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Journal of Food Protection, Vol. 59, No.8, 1996, Pages 864-868 Copyright ©, International Association of Milk, Food and Environmental Sanitarians
Production of the Mycotoxin Cyclopiazonic Acid by Penicillium commune on Solid Agar Media: Effects of Water Activity, Temperature, and Incubation Time
NCEBA GQALENI,t JOHN E. SMITH,l* JOHN LACEY,2 and GEORGE GETTINBy3 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/59/8/864/1666239/0362-028x-59_8_864.pdf by guest on 25 September 2021
lDepartment of Bioscience and Biotechnology, University of Strathclyde, 204 George Street, Glasgow G1 1XW,' 21nstitute of Arable Crops Research-Rothamsted, Harpenden, Herts, AL52JQ; and 3Department of Statistics and Modeling Science, University of Strathclyde, 26 Richmond Street, Glasgow G 1 1XH, UK
(MS# 95-242: Received 20 September I 995/Accepted 4 January 1996)
ABSTRACT nutrients on CPA production in submerged culture and found that both Czapek yeast autolysate and glucose-mannitol- The combined effects of water activity (aw), temperature, peptone supported high toxin production. Temperature and incubation time, and medium composition on cyclopiazonic acid water activity (aw) are regarded as the most important factors (CPA) production by an isolate of Penicillium commune was determining fungal growth and mycotoxin production (23, studied using a full-factorial experimental design. An analysis of 24). The effect of temperature on production of CPA by variance for the mycotoxin showed that there was a complex interaction between all these factors and that this affected the individual strains of P. camembertii in liquid culture has production of CPA. The minimum aw for CPA production (0.025 to been studied by LeBars (17, 18), who found that 25°C 0.238 J.lgml-1) in yeast extract agar was 0.90 whereas in Czapek favored high CPA yields by this fungus. yeast autolysate agar it was 0.85 (0.048 to 0.123 J.lg m1-1). The However, little is known of the quantitative effect of ~ temperature range for CPA production was 20 to 30°C. The on CPA production or how aw and temperature interact. Most optimum temperature for CPA production was 25°C. CPA was previous studies have been concerned with the toxicity of maximally produced (3.199 to 3.993 J.lgml-I) at an a of 0.996 w CPA and its occurrence in food and feedstuffs. This paper after 15 days of incubation. describes the effects of water activity, temperature, medium composition, and duration of incubation on the production Key words: Cyclopiazonic acid, Penicillium commune, factorial design, mycotoxin of CPA by an isolate of P. commune, a well-known producer of CPA, using a full-factorial experimental design.
Cyclopiazonic acid (CPA) is a toxic fungal secondary MATERIALS AND METHODS metabolite produced by several Penicillium and Aspergillus species (28). The oral toxicity of CPA is low but it occurs Experimental design naturally in several foods and feeds, including cheeses and Four temperatures (20, 25, 30, and 37°C), four water activities dried food products (4, 25, 27, 30, 31, 34). CPA mostly (0.85,0.90,0.95, and 0.996), four incubation periods (5, 10, 15, and causes necrosis of the liver or gastrointestinal tissue and 20 days), and two different substrates were used in this experiment. necrotic changes in the skeletal muscle and kidney (1, 5, 25, The two media used, yeast extract sucrose (YES) and Czapek yeast 29). It is quite toxic to chickens and has been shown to be autolysate (CYA) agars, have been widely used and are described mutagenic in the Ames test 6, 33). Among the penicillia, by Samson and van Reenen-Hoekstra (32). To determine the effect CPA has previously been known to be produced by P. of each or combinations of factors, a full-factorial experiment aurantiogriseum (P. cyclopium) (14, 26). However, P. com- design was used (7, 8). Measurements for each combination of mune Thorn is currently regarded to be the most common factors were carried out in triplicate. natural producer of CPA (9, 28) and all CPA-producing isolates previously identified as P. aurantiogriseum (P. Organism and culture methods cyclopium) have been assigned to P. commune (19,27). The Penicillium commune Thorn isolate used in this study had Large quantities of CPA have been produced in liquid been isolated from moldy, damp dwellings in Scotland and shown and solid substrate media with a high water content (13, 15, to be cytotoxic to human cell lines (20). It was grown on malt 16, 21, 30). Malik et al. (21) studied the effects of some extract agar at 25°C for 7 to 10 days until conidiation had occurred. Conidia were harvested using sterile glycerol-water solution con-
taining 0.1 % Tween, with the aw adjusted to the same aw as that of * Author for correspondence. Tel (+44) 141 552 4400 ext. 2085; Fax: the growth medium, according to the methods of Gervais et al. (10) (+44) 1415531181; Email: [email protected] and Gonzalez et al. (12). CYCLOPIAZONIC ACID PRODUCTION BY PENICILLIUM COMMUNE 865
Petri dishes containing agar medium (ca. 20 ml per dish), with Larger quantities (>0.1 Ilg ml-I) were determined by the the aw adjusted using glycerol as outlined by Dallyn and Fox (3), spectrophotometric method (2), but using methanol as the solvent were inoculated with 1 ml of a conidial suspension containing 106 instead of acetone. The limit of quantitation with this method was conidia, which was spread uniformly over the agar surface. The 0.05 Ilg ml-1 for 20 ml of agar. concentration of conidia had been adjusted using a Neubauer hemocytometer. Inoculated plates of the same aw were sealed in Statistical analyses sterile polyethylene containers, in which the awwas controlled by a The data were analyzed using the ANOVA and GLM com- glycerol-water solution prepared according to Gervais et al. (10) mands in the statistical software package MinitabR version 9.2 and as such acted as humidity chambers (22), and were incubated (Minitals Inc., State College, PA, USA). under appropriate experimental conditions. RESULTS Extraction and analysis of cyclopiazonic acid At the end of each experimental period, the contents of each Interaction of water activity, temperature, incubation time, test plate was macerated with 100 ml dichloromethane/methanol and type of medium on cyclopiazonic acid production by Downloaded from http://meridian.allenpress.com/jfp/article-pdf/59/8/864/1666239/0362-028x-59_8_864.pdf by guest on 25 September 2021 (80:20) in a Waring blender. The slurry was twice filtered through a Penicillium commune Buchner funnel using Whatman No. 1 filter paper and then The ANOVA for CPA (Table 1) showed that the most extracted following the method outlined by Chang-Yen and Bida- highly significant single factors (P < 0.(01) affecting CPA see (2). The final extract was concentrated, first by rotary evapora- production by this isolate of Penicillim commune were tempem- tion and then finally under a gentle stream of nitrogen. The ture, aw, and incubation time. Differences between the two media concentrate was made up to 2 ml with methanol and used to analyze were not significant. However, most two-, three-, and four-factor for CPA. interactions were highly significant (P O.OOl), but incubation Qualitative determination of CPA was achieved by thin-layer < chromatography (TLC) separation on silica gel G60 (20 by 20 cm) time *(medium and incubation time*medium*temperature (E. MerckAG, Darmstadt, Germany). The plates were first dipped were significant only at P < 0.05. The incubation in a 10% (wtlwt) solution of oxalic acid in methanol for 2 min and time*medium*aw interaction was significant at P < 0.01. after heating at 110°C for 2 min and cooling, the plates were Penicillium commune did not grow at 37°C irrespective spotted with 50 ,.d of the extract and developed in the solvent of aw, incubation period, or medium. However, at the lower mixture toluene:ethyl acetate:dichloro-methane:formic acid (70:50: temperatures tested the amount of CPA produced was 50:20) (11). CPA was viewed after spraying with Ehrlich's reagent BDH affected by all the variables investigated. The complex (Merck, Butterworth, U.K.) (2.0 g of p-dimethyl-aminobenzaldehyde in effects of aw, temperature, incubation time, and substrate on 100 ml of HC1)with subsequent development of a purple color. CPA production by P. commune are shown in Figure 1. Small quantities «0.1 Ilg ml-I) were quantified by compar- No CPA production occurred on YES agar at 0.85 aw at ing the intensity of spots from samples, developed on TLC as any temperature. However, on eYA agar at 0.85 a CPA was above, with that of CPA standards (Sigma Chemical Co., Poole, w U.K.) containing 0.005, 0.01, 0.02, 0.03, 0.04, and 0.05 Ilg mP only produced during the late incubation periods and at 20 l prepared from a spectrophotometric ally measured CPA solution in and 25°C. At 20°C production at 15 days was 0.048 flg ml- l methanol (log E = 4.31 at 284 nm; MW 336) (18). The detection and at 20 days, 0.098 flg ml- . At 25°C the production of limit of this method was 0.02 Ilg of CPA ml-1 for 20 ml of agar. CPA was 0.056 and 0.123 flg ml-I, respectively.
TABLE 1. Analysis of variance for cyclopiazonic acid production by Penicillium commune in a full factorial experiment
Source dfa Sum of squares Mean square pb
Days 3 15,410,543 5,136,848 30.44*** Medium 1 106,667 106,667 0.63 ns Temperature 3 102,743,112 34,247,704 202.95*** aw 3 73,576,496 24,525,498 145.34*** Days * Medium 3 1,589,956 529,985 3.14* Days * Temperature 9 18,659,658 2,073,295 12.29*** Days * aw 9 10,597,166 1,177,463 6.98*** Medium * Temperature 3 7,554,890 25,188,297 14.92*** Medium * aw 3 9,700,031 3,233,344 19.16*** Temperature * aw 9 68,644,608 7,627,179 45.20*** Days * Medium * Temperature 9 3,004,522 333,836 1.98* Days * Medium * aw 9 4,499,407 499,934 2.96** Days * Temperature * aw 27 26,901,816 996,364 5.90*** Medium * Temperature * aw 9 17,888,360 1,987,595 11.78*** Days * Medium * Temperature * aw 27 16,219,304 600,715 3.56*** Error 256 43,199,388 168,748 Total 383 420,295,904
a Degrees of freedom. b Variance ratio; ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. 866 GQALENI, SMITH, LACEY, AND GETTINBY
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TABLE 2. Interaction between water activity, temperature, and substrate determining the amount of the mycotoxin cyclopiazonic acid (CPA) produced by Penicillium commune after 15 days incubation
Concentration of CPA (flg ml-1)
Water activities of the media:o
Temperature 0.85 0.90 0.95 0.996 (0C) YES CYA YES CYA YES CYA YES CYA
20 0 0.048 0.093 0.110 0.972 1.564 2.174 2.392 25 0 0.056 0.238 0.347 1.836 2.589 3.199 3.993 30 0 0 0.062 0.080 0.560 0.786 1.462 1.960 a YES, yeast extract sucrose agar; CYA, Czapek yeast autolysate agar. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/59/8/864/1666239/0362-028x-59_8_864.pdf by guest on 25 September 2021
At 0.90 aw CPA production occurred as early as 5 days range (0.88 to 0.99) in poultry feed. The same workers (24) on both media at 20 and 25°C and increased to the optimum found that A. ochraceus, P. aurantiogriseum (as P. cyclo- at 25°C (15 days) of 0.238 Jlg ml-\ on YES agar and 0.347 pium) and P. verrucosum (as P. viridicatum) produced
Jlg ml-\ on CYA agar. At 30°C on both media CPA appeared ochratoxin A over a wide aw range (0.83 to 0.99) in Czapek after 10 days incubation and reached maximum production maize extract- or malt extract-based agar media. of only 0.062 and 0.080 Jlg ml-1 on YES and CYA agars, The temperature range for CPA production by P. respectively. commune was narrower (20 to 25°C) at 0.85 aw in CYA agar.
At 0.95 aw CPA production had increased greatly in both However at 0.90 to 0.996 aw CPA was produced in both media and over the whole incubation period. Maximum media at 20 to 30°C. The optimum temperature for CPA production was again at 15 days incubation and at 25°C: production was 25°C at all aw values. Similarly, LeBars (17, l 1.836 Jlg ml-\ on YES agar and 2.589 Jlg ml- on CYA agar. 18) demonstrated that the optimum temperature for CPA Even at 30°C CPA production at 5 days was 0.115 Jlg ml-\ production by P. camembertii was 25°C on Czapek solution on YES medium and 0.308 Jlg ml-\ on CYA medium. plus 16% sucrose and 2% yeast extract. Hermansen et al. Overall, maximum production of CPA occurred 0.966 (13), testing CPA production by several Penicillium and a and at 25°C on both media: 3.199 Jlg ml-\ on YES agar w Aspergillus strains using a range of different liquid media and 3.993 Jlg ml-\ on CYA agar. At 30°C and after 5 days of and temperatures, also found that the combinations of either incubation production of CPA was 0.198 Jlg ml-1 on YES Czapek yeast autolysate or glucose-mannitol-peptone and medium and 0.275 Jlg ml-1 on CYA agar, levels only 25°C gave the highest yields. approached after 15 days incubation at 25°C and 0.90 a • w At the end of the 20-day incubation period, the concen- The optimum temperature for CPA production was tration of CPA in both media was less than that at 15, except 25°C under all aw values and, with the exception of 0.85 aw, at 0.85 a • Northold et al. (23), conducting a relatively maximum production occurred at 15 days of incubation with w CPA levels decreasing with further incubation. The maxie similar study, found that the decrease of CPA was not a result mum levels of CPA production were determined by a of a shortage of oxygen supply in the cultures, since additional oxygen did not counter the decrease. This de- complex interaction between aw, incubation period, medium composition, and above all, temperature. In most cases CYA crease in mycotoxin concentrations with time in living agar favored slightly higher production levels of CPA than systems is well recognized but is still little understood (1,9). YES agar. Table 2 shows the levels of CPA produced after 15 Thus, in the present study, the ability to use a full- days of incubation at all experimental combinations. factorial design and analysis has again demonstrated the complex nature of mycotoxin formation in a living system DISCUSSION exposed to varying environmental conditions. CPA produc- tion in Penicillium commune was achieved only when the Water actIvIty, incubation time, and temperature all correct combination of temperature, aw, and substrate were significantly affected CPA production, and most two-, three-, present. The duration of exposure to these optimum condi- and four-factor interactions were also highly significant. The tions was also important with prolonged exposure (20 days) leading to a change in tl1e internal metabolic system leading medium (substrate) played an important role in the aw and temperature range for CPA production by P. commune. The to decreased CPA concentration. While incubation time was aw range for CPA production was narrower (0.90 to 0.996) critically important for aw values of 0.90 to 0.996 it was not on YES agar than on CYA agar (0.85 to 0.996). Northolt et so for an aw of 0.85 where CPA production was still al. (23) also found that penicillic acid (PA) production by A. increasing at 20 days. ochraceus, P. aurantiogriseum (as P. cyclopium) and P. The use of full-factorial experimental designs for myco- martensii was influenced by aw and type of substrate. On toxin production should assist in making more intelligent malt extract sucrose and malt extract glucose agars, the fungi predictions regarding the safe storage of foods and feed- had a narrow aw (0.97 to 0.99) for PA production, but a wide stuffs. 868 GQALENI, SMITH, LACEY, AND GETTINBY
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