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Effects of potassium sorbate and environmental factors on growth of tobacco spoilage fungi
Mycol. Res.
94 ( 7 ) :971-978 (1990)
971
Printed in Great Britain
Effects of potassium sorbate and environmental factors on growth of tobacco spoilage fungi
E. S. MUTASA*, N. M A G A N t A N D K. J. SEAL Biofechnology Centre, Cranfield lnstitufe of Technology, Cranfield, Bedford MK43 OAL
Effects of potassium sorbate and environmental factors on growth of tobacco spoilage hngi. Mycological Research 94 (7):971-978 (1990). Potassium sorbate was found to inhibit germination and growth of a range of spoilage fungi isolated from tobacco below pH 5. However, at and above pH 5 the efficacy of potassium sorbate was influenced by substrate composition and water activity (a,). Eurotium spp., Aspergillus flavus, A. fumigafus, A. niger, and Penicillium species all grew with 0.1-0.3% sorbate at 0.95 a, on malt, Virginia and Burley extract agars. However, at 085 a,, only Eurotium spp., A. flavus, P. chysogenum and P. viridicafum were able to grow in the presence of sorbate. At both high and low a, the tolerance of sorbate was influenced by substrate composition. The lag time for growth initiation in different concentrations of sorbate was significantly influenced by substrate composition for E. amstelodami and P. chysogenum but not for A. niger. Growth of E. amstelodami, A. flavus and P. chysogenum was significantly less on Burley extract agar than Virginia extract or malt agar over the range 0-754.90 a, at pH 5 and 6-5 and with 01+4% sorbate. Under some environmental conditions, growth of E. amsfelodami on tobacco extract agars was stimulated by sorbate. Key words: Growth inhibition, Potassium sorbate, Water activity, pH, Tobacco, Aspergillus, Penicillium.
Sorbic acid and its salts, especially potassium and calcium, are the most widely used preservatives in tobacco as well as in a wide range of food products against fungal spoilage. Sorbate is increasingly preferred because of its physiological inertness and organoleptic neutrality (Sofos & Busta, 1981). Maximum levels of sorbic acid currently permitted in tobacco are 0.5 % (w/w) in cigarette papers and hand-rolling tobacco and 1% in pipe tobacco (DHSS Report, 1989). However, the antimicrobial capabilities of sorbic acid and its salts are dependent on pH (Bandelin, 1958). It is most effective in its undissociated or unionized form at a pH below 4-75. The proportion of undissociated acid varies from 6-7% at pH 6.00 to 50% at pH 4.75 and 98% at pH 3.00 (Sofos & Busta, 1981). The dominant spoilage fungi of different flue-cured stored tobaccos varies with storage water content but includes Aspergillus spp., including A. niger, A. uersicolor, Eurotium spp. as well as Penicillium chysogenum, and P. viridicafum (Welty & Lucas, 1968; Mutasa, 1988). Many of these species, particularly Aspergillus and Eurofium spp., are xerophilic, and therefore able to grow at below 0.85 water activity (a,) (Ayerst, 1969; Pitt, 1975 ; Magan & Lacey, 1984). Although potassium sorbate is widely used as a preservative in tobacco there is very little information on its efficacy against the predominant spoilage fungi on interaction with a,, pH and tobacco type. Both * Present address: School of Biological Sciences, Queen Mary College, Mile End Road, London El 4NS.
t Corresponding author.
germination and growth of Aspergillus spp. have previously been found to vary considerably with concentration of other preservatives such as ammonium propionate formulations and with a, (Magan & Lacey, 1986a). The objectives of this study were to investigate the effect of up to 0.3% potassium sorbate on the dominant spoilage fungi of tobacco at both high and low a, and to determine the effect of interaction with pH and nutrient substrate on growth of some Aspergillus and Penicillium species.
MATERIALS A N D M E T H O D S Fungal isolates Eurotium amsfelodami Mangin (4 isolates), E. chevalieri Mangin, E. rubrum Konig, Spieckerrnan & Bremer (anamorph, Aspergillus rubrobrunneus Samson & Gams) (2 isolates), Aspergillus awamori Nakazawa, A. Jlnvus Link, A. fumigatus Fres. (2 isolates), A. niger van Tiegh. (10 isolates), A. sydowii (Bain. & Sart.) Thom & Church, A. versicolor Tiraboschi (5 isolates), Penicillium chysogenum Thorn (5 isolates), P. crustosum Thorn, P. viridicafum Westling, P. spinulosum Thorn, Scopulariopsis brevicaulis (Sacc.) Bain. and Syncephalastrum racemosum (Cohn) Schroter used in this study were all obtained from flue-cured tobaccos and represent the dominant fungi isolated from 5 different tobacco types (Mutasa, 1988). The identity of all species was confirmed by the CAB Intemafional Mycological Institute, Kew.
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Potassium sorbate and growth of tobacco spoilage fungi
Table 1. Concentrations of potassium sorbate allowing growth of fungal isolates on malt extract and two tobacco extract agars at two water activities Growth medium.. . Malt extract Virginia extract Burley extract Water activity.. . Fungal isolates E. E. E. E. E. E. E.
arnstelodami umsfelodami amstelodami amsfelodami chevalieri rubrum rubrum
0.96
0.85
0.96
0.85
0.96
0.3 0.3 N.G. 0.3 0.2 0.3 0.2
0.I
0.3 0.3 0.2 0.3 0.2 0.3 0.3
0. I 0.3 0.2 0.3 N.G. N.G.
N.G. N.G. N.G. N.G. N.G. N.G.
0.2 0.3 0.2 0.2 0.3 0.3
N.G.
0.3 N.G. 0.2
0.1
N.G. N.G.
0.1
0.3 0.3
0.3 0.3 0.3 0.3 0.2
0.1
0.2 0. I
N.G.
0.1
0.I
0.1
0.2 0.2 0.3
A. niger (3) A. niger A. niger A. niger A. niger A. nige~ A. niger A. niger A. awarnori
0.1 N.G. N.G. N.G. N.G. N.G. 0.3 0.1 0.1
0.3 N.G. 0.3 0.3 0.3 0.3 0.3 0.3 0.2
N.G. N.G. N.G. N.G. N.G. N.G. N.G.
A. flavus A. fumigatus A. fumigatus
N.G. N.G.
N.G.
0.1
0.1 0.1
N.G. N.G.
0.3 0.3 0.2 0.3 0.3 N.G. 0.3
N.G. N.G. N.G. N.G. N.G. N.G. N.G. N.G.
0.3 0. I 0.2 0.3 0.3 0.3
N.G.
N.G. 0.3
N.G. N.G.
0.3 0.2
0.1 0.1
A. versicolor A. versicolor A. versicolor A. versicolor A. versicolor A. sydowii
P. P. P. P. P. P. P. P.
chysogenum chrysogenum chrysogenurn chrysogenum chrysogenurn crwtosum viridicatum spinulosurn
S. racernosurn Sc. brevicaulis
N.G.
0.2 N.G. 0.3
0.85
N.G.
0.1
N.G. 0.1
0.1 N.G. N.G. N.G. N.G. N.G. N.G. N.G.
0.1
0.I
0.1
0. I 0.2
0.3 0.3 0.3
0.3 0.3 N.G. 0.1
N.G. N.G. N.G. N.G. N.G. N.G. N.G. N.G. 0.1 N.G. N.G.
0.3 0.3 0.3
N.G. N.G.
0. I
0.1
0.I
N.G. 0.3 N.G. 0.1 N.G.
0.2
0.1
N.G., no growth. A, Aspergillus; E, Eurotium; P, Penicillium; S, Syncephalasfrum; Sc, Scopulariopsis. (3). three isolates.
Water activity media Malt extract agar (3%) was modified by the addition of glycerol (Dallyn & Fox, 1980) to 0.96 and 0.85 a,. Two tobacco extract agars were prepared by the addition of ground Virginia (high sugar) or Burley (low sugar) tobacco to water and steaming the mixture for 30 min. The tobacco grounds were removed using a buchner filter funnel and filter paper (Whatman No I). These media were subsequently adjusted to 0.75, 0.80, 0.85, 0.90 and 0.96 a, with glycerol. The a , of all solidified media was checked with a Humidat IC I1 (Novasina AG, Switzerland).
Fungal growth in relation to sorbate concentration, pH and a , Filter sterilized potassium sorbate was added at levels of 0.1, 0.2, 0.3 and 0.4 % (w/v) to all three media previously adjusted to 0-96 and 0.85 a,. The pH was then adjusted with 1 MNaOH or 0.1 M-HCI to 4.5, 5.0 and 5.5. Non-bleeding indicator strips were used to check the pH of molten media and a flathead pH probe to determine the final pH of solidified media. Spores from 94 isolates of test fungi, grown for 7 to 14 d on either malt extract or malt yeast glucose (40%) agar, were
E. S. Mutasa, N. Magan and K. J. Seal
973
tested for their ability to grow in the presence of these concentrations of potassium sorbate. The plates were centrally inoculated with 2 ~1 aliquots of spore suspensions prepared in 0.1% distilled water agar. Ten replicates of each isolate and each treatment were prepared. The plates were sealed with parafilm and incubated at 25 OC until growth occurred, or for a maximum of 6 months. Outgrowth of at least 5 replicate colonies to a diameter of 10 and 5 mm at 0 9 6 and 0.85 a, respectively was used as the criterion for growth. The time required for growth was also monitored. To ensure that pH was not directly responsible for growth inhibition, all fungal isolates which tolerated at least 01% sorbate above pH 5 were tested for their ability to grow at pH 4.5. These experiments were carried out on malt extract agar buffered with 100 mM acetate. Plates were inoculated as described previously and 5 replicates incubated at 25' for up to one month. More detailed studies were subsequently carried out on E. amsfelodami, A. flavw and P. chysogenum which were able to grow with 0 . 2 4 . 3 % sorbate at 0.85 a,. Burley and Virginia extract agars were modified to 0.75-0.90 a,, and sorbate incorporated in the range 0.1 to 0.4%. The pH was adjusted to 5.0 and 6.5. Ten replicate plates per treatment were inoculated for each test fungus and incubated at 25' for up to 3 months. Radial growth rates were measured at regular intervals, appropriate to each a, level. Linear regression analyses of the mean radial extension rates were used to calculate the growth rates for each treatment. The standard errors of the means were calculated and confidence limits (at 0.98 level) attached.
RESULTS Fungal growth in presence of sorbate On malt extract agar all the fungal species and isolates were effectively inhibited by 0.1 % sorbate below pH 5, and by 0.2 or 0 3 % at pH 5. At high a , (pH greater than 5) a total of 38 isolates was able to grow in the presence of sorbate on malt extract agar, 32 on Virginia extract and 35 on Burley extract
agars (Table I). Lowering the substrate a, to 0.85 reduced the number of isolates able to grow in the presence of sorbate on each substrate as well as changing the sensitivity of some organisms. Members of the Eurotium group grew in the presence of 0.3% sorbate at 0.85 a, on all three substrates, although at 0.96 a , some isolates could only grow on media containing 0.1 % sorbate. More isolates of E. amsfelodami grew with 0.3 % sorbate in Burley than Virginia or malt extract agars at 0.85 a,. Members of the A. versicolor group were unable to grow in the presence of 0 3 % sorbate in malt extract agar at either 0-96 or 0.85 a,. Most other isolates required > 0.3% sorbate to inhibit growth at 0.96 a , on Virginia and Burley extract agars. However, at 0.85 a,, 0.3% sorbate was sufficient to inhibit most isolates but sensitivity to sorbate was greater on Virginia than on Burley extract agar (Table I). Almost all the A. niger isolates tested were able to grow in all three media at 0.96 a , with 0.3% sorbate, but at 0.85 a, seven of the ten isolates were inhibited by 0.1% sorbate. Growth of all the Penicilliurn spp. on malt extract agar at 0.96 a , was inhibited by 0.3 % sorbate but at 0.85 a, only two isolates of P. chrysogenum could grow with 0.1% sorbate. By contrast, on Virginia and Burley extract, many of the Penicillium species could grow at 0.96 a, with 0.3% sorbate but at 0.85 a, all were inhibited by 0.1 % sorbate on Virginia extract, but only half on Burley extract agar. O f the other species tested, A. fumigafus grew with 0 . 2 4 . 3 % sorbate on all media at 0.96 a, but only with 0.1 % sorbate at 0.85 a, on malt extract agar. A. fiavw was tolerant of 0-2+3 % sorbate at both 0.96 and 0-85 a, on both Virginia and Burley extract agars. S. racemusum and S. brevicaulis grew with 0.2-0.3 % sorbate at 0.96 a, and with 0.1 % at 0.85 a, on Burley extract agar but were more sensitive on Virginia extract and malt extract, respectively.
Interactions of sorbate concentration and substrate on growth initiation The time required for growth of species of E. amsfelodami, A.
Table 2. Lag times before growth of three isolates each of Eurotium amstelodami, Aspergillus niger and Penicilliurn chysogenum with up to 0.3% potassium sorbate on Virginia and Burley tobacco extract agars at 25' and 0.95 a, Time (d) Tobacco type.. . Virginia
Burley
Percentage sorbate (%) ... Species Isolate
0
E. amsfeiodami
I 2 3
4 4.5 4.5
9 13.5 11
> 28 > 28
> 28 > 28
9
A. niger
I 2 3
1.5 1.5 1.5
2 1.5 2
2 2 2
P. chysogenum
1 2 3
2 3 1.5
0.1
0.2
5
5
> 28
> 28
2
2
0.3
0
0.1
0.2
9
5 5 7
65 7.5 9
7 7 8
2.5 2.5 2
2 2 1.5
2 2 2
2.5 2.5 2
8.5
2.5 2.5 2.5
2.5 5.5 2.0
> 28 > 28
2.5
0.3
85
> 28 9.5 2.5 2 2 2.5
> 28
> 28
3
3
9 74
Potassium sorbate and growth of tobacco spoilage fungi Fig. I. Effects of water activity, pH and potassium sorbate concentration ( 0 ,0%; 0, 0.1%; A,0.2%; D, 0.3%) on growth of Eurotium amstelodami on Burley and Virginia tobacco extract agars at 25'. Bars represent confidence limits (0.98 level). Burley extract agar
Water activity
niger and P. chysogenum changed with both sorbate concentration and tobacco substrate. For example, isolates of E. amsfelodami had slightly longer lag times before growth on Burley than on Virginia tobacco extract agar (Table 2). Furthermore, while the time for growth initiation was increased by 0.1% sorbate, stimulation of growth by 0.2 and 0.3% sorbate resulted in shorter lag times. Growth was also inhibited much more markedly on Virginia extract at 0.2 and 0.3 % sorbate concentration when compared with Burley extract agar. The A. niger isolates used were tolerant of all sorbate concentrations tested on both Virginia and Burley extract agars with little difference in the observed times for growth initiation between controls and the 0.3 % sorbate treatment. P.
chrysogenum isolates were more variable in their response to increased sorbate concentrations and their lag times were in general longer on Virginia extract than Burley extract agar at 0.1 and 0.2% sorbate concentration.
Sorbate concentration, a,, pH and substrate effects on growth Detailed growth studies were carried out with A. amsfelodami, A. flnvus and P. chysogenum because of their ability to tolerate between 0.2 and 0.3% sorbate at 0.85 a, on Virginia or Burley extract agars. The effect of sorbate concentration, a, and pH on growth of A. amsfelodami on both Virginia and Burley extract is shown in Fig. 1. In the absence of sorbate,
E. S. Mutasa, N. Magan and K. J. Seal
975
Fig. 2. Effects of water activity, pH and potassium sorbate concentration (a,0%; 0, 0.1%; A,0.2%; W, 0.3%) on the growth of Aspergilltls flavus on Burley and Virginia tobacco extract agars at 25'. Bars represent confidence limits (0.98 level). Burley extract agar
extract agar
Water activity
growth was slower at pH 6.5 than at pH 5.0. Under similar test conditions but with 0.75-0.90 a, potassium sorbate was more inhibitory to growth at pH 5 than pH 6.5. On Burley extract of 0.85 a,, growth was decreased by half in the presence of between 0.1 and 0.3% sorbate. At 0.90 a, there was also a significant difference (P = 0.05) between the controls and treatments, but no difference in growth inhibition between 0.2 and 0.3% sorbate. O n Virginia extract growth rate was decreased significantly (P = 0.05) by 0.1 to 0.3% sorbate, particularly at pH 5.0. However, at pH 6.5 this was only true at 0.75 and 0.80 a,. Aspergillus flclvw did not grow on either tobacco extract agar below 0.80 a, (Fig. 2). It was most sensitive to sorbate on Burley extract agar at pH 5.0, with no growth in the
presence of 0.1 % sorbate below 0.90 a,. However, at pH 6.5, growth of A. flavus on Burley extract agar was significantly decreased by sorbate only at 0.90 a,. There was little change in growth rate with sorbate concentration. A. flavus grew slightly better on Virginia extract than on Burley extract agar and, at pH 5, growth of A.ficrvw was significantly reduced by 0.1% sorbate at both 0.85 and 0.90 a,. By contrast, at pH 6.5, A. flavus grew with 0.1-0.3% sorbate at 0.85 and 0.90 a,. Penicillium ckysogenum grew better and over a wider a, range on Virginia extract than Burley extract agar, particularly at pH 5 (Fig. 3). O n Burley extract agar and pH 5.0, growth only occurred in the presence of 0.1 % sorbate at 0.90 a,. At lower a, levels growth was completely inhibited. Whereas, at pH 6.5, P. chysogenum grew with up to 0.3 % sorbate and
9 76
Potassium sorbate and growth of tobacco spoilage fungi
Fig. 3. Effects of water activity, pH and potassium sorbate concentration (a,0%; 0, 0.1%; A,0.2%; 1, 0.3%)on the gowth of Penicillirim chrysogenum on Burley and Virginia tobacco extract agars at 25'. Bars represent confidence limits (0.98 level). Burley extract agar
0T75
0-80
0.85
0.90
0.75
0.80
0.85
0.90
Water activity
growth rate was only slightly decreased at 0.90 a,. On Virginia extract agar P. chrysogenum was more tolerant of sorbate at both pH 5.0 and pH 6.5. At pH 5.0 growth was inhibited by sorbate below 0.80 a,. However, even 0.3% sorbate only decreased the growth rate by about 50% at 0.90 a,. At pH 6.5,0.2 and 0.3 % sorbate completely inhibited growth of P. chysogenum at 0.75 and 0.80 a,. At 0.85 a, only 0.2 % sorbate significantly decreased the growth rate while at 0.90 a , none of the concentrations had any effect on growth of P. chrysogenum.
DISCUSSION Potassium sorbate effectively inhibited all fungi tested at pH 5.0, but was less inhibitory at higher pH. Its efficacy was
also profoundly influenced by interaction with substrate composition and water availability. Above pH 5.0, more isolates could grow with at least 0.1 % sorbate at 0.95 a, than at 0.85 a,. However, some species, in particular Eurofium spp., were able to grow in the presence of > 0.1% sorbate at low a,. Some species of E. rubrum and an isolate of E. arnsfelodami and A. niger only grew in the presence of sorbate at low a,. Such organisms may pose a particular problem in stored tobacco where the efficiency of the preservative may be decreased at low a,. By contrast, Liewen & Marth (1984) reported that Aspergillus species from cheese were unable to grow in the presence of up to 0.2% sorbate. However, their studies were carried out on a yeast malt agar at only high a, (0.995) and pH 5.5. The response of the fungal species and isolates to sorbate
977
E. S. Mutasa, N. Magan and K. J. Seal on tobacco extract agar suggested that in sorbate treated tobacco stored above 0.90 a , (30 %moisture content), spoilage may be predominantly caused by members of the A. niger group, in association with Penicillium spp. However, in tobacco stored below 0.85 a, (18 and 22% moisture content in Burley and Virginia, respectively) Eurotium spp. would be expected to predominate. Eurofium spp. are known to be able to grow at much lower a , levels than either A. niger or Penicilliurn spp. (Pitt, 1975; Magan & Lacey, 1984). The results obtained in this study are supported by those of Haas et.al. (1975) who found that at 0.85 a , E. rubrum was less sensitive to sorbate than A. niger. Studies of the efficacy of other common preservatives, such as ammonium propionate against Aspergillus spp., also showed that there were differences between species. For example, A. flavw, A. nidulans, A. niger and A. versicolor grew with 0.2% propionate at 0.995 to 0.90 a,, but only Eurotium spp. grew at 0.85 a , and the same propionate concentration (Magan & Lacey, 1986a). There was a complex interaction between sorbate concentration, a,, and the growth of E. arnstelodami, A. flnvus and P. chrysogenum at pH chosen to resemble closely the natural levels in Burley (6.9) and Virginia (4.9) tobaccos. All three fungi were more sensitive to sorbate at pH 5.0 and below 0.85 a , in both Burley and Virginia extract agars. Sorbic acid is most effective below pH 4.76, it pKa value, where the percentage undissociated ions are greatest (Lueck, 1980). At pH 6.0 and above, the percentage of undissociated ions is low and therefore the acid is less effective at inhibiting spoilage fungi. Furthermore, increasing sorbate concentration from 0.1 to 0.3 % at both high and low a , was not always beneficial and did not always decrease the growth rate. For example, growth of E. amstelodami on Virginia extract agar at pH 5.0 and 0.90 a , was significantly stimulated by increasing the sorbate concentration. It is possible that at increased concentrations of sorbate an interaction between the tobacco substrate and the undissociated ions could occur reducing the effectiveness of the preservative. The nutritional status of the tobacco type may therefore have an important influence on choosing the level of preservative to be used. It may perhaps be more appropriate to determine effective sorbate concentrations for different tobacco types and blends under different a , and temperature storage conditions. The observed variation in sorbate tolerance of individual species on different media suggested that substrate composition may have an important influence on the ability of fungi to grow in the presence of sorbate. This was supported by the fact that more species and isolates were able to grow in the presence of higher sorbate concentrations on the poorest medium, Burley extract agar, than on Virginia extract or the richer malt extract agar. Previous studies have shown that Eurotium spp., Paecilomyces variofii and spoilage yeasts are able to utilize preservatives such as ammonium propionate at both high and low a , (Lord ef al., 1981; Magan & Lacey, 1986 b). Sorbate metabolism by spoilage fungi may further influence the concentrations necessary to inhibit fungal growth. Preservatives may also i~fluence the production of mycotoxins by both Aspergillus and Penicillium species. For instance, ~roductionof aflatoxins by A.flavw was stimulated
by up to 0.2% propionic acid (Al-HilIi & Smith, 1979; Clevstrom et al., 1981) and by 0.025 % sorbic acid. The latter also stimulated production of T-2 Toxin by Fusarium acuminafurn (Gareis ef al., 1984) while higher concentrations were shown to inhibit mycotoxin production to varying degrees. Ray & Bullerman (1982) found that 1 % sorbate completely inhibited aflatoxin production, while 0.5 % caused 50% reduction and 0.1% caused only 10% inhibition. By contrast, in Aspergillus parasiticus, Penicillium commune and P. pafulurn mycotoxin production was almost completely inhibited by 0.1-0.15% sorbate (Bullerman, Schroeder & Park, 1984). This suggest that there may be complex interactions between growth of spoilage fungi in tobacco with sorbate concentration, a,, and pH. All these factors need to be borne in mind when considering the type and level of preservative required to prevent spoilage of tobacco during storage. We are grateful to Rothmans International for financial support.
REFERENCES AL-HILLI, A. L. & SMITH, J. E. (1979).Influence of propionic acid on growth and aflatoxin production by Aspergillus flavus. FEMS Microbiology Letters 6 , 367-370. AYERST, G. (1969). The effect of moisture and temperature on growth and spore germination in some fungi. 1ournal of Stored Products Research 5 , 127-141. BANDELIN, F. J. (1958). The effect of pH on the efficacy of various mould inhibiting compounds. 1ournal of the American Pharmaceutical Association 47,691-694. BULLERMAN, L. B., SCHROEDER, L. L. & PARK, K.-Y. (1984). Formation and control of mycotoxins in food. Journal of Food Protection 47,637-446. CLEVSTROM, G., GORANSSON, B., HLODVERSSON, R. & PETTERSSON, H. (1981).Aflatoxin formation in hay treated with formic acid and in isolated strains of Aspergillus flavus. Journal of Stored Products Research 17, 151-161. DALLYN, H. & FOX, A. (1980). Spoilage of materials of reduced water activity by xerophilic fungi. Society of Applied Bacferiology Technical Series 15 (ed. G. H. Gould & J. E. L. Cony), pp. 129-139. London : Academic Press. DEPARTMENT OF HEALTH AND SOCIAL SECURITY (1989). Report on additives to tobacco and cigarette papers. London Gazette. GAREIS, M., BAUER, J., VON MONTGELAS, A. & GEDEK, B. (1984). Stimulation of aflatoxin BI and T-2 toxin production by sorbic acid. Applied and Environmental Microbiology 47,416-418. HAAS, G. J., BENNET, D., HERMAN, E. B. & COLLETTE, D. (1975). Microbial stability of intermediate moisture foods. Food Product Development 9, 86-96. LIEWEN, M. B. & MARTH, E. H. (1984).Inhibition of Penicillia and Aspergilli by potassium sorbate. Journal of Food Protection 47, 554-5.56. LORD, K. A., LACEY, J., CAYLEY, G. R. & MANLOVE, R. (1981). Fatty acids as substrates and inhibitors of fungi from propionic acid-treated hay. Transactions of the British Mycological Society 77, 41-45. LUEK, E. (1980).Antimicrobial Food Additives. Berlin: Springer Vedag. MAGAN, N. & LACEY, J. (1984). Effect of temperature and pH on the water relations of field and storage fungi. Transactions of the British Mycological Society 82, 71-81.
Potassium sorbate and growth of tobacco spoilage fungi MAGAN, N. & LACEY, J. (1986a). The effects of two ammonium propionate formulations on growth in vitro of Aspergillus species isolated from hay. Ioumal of Applied Bacteriology 60,221-225. MAGAN, N. & LACEY, J. (1986 b). Water relations and metabolism of propionate in two yeasts from hay.]oumal of Applied Bacteriology 60,169-173.
MUTASA, E. S. (1988). Effect of water activity and potassium sorbate on growth of deteriogenic fungi from tobacco. Ph.D. Thesis, Cranfield Institute of Technology. PITT, J. I. (1975). Xerophilic fungi and spoilage of foods of plant
978 origin. In Water Relations of Foods (ed. R. B. Duckworth), pp. 273-307. London: Academic Press. RAY, L. L. & BULLERMAN, L. B. (1982). Preventing growth of potentially toxic moulds using antifungal agents. loumal of Food Protection 4 5 , 953-963.
SOFOS, J. F. & BUSTA, F. F. (1981). Antimicrobial activity of sorbate. Ioumal of Food Protection 44, 614-627. WELTY, R. E. & LUCAS, G. B. (1968). Fungi isolated from damaged flue-cured tobacco. Applied Microbiology 16,851-854.
(Received for publication 4 September 1989 and in revised form 7 December 1989)
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94 ( 7 ) :971-978 (1990)
971
Printed in Great Britain
Effects of potassium sorbate and environmental factors on growth of tobacco spoilage fungi
E. S. MUTASA*, N. M A G A N t A N D K. J. SEAL Biofechnology Centre, Cranfield lnstitufe of Technology, Cranfield, Bedford MK43 OAL
Effects of potassium sorbate and environmental factors on growth of tobacco spoilage hngi. Mycological Research 94 (7):971-978 (1990). Potassium sorbate was found to inhibit germination and growth of a range of spoilage fungi isolated from tobacco below pH 5. However, at and above pH 5 the efficacy of potassium sorbate was influenced by substrate composition and water activity (a,). Eurotium spp., Aspergillus flavus, A. fumigafus, A. niger, and Penicillium species all grew with 0.1-0.3% sorbate at 0.95 a, on malt, Virginia and Burley extract agars. However, at 085 a,, only Eurotium spp., A. flavus, P. chysogenum and P. viridicafum were able to grow in the presence of sorbate. At both high and low a, the tolerance of sorbate was influenced by substrate composition. The lag time for growth initiation in different concentrations of sorbate was significantly influenced by substrate composition for E. amstelodami and P. chysogenum but not for A. niger. Growth of E. amstelodami, A. flavus and P. chysogenum was significantly less on Burley extract agar than Virginia extract or malt agar over the range 0-754.90 a, at pH 5 and 6-5 and with 01+4% sorbate. Under some environmental conditions, growth of E. amsfelodami on tobacco extract agars was stimulated by sorbate. Key words: Growth inhibition, Potassium sorbate, Water activity, pH, Tobacco, Aspergillus, Penicillium.
Sorbic acid and its salts, especially potassium and calcium, are the most widely used preservatives in tobacco as well as in a wide range of food products against fungal spoilage. Sorbate is increasingly preferred because of its physiological inertness and organoleptic neutrality (Sofos & Busta, 1981). Maximum levels of sorbic acid currently permitted in tobacco are 0.5 % (w/w) in cigarette papers and hand-rolling tobacco and 1% in pipe tobacco (DHSS Report, 1989). However, the antimicrobial capabilities of sorbic acid and its salts are dependent on pH (Bandelin, 1958). It is most effective in its undissociated or unionized form at a pH below 4-75. The proportion of undissociated acid varies from 6-7% at pH 6.00 to 50% at pH 4.75 and 98% at pH 3.00 (Sofos & Busta, 1981). The dominant spoilage fungi of different flue-cured stored tobaccos varies with storage water content but includes Aspergillus spp., including A. niger, A. uersicolor, Eurotium spp. as well as Penicillium chysogenum, and P. viridicafum (Welty & Lucas, 1968; Mutasa, 1988). Many of these species, particularly Aspergillus and Eurofium spp., are xerophilic, and therefore able to grow at below 0.85 water activity (a,) (Ayerst, 1969; Pitt, 1975 ; Magan & Lacey, 1984). Although potassium sorbate is widely used as a preservative in tobacco there is very little information on its efficacy against the predominant spoilage fungi on interaction with a,, pH and tobacco type. Both * Present address: School of Biological Sciences, Queen Mary College, Mile End Road, London El 4NS.
t Corresponding author.
germination and growth of Aspergillus spp. have previously been found to vary considerably with concentration of other preservatives such as ammonium propionate formulations and with a, (Magan & Lacey, 1986a). The objectives of this study were to investigate the effect of up to 0.3% potassium sorbate on the dominant spoilage fungi of tobacco at both high and low a, and to determine the effect of interaction with pH and nutrient substrate on growth of some Aspergillus and Penicillium species.
MATERIALS A N D M E T H O D S Fungal isolates Eurotium amsfelodami Mangin (4 isolates), E. chevalieri Mangin, E. rubrum Konig, Spieckerrnan & Bremer (anamorph, Aspergillus rubrobrunneus Samson & Gams) (2 isolates), Aspergillus awamori Nakazawa, A. Jlnvus Link, A. fumigatus Fres. (2 isolates), A. niger van Tiegh. (10 isolates), A. sydowii (Bain. & Sart.) Thom & Church, A. versicolor Tiraboschi (5 isolates), Penicillium chysogenum Thorn (5 isolates), P. crustosum Thorn, P. viridicafum Westling, P. spinulosum Thorn, Scopulariopsis brevicaulis (Sacc.) Bain. and Syncephalastrum racemosum (Cohn) Schroter used in this study were all obtained from flue-cured tobaccos and represent the dominant fungi isolated from 5 different tobacco types (Mutasa, 1988). The identity of all species was confirmed by the CAB Intemafional Mycological Institute, Kew.
972
Potassium sorbate and growth of tobacco spoilage fungi
Table 1. Concentrations of potassium sorbate allowing growth of fungal isolates on malt extract and two tobacco extract agars at two water activities Growth medium.. . Malt extract Virginia extract Burley extract Water activity.. . Fungal isolates E. E. E. E. E. E. E.
arnstelodami umsfelodami amstelodami amsfelodami chevalieri rubrum rubrum
0.96
0.85
0.96
0.85
0.96
0.3 0.3 N.G. 0.3 0.2 0.3 0.2
0.I
0.3 0.3 0.2 0.3 0.2 0.3 0.3
0. I 0.3 0.2 0.3 N.G. N.G.
N.G. N.G. N.G. N.G. N.G. N.G.
0.2 0.3 0.2 0.2 0.3 0.3
N.G.
0.3 N.G. 0.2
0.1
N.G. N.G.
0.1
0.3 0.3
0.3 0.3 0.3 0.3 0.2
0.1
0.2 0. I
N.G.
0.1
0.I
0.1
0.2 0.2 0.3
A. niger (3) A. niger A. niger A. niger A. niger A. nige~ A. niger A. niger A. awarnori
0.1 N.G. N.G. N.G. N.G. N.G. 0.3 0.1 0.1
0.3 N.G. 0.3 0.3 0.3 0.3 0.3 0.3 0.2
N.G. N.G. N.G. N.G. N.G. N.G. N.G.
A. flavus A. fumigatus A. fumigatus
N.G. N.G.
N.G.
0.1
0.1 0.1
N.G. N.G.
0.3 0.3 0.2 0.3 0.3 N.G. 0.3
N.G. N.G. N.G. N.G. N.G. N.G. N.G. N.G.
0.3 0. I 0.2 0.3 0.3 0.3
N.G.
N.G. 0.3
N.G. N.G.
0.3 0.2
0.1 0.1
A. versicolor A. versicolor A. versicolor A. versicolor A. versicolor A. sydowii
P. P. P. P. P. P. P. P.
chysogenum chrysogenum chrysogenurn chrysogenum chrysogenurn crwtosum viridicatum spinulosurn
S. racernosurn Sc. brevicaulis
N.G.
0.2 N.G. 0.3
0.85
N.G.
0.1
N.G. 0.1
0.1 N.G. N.G. N.G. N.G. N.G. N.G. N.G.
0.1
0.I
0.1
0. I 0.2
0.3 0.3 0.3
0.3 0.3 N.G. 0.1
N.G. N.G. N.G. N.G. N.G. N.G. N.G. N.G. 0.1 N.G. N.G.
0.3 0.3 0.3
N.G. N.G.
0. I
0.1
0.I
N.G. 0.3 N.G. 0.1 N.G.
0.2
0.1
N.G., no growth. A, Aspergillus; E, Eurotium; P, Penicillium; S, Syncephalasfrum; Sc, Scopulariopsis. (3). three isolates.
Water activity media Malt extract agar (3%) was modified by the addition of glycerol (Dallyn & Fox, 1980) to 0.96 and 0.85 a,. Two tobacco extract agars were prepared by the addition of ground Virginia (high sugar) or Burley (low sugar) tobacco to water and steaming the mixture for 30 min. The tobacco grounds were removed using a buchner filter funnel and filter paper (Whatman No I). These media were subsequently adjusted to 0.75, 0.80, 0.85, 0.90 and 0.96 a, with glycerol. The a , of all solidified media was checked with a Humidat IC I1 (Novasina AG, Switzerland).
Fungal growth in relation to sorbate concentration, pH and a , Filter sterilized potassium sorbate was added at levels of 0.1, 0.2, 0.3 and 0.4 % (w/v) to all three media previously adjusted to 0-96 and 0.85 a,. The pH was then adjusted with 1 MNaOH or 0.1 M-HCI to 4.5, 5.0 and 5.5. Non-bleeding indicator strips were used to check the pH of molten media and a flathead pH probe to determine the final pH of solidified media. Spores from 94 isolates of test fungi, grown for 7 to 14 d on either malt extract or malt yeast glucose (40%) agar, were
E. S. Mutasa, N. Magan and K. J. Seal
973
tested for their ability to grow in the presence of these concentrations of potassium sorbate. The plates were centrally inoculated with 2 ~1 aliquots of spore suspensions prepared in 0.1% distilled water agar. Ten replicates of each isolate and each treatment were prepared. The plates were sealed with parafilm and incubated at 25 OC until growth occurred, or for a maximum of 6 months. Outgrowth of at least 5 replicate colonies to a diameter of 10 and 5 mm at 0 9 6 and 0.85 a, respectively was used as the criterion for growth. The time required for growth was also monitored. To ensure that pH was not directly responsible for growth inhibition, all fungal isolates which tolerated at least 01% sorbate above pH 5 were tested for their ability to grow at pH 4.5. These experiments were carried out on malt extract agar buffered with 100 mM acetate. Plates were inoculated as described previously and 5 replicates incubated at 25' for up to one month. More detailed studies were subsequently carried out on E. amsfelodami, A. flavw and P. chysogenum which were able to grow with 0 . 2 4 . 3 % sorbate at 0.85 a,. Burley and Virginia extract agars were modified to 0.75-0.90 a,, and sorbate incorporated in the range 0.1 to 0.4%. The pH was adjusted to 5.0 and 6.5. Ten replicate plates per treatment were inoculated for each test fungus and incubated at 25' for up to 3 months. Radial growth rates were measured at regular intervals, appropriate to each a, level. Linear regression analyses of the mean radial extension rates were used to calculate the growth rates for each treatment. The standard errors of the means were calculated and confidence limits (at 0.98 level) attached.
RESULTS Fungal growth in presence of sorbate On malt extract agar all the fungal species and isolates were effectively inhibited by 0.1 % sorbate below pH 5, and by 0.2 or 0 3 % at pH 5. At high a , (pH greater than 5) a total of 38 isolates was able to grow in the presence of sorbate on malt extract agar, 32 on Virginia extract and 35 on Burley extract
agars (Table I). Lowering the substrate a, to 0.85 reduced the number of isolates able to grow in the presence of sorbate on each substrate as well as changing the sensitivity of some organisms. Members of the Eurotium group grew in the presence of 0.3% sorbate at 0.85 a, on all three substrates, although at 0.96 a , some isolates could only grow on media containing 0.1 % sorbate. More isolates of E. amsfelodami grew with 0.3 % sorbate in Burley than Virginia or malt extract agars at 0.85 a,. Members of the A. versicolor group were unable to grow in the presence of 0 3 % sorbate in malt extract agar at either 0-96 or 0.85 a,. Most other isolates required > 0.3% sorbate to inhibit growth at 0.96 a , on Virginia and Burley extract agars. However, at 0.85 a,, 0.3% sorbate was sufficient to inhibit most isolates but sensitivity to sorbate was greater on Virginia than on Burley extract agar (Table I). Almost all the A. niger isolates tested were able to grow in all three media at 0.96 a , with 0.3% sorbate, but at 0.85 a, seven of the ten isolates were inhibited by 0.1% sorbate. Growth of all the Penicilliurn spp. on malt extract agar at 0.96 a , was inhibited by 0.3 % sorbate but at 0.85 a, only two isolates of P. chrysogenum could grow with 0.1% sorbate. By contrast, on Virginia and Burley extract, many of the Penicillium species could grow at 0.96 a, with 0.3% sorbate but at 0.85 a, all were inhibited by 0.1 % sorbate on Virginia extract, but only half on Burley extract agar. O f the other species tested, A. fumigafus grew with 0 . 2 4 . 3 % sorbate on all media at 0.96 a, but only with 0.1 % sorbate at 0.85 a, on malt extract agar. A. fiavw was tolerant of 0-2+3 % sorbate at both 0.96 and 0-85 a, on both Virginia and Burley extract agars. S. racemusum and S. brevicaulis grew with 0.2-0.3 % sorbate at 0.96 a, and with 0.1 % at 0.85 a, on Burley extract agar but were more sensitive on Virginia extract and malt extract, respectively.
Interactions of sorbate concentration and substrate on growth initiation The time required for growth of species of E. amsfelodami, A.
Table 2. Lag times before growth of three isolates each of Eurotium amstelodami, Aspergillus niger and Penicilliurn chysogenum with up to 0.3% potassium sorbate on Virginia and Burley tobacco extract agars at 25' and 0.95 a, Time (d) Tobacco type.. . Virginia
Burley
Percentage sorbate (%) ... Species Isolate
0
E. amsfeiodami
I 2 3
4 4.5 4.5
9 13.5 11
> 28 > 28
> 28 > 28
9
A. niger
I 2 3
1.5 1.5 1.5
2 1.5 2
2 2 2
P. chysogenum
1 2 3
2 3 1.5
0.1
0.2
5
5
> 28
> 28
2
2
0.3
0
0.1
0.2
9
5 5 7
65 7.5 9
7 7 8
2.5 2.5 2
2 2 1.5
2 2 2
2.5 2.5 2
8.5
2.5 2.5 2.5
2.5 5.5 2.0
> 28 > 28
2.5
0.3
85
> 28 9.5 2.5 2 2 2.5
> 28
> 28
3
3
9 74
Potassium sorbate and growth of tobacco spoilage fungi Fig. I. Effects of water activity, pH and potassium sorbate concentration ( 0 ,0%; 0, 0.1%; A,0.2%; D, 0.3%) on growth of Eurotium amstelodami on Burley and Virginia tobacco extract agars at 25'. Bars represent confidence limits (0.98 level). Burley extract agar
Water activity
niger and P. chysogenum changed with both sorbate concentration and tobacco substrate. For example, isolates of E. amsfelodami had slightly longer lag times before growth on Burley than on Virginia tobacco extract agar (Table 2). Furthermore, while the time for growth initiation was increased by 0.1% sorbate, stimulation of growth by 0.2 and 0.3% sorbate resulted in shorter lag times. Growth was also inhibited much more markedly on Virginia extract at 0.2 and 0.3 % sorbate concentration when compared with Burley extract agar. The A. niger isolates used were tolerant of all sorbate concentrations tested on both Virginia and Burley extract agars with little difference in the observed times for growth initiation between controls and the 0.3 % sorbate treatment. P.
chrysogenum isolates were more variable in their response to increased sorbate concentrations and their lag times were in general longer on Virginia extract than Burley extract agar at 0.1 and 0.2% sorbate concentration.
Sorbate concentration, a,, pH and substrate effects on growth Detailed growth studies were carried out with A. amsfelodami, A. flnvus and P. chysogenum because of their ability to tolerate between 0.2 and 0.3% sorbate at 0.85 a, on Virginia or Burley extract agars. The effect of sorbate concentration, a, and pH on growth of A. amsfelodami on both Virginia and Burley extract is shown in Fig. 1. In the absence of sorbate,
E. S. Mutasa, N. Magan and K. J. Seal
975
Fig. 2. Effects of water activity, pH and potassium sorbate concentration (a,0%; 0, 0.1%; A,0.2%; W, 0.3%) on the growth of Aspergilltls flavus on Burley and Virginia tobacco extract agars at 25'. Bars represent confidence limits (0.98 level). Burley extract agar
extract agar
Water activity
growth was slower at pH 6.5 than at pH 5.0. Under similar test conditions but with 0.75-0.90 a, potassium sorbate was more inhibitory to growth at pH 5 than pH 6.5. On Burley extract of 0.85 a,, growth was decreased by half in the presence of between 0.1 and 0.3% sorbate. At 0.90 a, there was also a significant difference (P = 0.05) between the controls and treatments, but no difference in growth inhibition between 0.2 and 0.3% sorbate. O n Virginia extract growth rate was decreased significantly (P = 0.05) by 0.1 to 0.3% sorbate, particularly at pH 5.0. However, at pH 6.5 this was only true at 0.75 and 0.80 a,. Aspergillus flclvw did not grow on either tobacco extract agar below 0.80 a, (Fig. 2). It was most sensitive to sorbate on Burley extract agar at pH 5.0, with no growth in the
presence of 0.1 % sorbate below 0.90 a,. However, at pH 6.5, growth of A. flavus on Burley extract agar was significantly decreased by sorbate only at 0.90 a,. There was little change in growth rate with sorbate concentration. A. flavus grew slightly better on Virginia extract than on Burley extract agar and, at pH 5, growth of A.ficrvw was significantly reduced by 0.1% sorbate at both 0.85 and 0.90 a,. By contrast, at pH 6.5, A. flavus grew with 0.1-0.3% sorbate at 0.85 and 0.90 a,. Penicillium ckysogenum grew better and over a wider a, range on Virginia extract than Burley extract agar, particularly at pH 5 (Fig. 3). O n Burley extract agar and pH 5.0, growth only occurred in the presence of 0.1 % sorbate at 0.90 a,. At lower a, levels growth was completely inhibited. Whereas, at pH 6.5, P. chysogenum grew with up to 0.3 % sorbate and
9 76
Potassium sorbate and growth of tobacco spoilage fungi
Fig. 3. Effects of water activity, pH and potassium sorbate concentration (a,0%; 0, 0.1%; A,0.2%; 1, 0.3%)on the gowth of Penicillirim chrysogenum on Burley and Virginia tobacco extract agars at 25'. Bars represent confidence limits (0.98 level). Burley extract agar
0T75
0-80
0.85
0.90
0.75
0.80
0.85
0.90
Water activity
growth rate was only slightly decreased at 0.90 a,. On Virginia extract agar P. chrysogenum was more tolerant of sorbate at both pH 5.0 and pH 6.5. At pH 5.0 growth was inhibited by sorbate below 0.80 a,. However, even 0.3% sorbate only decreased the growth rate by about 50% at 0.90 a,. At pH 6.5,0.2 and 0.3 % sorbate completely inhibited growth of P. chysogenum at 0.75 and 0.80 a,. At 0.85 a, only 0.2 % sorbate significantly decreased the growth rate while at 0.90 a , none of the concentrations had any effect on growth of P. chrysogenum.
DISCUSSION Potassium sorbate effectively inhibited all fungi tested at pH 5.0, but was less inhibitory at higher pH. Its efficacy was
also profoundly influenced by interaction with substrate composition and water availability. Above pH 5.0, more isolates could grow with at least 0.1 % sorbate at 0.95 a, than at 0.85 a,. However, some species, in particular Eurofium spp., were able to grow in the presence of > 0.1% sorbate at low a,. Some species of E. rubrum and an isolate of E. arnsfelodami and A. niger only grew in the presence of sorbate at low a,. Such organisms may pose a particular problem in stored tobacco where the efficiency of the preservative may be decreased at low a,. By contrast, Liewen & Marth (1984) reported that Aspergillus species from cheese were unable to grow in the presence of up to 0.2% sorbate. However, their studies were carried out on a yeast malt agar at only high a, (0.995) and pH 5.5. The response of the fungal species and isolates to sorbate
977
E. S. Mutasa, N. Magan and K. J. Seal on tobacco extract agar suggested that in sorbate treated tobacco stored above 0.90 a , (30 %moisture content), spoilage may be predominantly caused by members of the A. niger group, in association with Penicillium spp. However, in tobacco stored below 0.85 a, (18 and 22% moisture content in Burley and Virginia, respectively) Eurotium spp. would be expected to predominate. Eurofium spp. are known to be able to grow at much lower a , levels than either A. niger or Penicilliurn spp. (Pitt, 1975; Magan & Lacey, 1984). The results obtained in this study are supported by those of Haas et.al. (1975) who found that at 0.85 a , E. rubrum was less sensitive to sorbate than A. niger. Studies of the efficacy of other common preservatives, such as ammonium propionate against Aspergillus spp., also showed that there were differences between species. For example, A. flavw, A. nidulans, A. niger and A. versicolor grew with 0.2% propionate at 0.995 to 0.90 a,, but only Eurotium spp. grew at 0.85 a , and the same propionate concentration (Magan & Lacey, 1986a). There was a complex interaction between sorbate concentration, a,, and the growth of E. arnstelodami, A. flnvus and P. chrysogenum at pH chosen to resemble closely the natural levels in Burley (6.9) and Virginia (4.9) tobaccos. All three fungi were more sensitive to sorbate at pH 5.0 and below 0.85 a , in both Burley and Virginia extract agars. Sorbic acid is most effective below pH 4.76, it pKa value, where the percentage undissociated ions are greatest (Lueck, 1980). At pH 6.0 and above, the percentage of undissociated ions is low and therefore the acid is less effective at inhibiting spoilage fungi. Furthermore, increasing sorbate concentration from 0.1 to 0.3 % at both high and low a , was not always beneficial and did not always decrease the growth rate. For example, growth of E. amstelodami on Virginia extract agar at pH 5.0 and 0.90 a , was significantly stimulated by increasing the sorbate concentration. It is possible that at increased concentrations of sorbate an interaction between the tobacco substrate and the undissociated ions could occur reducing the effectiveness of the preservative. The nutritional status of the tobacco type may therefore have an important influence on choosing the level of preservative to be used. It may perhaps be more appropriate to determine effective sorbate concentrations for different tobacco types and blends under different a , and temperature storage conditions. The observed variation in sorbate tolerance of individual species on different media suggested that substrate composition may have an important influence on the ability of fungi to grow in the presence of sorbate. This was supported by the fact that more species and isolates were able to grow in the presence of higher sorbate concentrations on the poorest medium, Burley extract agar, than on Virginia extract or the richer malt extract agar. Previous studies have shown that Eurotium spp., Paecilomyces variofii and spoilage yeasts are able to utilize preservatives such as ammonium propionate at both high and low a , (Lord ef al., 1981; Magan & Lacey, 1986 b). Sorbate metabolism by spoilage fungi may further influence the concentrations necessary to inhibit fungal growth. Preservatives may also i~fluence the production of mycotoxins by both Aspergillus and Penicillium species. For instance, ~roductionof aflatoxins by A.flavw was stimulated
by up to 0.2% propionic acid (Al-HilIi & Smith, 1979; Clevstrom et al., 1981) and by 0.025 % sorbic acid. The latter also stimulated production of T-2 Toxin by Fusarium acuminafurn (Gareis ef al., 1984) while higher concentrations were shown to inhibit mycotoxin production to varying degrees. Ray & Bullerman (1982) found that 1 % sorbate completely inhibited aflatoxin production, while 0.5 % caused 50% reduction and 0.1% caused only 10% inhibition. By contrast, in Aspergillus parasiticus, Penicillium commune and P. pafulurn mycotoxin production was almost completely inhibited by 0.1-0.15% sorbate (Bullerman, Schroeder & Park, 1984). This suggest that there may be complex interactions between growth of spoilage fungi in tobacco with sorbate concentration, a,, and pH. All these factors need to be borne in mind when considering the type and level of preservative required to prevent spoilage of tobacco during storage. We are grateful to Rothmans International for financial support.
REFERENCES AL-HILLI, A. L. & SMITH, J. E. (1979).Influence of propionic acid on growth and aflatoxin production by Aspergillus flavus. FEMS Microbiology Letters 6 , 367-370. AYERST, G. (1969). The effect of moisture and temperature on growth and spore germination in some fungi. 1ournal of Stored Products Research 5 , 127-141. BANDELIN, F. J. (1958). The effect of pH on the efficacy of various mould inhibiting compounds. 1ournal of the American Pharmaceutical Association 47,691-694. BULLERMAN, L. B., SCHROEDER, L. L. & PARK, K.-Y. (1984). Formation and control of mycotoxins in food. Journal of Food Protection 47,637-446. CLEVSTROM, G., GORANSSON, B., HLODVERSSON, R. & PETTERSSON, H. (1981).Aflatoxin formation in hay treated with formic acid and in isolated strains of Aspergillus flavus. Journal of Stored Products Research 17, 151-161. DALLYN, H. & FOX, A. (1980). Spoilage of materials of reduced water activity by xerophilic fungi. Society of Applied Bacferiology Technical Series 15 (ed. G. H. Gould & J. E. L. Cony), pp. 129-139. London : Academic Press. DEPARTMENT OF HEALTH AND SOCIAL SECURITY (1989). Report on additives to tobacco and cigarette papers. London Gazette. GAREIS, M., BAUER, J., VON MONTGELAS, A. & GEDEK, B. (1984). Stimulation of aflatoxin BI and T-2 toxin production by sorbic acid. Applied and Environmental Microbiology 47,416-418. HAAS, G. J., BENNET, D., HERMAN, E. B. & COLLETTE, D. (1975). Microbial stability of intermediate moisture foods. Food Product Development 9, 86-96. LIEWEN, M. B. & MARTH, E. H. (1984).Inhibition of Penicillia and Aspergilli by potassium sorbate. Journal of Food Protection 47, 554-5.56. LORD, K. A., LACEY, J., CAYLEY, G. R. & MANLOVE, R. (1981). Fatty acids as substrates and inhibitors of fungi from propionic acid-treated hay. Transactions of the British Mycological Society 77, 41-45. LUEK, E. (1980).Antimicrobial Food Additives. Berlin: Springer Vedag. MAGAN, N. & LACEY, J. (1984). Effect of temperature and pH on the water relations of field and storage fungi. Transactions of the British Mycological Society 82, 71-81.
Potassium sorbate and growth of tobacco spoilage fungi MAGAN, N. & LACEY, J. (1986a). The effects of two ammonium propionate formulations on growth in vitro of Aspergillus species isolated from hay. Ioumal of Applied Bacteriology 60,221-225. MAGAN, N. & LACEY, J. (1986 b). Water relations and metabolism of propionate in two yeasts from hay.]oumal of Applied Bacteriology 60,169-173.
MUTASA, E. S. (1988). Effect of water activity and potassium sorbate on growth of deteriogenic fungi from tobacco. Ph.D. Thesis, Cranfield Institute of Technology. PITT, J. I. (1975). Xerophilic fungi and spoilage of foods of plant
978 origin. In Water Relations of Foods (ed. R. B. Duckworth), pp. 273-307. London: Academic Press. RAY, L. L. & BULLERMAN, L. B. (1982). Preventing growth of potentially toxic moulds using antifungal agents. loumal of Food Protection 4 5 , 953-963.
SOFOS, J. F. & BUSTA, F. F. (1981). Antimicrobial activity of sorbate. Ioumal of Food Protection 44, 614-627. WELTY, R. E. & LUCAS, G. B. (1968). Fungi isolated from damaged flue-cured tobacco. Applied Microbiology 16,851-854.
(Received for publication 4 September 1989 and in revised form 7 December 1989)
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