media only occurred below a, 0.93 with a lower limit of 0.85. Patulin production was found to be most dependent on a, for, although growth of P. expansum was observed above 0.85, the toxin was not detected until 0.95. This high a, requirement for patulin production has been observed by other workers (Northolt et al. 1978; Roland & Beuchat 1984).

Foodstuffs provide a complex nutritional medium and allow for physical interactions such as a, and pH to affect growth and toxin pro- duction of fungi. This may account for differ- ences observed for limits of toxin production on the bread analogue compared to synthetic sub- strates. This would suggest that, where possible, natural substrates should be used to investigate factors affecting the production of mycotoxins in foodstuffs.

a, , p H and mycotoxin production 125 Ochratoxin A I LSD

Pa tuli n 8 r

I LSD

0, 0.8- 0.74 4.0

Steriamatocvstm

Fig. 1. Production of ochratoxin A and citrinin by Penicillium uiridicatum, patulin by Penicillium expansum and sterigmatocystin by Aspergillus uersicolor on a bread analogue substrate at varying water activities and pHs. 0, Toxin production after 7 d incubation at 25°C; D, toxin production after 10 d incubation at 25°C.

References

DAMOGLOU, A.P., DOWNEY, G.A. & SHANNON, W. 1984 The production of ochratoxin A and citrinin in barley. Journal of Science of Food and Agriculture 35,395400.

DAMOGLOU, A.P., CAMPBELL, D.S. & BUTTON, J.E. 1985 Some factors governing the production of patulin in apples. Food Microbiology 2, 3-10.

JARVIS, B. 1971 Factors affecting the production of mycotoxins. Journal of Applied Bacteriology 34,

NORTHOLT, M.D. & VAN EGMOND, H.P. 1981 Limits of water activity and temperature for the production of some mycotoxins. Proceedings of the 4th meeting on mycotoxins and animal disease, Weybridge, England, pp. 106108.

NORTHOLT, M.D. & BULLERMAN, L.B. 1982 Preven- tion of mould growth and toxin production through control of environmental conditions. Journal of Food Protection 45, 519-526.

199-21 3.

NORTHOLT, M.D., VAN EGMOND, H.P. & PAULSCH, W.E. 1978 Patulin production by some fungal species in relation to water activity and tem- perature. Journal of Food Protection 41,885-890.

NORTHOLT, M.D., VAN EGMOND, H.P. & PAULSCH, W.E. 1979 Ochratoxin A production by some fungal species in relation to water activity and tem- perature. Journal of Food Protection 42,47&484.

PATTERSON, M.F. 1984 PhD thesis, Queen’s University of Belfast, UK.

ROBINSON, R.A. & STOKES, R.H. 1955 Electrolyte Solu- tions, p. 490. London: Butterworth.

ROLAND, J.O. & BEUCHAT, L.R. 1984 Influence of tem- perature and water activity on growth and patulin production by Byssochlamys niuea in apple juice. Applied and Enuironmental Microbiology 41, 205- 207.

STACK, M. & RODRICKS, J.V. 1971 Method for analysis and chemical confirmation of sterigmatocy- stin. Journal of the Association of Official Analytical Chemists 54, 8690.