Food Microbiology 28 (2011) 1463e1467. Contents lists available at ScienceDirect. Food Microbiology. journal homepage:

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1 Food Microbiology 28 (2011) 1463e1467 Contents lists available at ScienceDirect Food Microbiology journal homepage: Stimulating effect of sorbitol and xylitol on germination and growth of some xerophilic fungi A. Patriarca a, *, G. Larumbe a, M.P. Buera b, G. Vaamonde a a Laboratorio de Microbiología de Alimentos, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Intendente Güiraldes 2160, Pabellón II, 3 Piso, Ciudad Universitaria C1428EGA, Buenos Aires, Argentina b Departamento de Industrias y Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria C1428EGA, Buenos Aires, Argentina article info abstract Article history: Received 17 January 2011 Received in revised form 26 July 2011 Accepted 29 July 2011 Available online 5 August 2011 Keywords: Xerophilic fungi Sorbitol Xylitol Germination Growth Sorbitol and xylitol are polyols often used in foods as naturally occurring sugar substitutes. They provide sweet taste and reduced calories in products of intermediate moisture. This type of food is susceptible to spoilage by xerophilic molds which affect shelf life of foods and produce significant losses. The aim of the present work was to study the effect of glycerol, sorbitol and xylitol on the germination and growth of four xerophilic fungi at different temperatures and water activity levels. Penicillium chrysogenum, Wallemia sebi, Eurotium chevalieri and Eurotium repens were cultivated on malt extract agar with the addition of the respective polyols and a w adjusted to 0.85, 0.88, 0.90 and Incubation was made at 25, 30 and 35 C. Results of the present study demonstrated that sorbitol and xylitol affect the growth kinetics of the four fungal species. The observed tendency was that these solutes shortened the germination times and increased the growth rates. The effect of each solute depended on the fungal species and the a w / temperature combinations. At lower a w the influence was more evident on the germination times while the effect on growth rates was more pronounced at higher a w levels. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Sorbitol and xylitol are members of a group of sweeteners, called sugar polyols, which are often used in foods as naturally occurring sugar substitutes. They provide sweet taste and reduced calories in a wide variety of products including sugar-free candies, chewing gums, frozen desserts, cookies, icings and fillings, as well as oral care products, such as toothpaste and mouthwash. Xylitol is also used in pharmaceuticals formulations to provide sweet taste. The range of xylitol and/or sorbitol consumption could be up to 10 g/day (Soderling et al., 1988; Makinen et al., 1995). Most products (gums, candies) contain a total polyol lower than 2% (Giese, 1993; Burt, 2006). In addition, sorbitol is used as humectant in the production of confectionery, baked goods and chocolate, where products tend to become dry or harden. The moisture-stabilizing and textural properties of sorbitol protect these products from drying and maintain their initial freshness during storage. Intermediate moisture products are susceptible to spoilage by xerophilic molds, * Corresponding author. Tel./fax: þ address: andreap@qo.fcen.uba.ar (A. Patriarca). that are capable of growing under dry conditions or in the presence of high levels of solutes such as salts or sugars (Hocking, 1991). Most filamentous xerophiles belong to the genera Aspergillus and Penicillium or are teleomorphs of Aspergillus such as Eurotium and Emericella (Abellana et al., 1999a). Eurotium species are probably the most destructive of all xerotolerant fungi (Abellana et al., 1999b). Eurotium chevalieri and Eurotium repens are the major cause of spoilage of a confectionary product very popular in Argentina ( alfajor ) consisting of chocolate coated sandwich cookies filled with milk caramel. Xerophilic fungi are responsible of shortening the mold free-shelf life of this confection, mainly in summer, when ambient temperature raises to ca 30 C. Sorbitol is used as humectant in this product. Wallemia sebi is another xerophilic mold able to grow at a wide range of a w (Patriarca et al., 2001). When present, growth of these organisms affects shelf life of foods producing significant losses (Pitt and Hocking, 1997; Roessler and Ballenger, 1996). Temperature and water activity (a w ) are recognized as the major factors that control germination and growth of fungi in foods. Many previous studies have examined the effect of these parameters and their interactions on the growth of xerophilic molds (Magan and Lacey, 1988; Abellana et al., 1999a,b; Gock et al., 2003). Water activity is probably the most important environmental factor /$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi: /j.fm

2 1464 A. Patriarca et al. / Food Microbiology 28 (2011) 1463e1467 determining whether and at which rate a microorganism will grow on intermediate moisture food (Guynot et al., 2002). However, the use of this parameter as the sole predictor for product stability and safety has been questioned because different solutes (glycerol, salts and sugars) exert different effects on microorganisms at the same a w (Huang et al., 2009). Nonionic solutes such as glycerol, sorbitol and glucose are less inhibitory than the ionic solute NaCl (Beuchat and Pitt, 1990; Lahlali et al., 2006; Sautour et al., 2001). Hocking and Pitt (1979) recommended the use of glycerol to adjust the a w of a medium because it is the solute with less adverse effects on fungal growth. Another advantage of glycerol is that it is possible to obtain lower a w values than with NaCl or sugars. Taking into account the use of the sugar polyols as ingredients in low moisture foods, the aim of the present work was to study the effect of some of the most employed ones (glycerol, sorbitol and xylitol), on germination and growth of some xerophilic fungi of significance in food deterioration at different temperatures and water activity levels. 2. Material and methods 2.1. Fungi The four fungi strains used in this study were isolated in our laboratory from spoiled low moisture foods: Penicillium chrysogenum (from cocoa powder), W. sebi (from milk caramel), E. chevalieri (from chocolate pudding) and E. repens (from vanilla pudding). All strains belong to the Buenos Aires Facultad de Ciencias Culture Collection (BAFC) Media The basal medium contained 2% malt extract, 0.1% peptone, 2% glucose and glycerol in the required concentrations to adjust a w to 0.85, 0.88, 0.90 and To study the effect of the sugar polyols, glycerol was in part substituted for either sorbitol or xylitol at 10% w/w. Individual calibration curves were made to calculate the amount of glycerol to be added to basal media with 10% of each polyol to obtain the desired final a w level. Water activity values were checked using a water activity meter with an accuracy of a w units (AquaLab, Decagon Devices, Pullman, WA, USA) Inoculation and incubation Spores of each strain coming from 7-day cultures in G25N agar (Pitt and Hocking, 1997)at25 C were collected with an inoculation needle and were placed in a hemolysis tube containing 0.5 ml of Tween 80 solution (0.05%) of a w adjusted with glycerol at the same level of the subsequent studies (Nanguy et al., 2010). After homogenizing, the suspension was counted using a hematocytometer. Under these conditions the inoculum concentrations varied between 1 and spores ml 1. Petri plates with the media containing only glycerol, or a combination of glycerol and sorbitol or xylitol at the different a w levels were inoculated centrally with a calibrated loop containing 2 ml of spore suspension. The plates were incubated at 25, 30 and 35 C for a maximum period of 100 days. To minimize the transfer of water to or from the media, the plates were placed inside plastic bags containing a recipient with glycerol solutions adjusted to the corresponding a w Germination times and growth rates To evaluate germination, Petri plates were initially examined twice a day under transmitted light microscope (100), diminishing the periodicity up to three times a week in the final stages of the incubation period. The criterion for defining germination time was the production of a germination tube of longitude equal to the length of the conidia, in at least 50% of the inoculum (Hocking and Miscamble, 1995). The radial mycelial growth was determined by periodical measurement of two right-angled diameters of the colonies. Radial mycelial growth vs. time was plotted and radial growth rates (mm day 1 ) were evaluated from the slope by linear regression (Patriarca et al., 2001). All the experiments were performed in quadrupled. An average radial growth rate (k r ) was calculated from the different independent experiment for each set of conditions Statistical analysis The effect of a w, temperature, and their interaction on germination time and growth rate of xerophilic fungi was examined by ANOVA using Statistica software v6.0 (StatSoft, Inc., 1984e2001, Tulsa, OK, USA). Comparisons of means were conducted by LSD test of significant difference (p < 0.05). 3. Results Germination times and growth rates of the four xerophilic fungi at different temperatures and water activity levels in media adjusted with glycerol as single solute were compared with those obtained in presence of sorbitol or xylitol (10% w/w) in the same environmental conditions (Tables 1 and 2). ANOVA showed that all factors, polyol, temperature, water activity, and their interactions, significantly influenced on the germination time and growth rate (p < ) of each xerophilic fungus P. chrysogenum This fungus was not able to germinate after 100 days at 35 C and a w 0.85 or 0.88 when glycerol was the single solute (Table 1). Sorbitol addition allowed germination in these environmental conditions after 5.8 and 8 days respectively. In presence of xylitol the fungus germinated at 35 C and a w 0.88 but not at a w At the other temperatures and these two a w levels the presence of sorbitol and xylitol produced a decrease in germination time being this effect more evident with xylitol. This effect was not observed at a w 0.93, where the germination times were shorter (1 day) in presence of any solute, and at a w 0.90, where the only significant difference was observed at 35 C. Xylitol significantly increased growth rate of P. chrysogenum in comparison with glycerol at 25 C, except at 0.85 (Table 2). At 30 C growth rates varied in the same way (glycerol < sorbitol < xylitol) in the a w range 0.88e0.90. However, at this temperature and a w 0.93, the fungus grew faster in presence of sorbitol instead of the other solutes. As was pointed out, P. chrysogenum was not able to grow at 35 Canda w < 0.90 when a w vas adjusted only with glycerol, while in presence of sorbitol the fungus could grow in these conditions. With xylitol, at the same temperature, growth was detected at a w 0.88 but not at a w The most favorable a w level for the growth of this fungus was 0.93, with an optimum temperature depending on the polyol present in the medium. The combination of 35 C and 0.93 a w was the only one at which this fungus grew significantly faster in presence of glycerol than with xylitol W. sebi This fungus was not able to germinate after 100 days at 35 C and a w 0.85 and 0.88 in presence of xylitol (Table 1). However, at the same temperature and a w 0.90 and 0.93 xylitol produced a pronounced decrease of germination times compared to the other

3 A. Patriarca et al. / Food Microbiology 28 (2011) 1463e Table 1 Germination times (days) for xerophilic fungi in presence of different polyols at various a w and temperatures ( C). * a w Germination time (days) Glycerol Sorbitol Xylitol P. chrysogenum c 5 c >100 a 3 e 4 d 5.8 c 3 e 3 e >100 a c 8 b >100 a 4 d 4 d 8 b 1 gh 1 gh 1 gh fg 1 gh 3.5 de 2 f 1 gh 2 f 1.3 fgh 1 gh 1.1 fgh gh 1 gh 1 gh 1 gh 1 gh 1 gh 0.5 h 0.8 gh 0.5 h W. sebi i 10 h 14 e 5.8 k 5.3 l 10 h 8.3 i 11 g >100 a l 7 j 13 f 4 m 3 n 13 f 2 o 1 q >100 a n 3.3 n 17 d 3 n 2 o 17 d 1.5 p 1.5 p 3 n l 3 n 21 c 3 n 3 n 23 b 0.8 q 0.8 q 1 q E. chevalieri b 3.8 de 2 h 5 b 3 f 5 b 4 d 3 f 2 h d 3.5 e 1 ij 3 f 3 f 3 f 1 ij 1 ij 1 ij f 2 h 1 ij 3 f 1 ij 4.5 c 1.2 i 1 ij 0.6 jk h 2 h 8.3 a 2.5 g 3 f 1 ij 1 ij 0.8 ijk 0.4 k E. repens f 5 g >100 a 5 g 4 h 4 h 3 i 8 d 14 c e 4 h 16 b 3 i 3 i 3 i 1 lm 2 j 2.3 j j 1 lm 3 i 2 j 2 j 1.5 k 1.3 kl 1.3 kl 0.5 n j 1 lm 5 g 2 j 1 lm 1 lm 0.8 mn 0.8 mn 0.8 mn * Values with the same superscript within each fungal species are not significantly different (p < 0.05). polyols. The opposite effect was observed with the other solutes, germination times increased at higher a w levels. At 25 and 30 C the mold germinated faster in presence of sorbitol than with glycerol, except at 0.90 a w and 25 C and 0.93 a w and 30 C, where no significant differences were observed between both solutes. Xylitol also produced a reduction in germination times in comparison with glycerol and sorbitol at 25 and 30 C and a w > W. sebi is a slow growing fungus; its growth rates are lower than those of the other fungi at all environmental conditions tested (Table 2). This fungus did not grow at 35 C regardless the a w and the solute. Both sugar polyols increased growth rates at 25 and 30 C at some of the a w levels evaluated. The effect of sorbitol was significant at lower a w levels, while xylitol significantly increased growth rates at a w > 0.85 compared to the other solutes E. chevalieri Sorbitol demonstrated little influence on germination times of E. chevalieri at 25 C (Table1). At 30 C, the presence of sorbitol resulted in a reduction on germination times. However, at 35 C this polyol increased the germination time at all a w levels, except at Xylitol significantly reduced the germination times, at 25 and 30 C; at 35 C this reduction was only observed at 0.93 a w. At 0.93 a w and 35 C the germination time was strongly reduced by both polyols in comparison to glycerol. As it is shown in Table 2, this fungus grew faster in presence of sorbitol at 25 C, except for 0.85 a w, and at 30 C with the exception of 0.90 a w. The effect of xylitol did not show a clear tendency at these temperatures; the only increase in growth rate was observed at 0.88 a w.at35 C growth rates decreased in presence of sorbitol at all a w levels and was not significantly affected in presence of xylitol, except at 0.90 a w where the fungus grew faster than in presence of the other solutes E. repens Sorbitol exhibited a marked effect on germination times for this fungus. It significantly reduced the germination times at lower a w levels (0.85 and 0.88) and the three temperatures, and also at 35 C for all the a w levels. At this temperature and 0.85 a w sorbitol decreased the germination time of E. repens to 4 days, while the fungus was unable to germinate in 100 days at the same conditions Table 2 Growth rates (mm/day) for xerophilic fungi in presence of different polyols at various a w and temperatures ( C). * a w Growth rate (mm/day) Glycerol Sorbitol Xylitol P. chrysogenum lmn 1.14 lmn 0.00 p 1.21 lmn 1.27 lmn 1.01 mno 1.13 lmn 1.24 lmn 0.00 p lmno 0.85 no 0.00 p 1.00 mno 1.05 lmno 1.16 lmn 1.98 ij 1.97 ij 0.56 o klm 1.55 jkl 1.47 jklm 1.86 ijk 2.19 hi 2.59 gh 2.98 fg 3.24 ef 2.17 hi ef 3.46 ef 6.56 ab 3.57 e 6.81 a 6.19 b 5.01 c 4.59 cd 4.39 d W. sebi mn 0.12 kl 0.00 n 0.21 ghi 0.23 fgh 0.00 n 0.05 mn 0.09 klm 0.00 n kl 0.12 kl 0.00 n 0.08 lm 0.18 hij 0.00 n 0.29 de 0.35 bc 0.00 n ghi 0.13 jk 0.00 n 0.28 def 0.18 ij 0.00 n 0.35 bc 0.32 cd 0.00 n bc 0.25 efg 0.00 n 0.39 b 0.22 ghi 0.00 n 0.46 a 0.39 b 0.00 n E. chevalieri c 4.41 p 4.63 op 8.66 ij def 2.46 q 3.89 p 4.49 p 5.43 no p 3.88 p 8.50 ijk 8.38 jk 9.55 fgh 2.02 q 5.76 n 6.78 m 8.49 ijk m 9.85 defg 7.81 kl b 9.03 hij 4.39 p 6.72 m 7.80 kl d c 9.70 efgh 9.81 defg b a 4.27 p 7.19 lm 9.25 ghi de E. repens op 3.71 o 0.00 q 6.82 hi 6.07 ij 4.12 mno 3.69 o 4.15 mno 0.00 q no 4.55 lmn 0.00 q 7.15 h bc 8.24 g 6.48 hij 7.94 g 5.72 jk kl 4.85 lm 2.69 p 9.22 f cd 9.20 f 8.13 g 8.47 fg 6.18 ij h de 4.83 lm b e 8.13 g d a 5.12 kl * Values with the same superscript within each fungal species are not significantly different (p < 0.05).

4 1466 A. Patriarca et al. / Food Microbiology 28 (2011) 1463e1467 when glycerol was the only solute. A 0.85 a w level and 30 C was the only condition at which germination time increased in presence of xylitol in comparison to glycerol. At all the other a w and temperature combinations germination time was shorter or not significantly different to that observed for glycerol (Table 1). Growth rates at 25 C were practically twofold in presence of sorbitol in comparison with glycerol at all a w levels (Table 2). Growth rates also increased in presence of xylitol at 25 C and a w >0.85. Both polyols resulted in an increment of growth rates at 30 C in the whole a w range, except for sorbitol at 0.93 a w and xylitol at 0.85 a w where no significant differences were observed when compared to glycerol. At 35 C the fungus was not able to grow at a w 0.85 and 0.88 when glycerol was the only solute. However, at the same temperature and in presence of sorbitol, growth was quite fast even at the lowest a w level. In presence of xylitol, the fungus grew at a w > 0.85 although growth rates were lower than in presence of sorbitol. 4. Discussion Fungal isolates used in the present study belong to the most widespread and significant species in deterioration of low moisture foods (Pitt and Hocking, 1997). All of them were able to grow in the studied range of a w. P. chrysogenum, known as one of the most xerophilic Penicillia, was able to grow in the whole a w range, except at 35 C which is not a favorable temperature for this fungus. At this temperature the mold did not germinate when a w was adjusted with glycerol at a w 0.85 and 0.88 but was able to germinate in presence of sorbitol and xylitol. Sautour et al. (2001) found no significant difference between a glycerol and a sorbitol model based on germination of P. chrysogenum. In the present study, sorbitol stimulated P. chrysogenum germination, and the effect was more evident in suboptimal environmental conditions. W. sebi grew very slowly under all experimental conditions, in concordance with results reported by Patriarca et al. (2001) and Gock et al. (2003). At35 C spore germination was not followed by mycelial growth. At 25 and 30 C the presence of sugar polyols produced a decrease in germination times and an increase of the growth rates, mainly xylitol at higher a w (0.90 and 0.93). Beuchat and Pitt (1990) studied the influence of glucose, sorbitol and NaCl at a w from 0.82 to 0.97 on colony formation by sublethally heatstressed W. sebi conidia and, in agreement with our results, they reported that colony diameters were generally largest on media containing sorbitol. Both polyols showed significant effects on germination times of Eurotium species, with a resulting decrease in most of the conditions of the present work, especially with xylitol. At 25 and 30 C sorbitol considerably increased the growth rate of both species at most of the a w levels. A similar effect was observed with xylitol, but only on E. repens. Results of the present study demonstrated that sugar polyols, sorbitol and xylitol, affect the growth kinetics of the four fungal species considered. The tendency observed was that these solutes shortened the germination times and increased the growth rates. The effect of each solute depended on the fungal species and the a w /temperature combinations. At lower a w the influence was more evident on the germination times while the effect on growth rates was more pronounced at higher a w levels. It could be hypothesized that polyols can act as compatible solutes, as was mentioned by other authors (Hallsworth et al., 1998; Ramos et al., 1999), and hence enhance germination of the stressed conidia at low water availability. The effect is less evident at higher a w where the fungi have not difficulty to germinate. On the other hand, at high a w levels at which the fungi are able to grow these solutes could be used as sources of carbon, resulting in a faster growth. Another fact that should be considered is the interaction of water with the solid matrices. Recently, Huang et al. (2009) studied the effect of the matric potential of glycerol agar media with different agar concentrations on the germination and growth of xerophilic and non-xerophilic fungi. These authors found that the interaction between the agar-water gel and glycerol can affect the gel structure and, consequently, modify the matric potential and the water status in the solid culture media. In a similar way, the presence of polyols in lowered a w foods could have influence on fungal growth as a consequence of affecting the matric potential. Further studies are necessary to clarify the mode of action of these compounds. However, their effect on fungal growth kinetics observed in the present work should be taken into account when these additives are included in the formulation of a w reduced foods in which shelf life depends on germination and growth of xerophilic fungi. Acknowledgments Financial support of Universidad de Buenos Aires is acknowledged. The authors of the present work are members of CONICET. References Abellana, M., Benedí, J., Sanchis, V., Ramos, A.J., 1999a. Water activity and temperature effects on germination and growth of Eurotium amstelodami, E. chevalieri and E. herbariorum isolates from bakery products. Journal of Applied Microbiology 87, 371e380. Abellana, M., Magrí, X., Sanchis, V., Ramos, A.J., 1999b. Water activity and temperature effects on growth of Eurotium amstelodami, E.chevalieri and E. herbariorum on a sponge cake analogue. International Journal of Food Microbiology 52, 97e103. 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