Production of the fungal biocontrol agent Epicoccum nigrum by solid substrate fermentation: effect of water activity on accumulation of compatible solutes

Epicoccum nigrum conidia were produced by solid fermentation on wheat grains (cv. Rendeveaux and Brigadier) at different water activities (a_w). Conidial production was highest at high a_w(0.996) than at reduced a_w (0.98). However, conidial production at reduced a_w was improved when the a_w of the substrate was adjusted with a mixture of glycerol and water. Maximum levels ofconidiation were 7–11 × 10⁶ conidia g⁻¹ grain. The a_w of the solid substrate affected the pattern of accumulation of compatible solutes in the conidia. Mannitol was the main polyol in all conidialtypes. However, the amounts of mannitol were higher in conidia produced at high a_w. At reduced a_w the conidia of E. nigrum accumulated moreglycerol, which is more efficient in the osmorregulation proccess than mannitol. Arabitol accumulated in low amounts, specifically in conidia produced at the lower a_w, on cv. Rendeveaux but not on cv. Brigadier. Trehalose was detected in higher amounts in cv. Rendeveaux than in cv. Brigadier, andthe amounts were higher in conidia produced at high a_w. A significant amount of endogenous solutes was detected in the washing liquid used for the separation of the conidia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Survival and Filamentation of Salmonella enterica Serovar Enteritidis PT4 and Salmonella enterica Serovar Typhimurium DT104 at Low Water Activity

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a_w). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a_w for long periods, but minimum humectant concentrations of 8% NaCl (a_w, 0.95), 96% sucrose (a_w, 0.94), and 32% glycerol (a_w, 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a_w, incubation at 37°C resulted in more rapid loss of viability than incubation at 21°C. At a_w values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 μm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a_w conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a_w highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a_w (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a_w storage. If Salmonella strains form filaments in food products that have low a_w values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.     

Optimization of erythritol and glycerol accumulation in conidia of Beauveria bassiana by solid-state fermentation, using response surface methodology

Entomopathogenic fungi are widely produced for use as mycoinsecticides. Therefore, improvement of the shelf life of fungal propagules under good and adverse conditions should be a pre-requisite of their production. In order to improve conidial physiology as well as mycoinsecticide efficiency, culture conditions may be varied. The Doehlert design was used to generate response surfaces with an estimation of the parameters of the quadratic model allowing the study of three different factors at a different number of levels. This experimental design was applied to optimize water activity (a _w), pH, and fermentation time for Beauveria bassiana conidial production and accumulation of polyols in solid-state fermentation. Thus, it was possible to identify the region in the experimental range in which the optimum values of these parameters were simultaneously achieved. Maximal conidia production was achieved at pH 5–6 and a _w=0.999. Under these conditions, polyol accumulation was 3 mg erythritol/g conidia and 29.6 mg glycerol/g conidia. However, maximal polyol accumulation was achieved at pH 4.5 and a _w 0.950; erythritol production increased 33-fold and glycerol production 4.5-fold. Under these conditions conidia production was 1,000 times lower. The possibilities of increasing the quality of the biocontrol agent without neglecting yield are discussed.     

Salmonella spp. are affected by different levels of water activity in closed microcosms

Controlling water activity (a _w) can significantly impact the growth of Salmonella in poultry litter and manure — a phenomenon that was studied quantitatively using two common serotypes of Salmonella. The quantitative effect of changes in levels of a _w on Salmonella populations was determined using inoculated, frosted glass rectangles placed in closed chambers (microcosms). Glass rectangles with known concentrations of Salmonella enteritidis and S. brandenburg were placed in microcosms maintained at an a _w level of 0.893 for 24 h at room temperature (RT) and then transferred to other microcosms maintained at the same temperature but with higher a _w levels (0.932 and 0.987). Salmonella populations on the slides were quantified at 4, 18, 24, and 48 h. Slightly elevated levels of a _w (<0.1, i.e., 10% equilibrium relative humidity) for 24 h resulted in a 100-fold increase in counts of Salmonella. The data also suggested that in vitro adaptation to dry environments may occur when the organisms are exposed to alternating levels of relatively high and low (0.987 and 0.893) levels of a _w. Any increased tolerance of Salmonella to reduced levels of a _w could be the result of physico-chemical changes in the organism due to selective environmental pressure, formation of a protective biofilm, and/or entry into a dormant state. Results from this study are compatible with those from previously reported on-farm surveys, reinforcing the contention that maintaining a _w below 0.85 in and around litter/manure surfaces in poultry or livestock bedding areas may be a critical factor in safe production of food. Journal of Industrial Microbiology & Biotechnology (2001) 26, 222–225. Received 18 May 2000/ Accepted in revised form 24 January 2001     

Zearalenone, deoxynivalenol and fumonisins in mixed-feed for laying hens

Fusarium toxins are secondary metabolites produced byfungi of these genera in many commodities under certain conditions. A study was carried out to investigate the co-occurrence of zearalenone (ZEN), deoxynivalenol (DON) and fumonisins (FB₁ and FB₂) in 52 samples of mixed-feed for poultry contaminated withFusarium verticillioides. The zearalenone and deoxynivalenol were checked using immunoaffinity column and the extraction of fumonisin was performed by strong anion exchange (SAX) solid phase column. Detection and quantification were determined by high performance liquid chromatography (HPLC). The limit of detection was 5 μg/kg for ZEN, 100 μg/kg for DON and 50 and 100 μg/kg for FB₁ and FB₂ respectively.Fusarium toxins were detected in 20 samples. Sixteen samples were positive for ZEN (30.7%) presenting levels that ranged from 7.4 μg/kg to 61.4 μg/kg (mean=27.0 μg/kg). 13.5% of the samples presented contaminations of DON, with levels ranging from 100.0 μg/kg to 253 μg/kg (mean=l18.07 μg/kg). FB₁ was detected in 19.2% of samples, with levels ranging from 50.0 μg/kg to 110.0 μg/kg (mean=73.6 μg/kg). FB2 was not detected in any sample. In positive samples simultaneously contamination with two or three mycotoxins were detected in 9 of them (17.3%).     

The Influence of Water Activity and Temperature on Germination,Growth and Sporulation of <Emphasis Type="Italic">Stachybotrys chartarum</Emphasis> Strains

The objectives were to determine the influence of water activity (a_w, 0.997–0.92) and temperature (10–37°C) and their interactions on conidial germination, mycelial growth and sporulation of two strains of Stachybotrys chartarum in vitro on a potato dextrose medium. Studies were carried out by modifying the medium with glycerol and either spread plating with conidia to evaluate germination and germ tube extension or centrally inoculating treatment media for measuring mycelial growth rates and harvesting whole colonies for determining sporulation. Overall, germination of conidia was significantly influenced by a_w and temperature and was fastest at 0.997–0.98 a_w between 15 and 30°C with complete germination within 24 h. Germ tube extension was found to be most rapid at similar a_w levels and 25–30°C. Mycelial growth rates of both strains were optimal at 0.997 a_w between 25 and 30°C, with very little growth at 37°C. Sporulation was optimum at 30°C at 0.997 a_w. However, under drier conditions, this was optimum at 25°C. This shows that there are differences in the ranges of a_w x temperature for germination and growth and for sporulation. This may help in understanding the role of this fungal species in damp buildings and conditions under which immune-compromised patients may be at risk when exposed to such contaminants in the indoor air environment.     

Effect of Water Activity on Enterotoxin B Production and Growth of Staphylococcus aureus

Staphylococcus aureus C-243, an enterotoxin B-producing strain, was cultured on media adjusted to various water activity (a_w) levels by means of two different solute systems. Total numbers and rate of growth were diminished at low a_w levels, and enterotoxin synthesis was extremely sensitive to reduction in a_w. A reduction of a_w from 0.99 to 0.98 in one medium and from 0.99 to 0.97 in the other medium resulted in extremely low levels of enterotoxin in spent culture media despite the attainment of high numbers of staphylococci.     

Adaptation of Pseudomonas fluorescens to Low Levels of Water Activity Produced by Different Solutes

A culture of Pseudomonas fluorescens which grew at a minimal a_w of 0.97 in a glucose-salts broth adjusted to that level with NaCl, KCl, or glucose, and at a_w 0.9725 with Na₂SO₄, was adapted to grow at lower a_w levels produced by these solutes. Adaptation to growth at a low a_w produced by means of one solute did not necessarily result in growth at that a_w when another solute was employed to lower the a_w. The culture adapted to grow at a_w 0.9650 produced by addition of glucose could grow equally well in broth adjusted to that a_w with NaCl or KCl, but not Na₂SO₄. NaCl and KCl permitted growth at the lowest a_w level (0.9625), and the culture adapted in KCl did equally well in NaCl.     

Natural occurrence of mycotoxins and mycoflora of argentinien popcorn

Argentina is the first popcorn exporting country worldwide. In 1997-1998 harvest season, 40,000 ha were sown with a production of 125,000 tons; 120,000 tons of this production were exported to more than 40 countries. The objectives of this study were to isolate and to identify the fungi present in this cereal and to assess the occurrence of mycotoxins in freshly harvested popcorn in Buenos Aires province, Argentina, in 1999. All popcorn samples showed fungal contamination. A total of 4,211 isolates were recovered from popcorn kernel samples. The prevalent species isolated wasFusarium verticillioides followed byPenicillium funiculosum, F. graminearum andAlternaria alternata. No aflatoxins, zearalenone, deoxynivalenol or ochratoxin A were detected in the 42 popcorn simples. All samples but one were contaminated with fumonisins (FB) in a range in ug/kg of (not detected-529) for FB₁; (not detected-216) for FB₂ and (not detected-103) for FB₃. Fumonisin contamination levels in Argentinean popcorn were lower than observed in flint corn. No significant differences in fungal and fumonisins contamination levels were observed in the different tested hybrids.     

Aflatoxin and Ochratoxin — a contamination of dried figs (Ficus carina L) from the 1988 crop

The study examines the occurrence of aflatoxin and ochratoxin A in the !988 dried figs crop. Mycotoxin content, moisture, and a_w (water activity) were analyzed in a total of 103 fig samples collected from various orchards and different stages of fig processing. Aflatoxins (B₁, B₂, G₁, and G₂) were present in 29% of the samples examined at 0.5–63.0, 0.5–37.7, 0.5–78.3, and 0.5–12.5μ/kg, respectively. Ochratoxin A was detected in only 3% of the samples at 5.2–8.3 μ/kg. The moisture (and a_w) values of the fruits were found suitable for mycotoxin formation in firm ripened and shrivelled figs.     

Fusarium Head Blight Reactions and Accumulation of Deoxynivalenol (DON) and Some of its Derivatives in Kernels of Wheat, Triticale and Rye

Fifty-three commercially grown cultivars and germplasm lines of winter triticale (n = 18), wheat (n = 13), and rye (n = 5) and spring triticale (n = 8), wheat (n = 7) and rye (n = 2) were inoculated at mid anthesis with a spore suspension consisting of a mixture of Fusarium culmorum, Fusarium avenaceum and Fusarium graminearum isolates of known toxinogenic activity. Reactions to Fusarium head blight were measured as disease severity, reductions of kernel number/head, kernel weight/head and 1000 kernel weight, number of Fusarium-damaged kernels and kernel content of deoxynivalenol (DON) and its acetyl-derivatives 3-AcDON, 15-AcDON, and moniliformin. None of the cereal genotypes was completely resistant to Fusarium head blight. Wheat suffered from the largest kernel weight reductions, and accumulated the largest amounts of deoxynivalenol (up to 39.5 mg/kg) and 3AcDON (up to 6.0 mg/kg) in kernels. Deoxynivalenol was not detected in grain samples of winter rye cv. Dańkowskie Z?ote, and spring rye cv. Ludowe. 15-AcDON was only detected in genotypes of triticale, and 3AcDON only in a few genotypes of winter wheat and rye. Moniliformin was detected at low concentrations (up to 0.092 mg/kg) in kernels of some genotypes selected for the mycotoxin analysis. A moderately strong Pearson correlation was found between head blight severity parameters and the accumulation of deoxynivalenol and its derivatives in grain of the cereal genotypes studied. Fusarium head blight severity parameters were correlated with the percentage of Fusarium-damaged kernels and reductions of yield components. However, some head blight-susceptible genotypes realized their potential yields, but accumulated high levels of mycotoxins in kernels. Both Fusarium head blight resistant and susceptible genotypes of the three cereal species accumulated deoxynivalenol in kernels. This finding suggests that the system regulating deoxynivalenol accumulation may be independent of Fusarium head blight reaction.     

Water relations of polyol accumulation by Zygosaccharomyces rouxii in continuous culture

Summary Glycerol and arabitol were the main polyols accumulated by Zygosaccharomyces rouxii in continuous culture but the intracellular and extracellular concentrations of the polyols varied with the dilution rate and osmoticum used to adjust the water activity (a_w) to 0.960. When the a_w was adjusted with NaCl, glycerol was the main polyol accumulated intracellularly whereas glycerol and arabitol were accumulated when polyethylene glycol (PEG) 400 was used. The extracellular glycerol and arabitol concentrations at 0.960 a_w (NaCl or PEG 400) were similar or decreased relative to cultures at 0.998 a_w. Compared to steady-state cultivation at 0.998 a_w, the yeast retained at 0.960 a_w (NaCl or PEG 400) a greater proportion of the total glycerol intracellularly against an increased concentration ratio without significantly greater production of glycerol. Arabitol was only significant in osmoregulation when cultivated at 0.960 a_w (PEG 400). The intracellular glycerol concentration was insufficient to balance the a_w across the membrane, but an equilibrium could be achieved under certain conditions if arabitol was also osmotically active. Offprint requests to: P. J. van Zyl     

Influence of Reduced Water Activity on Lactose Metabolism by Lactococcus lactis subsp. cremoris at Different pH Values

The influence of reduced water activity (a_w) on lactose metabolism by Lactococcus lactis subsp. cremoris 2254 and 2272 was studied at different pH values. In control incubations (a_w, 0.99) with nongrowing cells in pH-controlled phosphate buffer, the levels of carbon recovered as l-(+)-lactate were 92% at pH 6.1 and 5.3 and 78% at pH 4.5. However, the levels of recovery decreased to ∼50% at all pH values tested when the a_w was 0.88 (with glycerol as the humectant). When growing cells in broth controlled at pH 6.3 were used, a reduction in the a_w from 0.99 to 0.96 resulted in a decrease in the level of lactose carbon recovered as l-(+)-lactate from 100 to 71%. Low levels of l-(+)-lactate carbon recovery (<50%) were also observed with cells resuspended in pH-uncontrolled reconstituted skim milk at a_w values of 0.99 and 0.87 and in young cheese curds. The missing lactose carbon could not be accounted for by acetate, ethanol, formate, acetaldehyde, or pyruvate. Attempts were made to determine where the missing lactose carbon was diverted to under the stress conditions used. Some of the missing lactose carbon was recovered as galactose (0.1 to 2.5 mM) in culture supernatants. Decreasing either the a_w or the pH resulted in increased galactose accumulation by nongrowing cells; adjusting both environmental factors together potentiated the effect. The sensitivities of the two lactococcal strains tested were different; strain 2272 was more prone to accumulate galactose under stress conditions. A methyl pentose(s) and additional galactose were found in acid-hydrolyzed supernatants from cultures containing both growing and nongrowing cells, indicating that a saccharide(s) rich in these components was formed by lactococci under low-a_w and low-pH stress conditions.Water activity (a_w) affects the growth, physiology, and metabolism of microorganisms and their resistance to inimical agents (22, 54). A number of microorganisms respond to a low-a_w environment by intracellular accumulation of low-molecular-weight compatible solutes, such as amino acids, amino acid derivatives, trehalose, and polyols (6, 12). These compatible solutes restore turgor pressure and membrane tension to levels very similar to those that occur before osmotic upshift (12), preserve enzyme activity and protein stability, and maintain the integrity and stability of membranes and nucleic acids (6).There have been several reports concerning the accumulation of compatible solutes in lactic acid bacteria. Hutkins et al. (26) found that betaine was accumulated by an osmotolerant strain of Lactobacillus acidophilus, while Molenaar et al. (38) reported that Lactococcus lactis subsp. lactis ML3 contained high levels of proline or betaine when it was grown under osmotic stress conditions in complex media. Lactobacillus plantarum, however, accumulated not only betaine and proline, but also carnitine, glutamic acid, and trehalose when it was cultured in a complex medium having a reduced a_w (29, 30, 36). The accumulation of betaine enhanced the survival of several lactic acid bacteria subjected to drying conditions (28, 30).The influence of reduced a_w on substrate metabolism and product formation by lactic acid bacteria has received little attention. Optimal production of lactic acid by some dairy lactic acid bacteria occurs at a_w values of 1.0 to 0.95, and production declines dramatically as the a_w is decreased, which is consistent with growth inhibition, whereas diacetyl production by some lactic acid bacteria increases with decreasing a_w and optimal diacetyl production occurs at a_w values of 0.95 to 0.97 (51, 52). Bassit et al. (1) studied the influence of a_w on the metabolism of Streptococcus diacetylactis (now Lactococcus lactis subsp. lactis var. diacetylactis) and found that decreasing the a_w decreased lactose consumption and lactic acid formation and inhibited growth. Blickstad (3) observed no significant variations in the formation of end products by two meat lactobacilli at different a_w values (0.99 to 0.94).Lactose is the major carbohydrate present in milk and young cheeses, and metabolism of lactose by starters during curd manufacture and early ripening is important for acid development. The cheese a_w decreases during manufacture and ripening as a result of dehydration, salting, and the production of water-soluble solutes from glycolysis, proteolysis, and lipolysis; the cheese a_w values range from 0.70 for extrahard cheeses to 0.99 for fresh, soft cheeses, such as cottage cheese, while semihard cheeses have a_w values of around 0.90 (33, 41). The cheese pH also decreases during manufacture and ripening (19). We describe here the influence of low a_w values on lactose metabolism by both growing and nongrowing cells of Lactococcus lactis subsp. cremoris at different pH values in buffer, broth, and reconstituted skim milk (RSM) with and without pH control. Cheese experiments to determine lactose utilization during ripening were also linked with our in vitro studies.     

Aflatoxin B1, zearalenone and deoxynivalenol production on irradiated corn kernels: Influence of inoculum size

The influence of inoculum size on aflatoxin B₁ (AFB₁), zearalenone (ZEN) and deoxynivalenol (DON) production was examined on irradiated corn kernels. Spore concentrations were determined in serial dilutions and adjusted to 10,10²,10³,10⁵ and 10⁶ spores/ml. Aflatoxin B₁ production was dependent on the inoculum size. The high levels of aflatoxin B₁ produced byA. parasiticus (21 and 30 mg/kg) were obtained with 10² and 10³ spores/ml after 35 and 20 days incubation. There was no spore concentration influence on zearalenone and deoxynivalenol production after 10, 20 and 35 days incubation. At 28°C and 0.97 water activity (aw), the mean levels of zearalenone production were 382, 267 and 520 μg/kg and the mean levels on deoxynivalenol production were 697,465 and 782 μg/kg.     

Moisture Requirements for Growth and Metabolite Production by Lactic Acid Bacteria

The effect of water activity (a_w) reduction on growth and acid and diacetyl production by three lactic streptococci was studied. In addition, the influence of low moisture conditions on several bacteria of significance in the fermentation of sauerkraut was examined. The minimal a_w supporting growth of dairy lactics was 0.93 in a medium adjusted with glycerol. Media adjusted with sucrose generally were more inhibitory than those in which glycerol was the humectant. Titratable acidity, although not related to the type of humectant, did depend on the a_w of the medium and was directly related to the extent of growth. Diacetyl concentration increased in cultures of reduced a_w when the media were adjusted with both humectants; however, the effect was greatest with glycerol. A lactic strain associated with sauerkraut fermentation appeared to grow at a lower minimal a_w in a glycerol-adjusted medium than in a system adjusted with NaCl; however, none of the sauerkraut organisms grew at a_w levels of <0.95 when NaCl was the solute. Acid production appeared to be related to the presence and extent of growth at all of the a_w levels studied.     

Modelling the effect of water activity and temperature on growth rate and aflatoxin production by two isolates of Aspergillus flavus on paddy

Aims: This study was conducted to characterize the growth of and aflatoxin production by Aspergillus flavus on paddy and to develop kinetic models describing the growth rate as a function of water activity (a_w) and temperature. Methods and Results: The growth of A. flavus on paddy and aflatoxin production were studied following a full factorial design with seven a_w levels within the range of 0·82–0·99 and seven temperatures between 10 and 43°C. The growth of the fungi, expressed as colony diameter (mm), was measured daily, and the aflatoxins were analysed using HPLC with a fluorescence detector. The maximum colony growth rates of both isolates were estimated by fitting the primary model of Baranyi to growth data. Three potentially suitable secondary models, Rosso, polynomial and Davey, were assessed for their ability to describe the radial growth rate as a function of temperature and a_w. Both strains failed to grow at the marginal temperatures (10 and 43°C), regardless of the a_w studied, and at the a_w level of 0·82, regardless of temperature. Despite that the predictions of all studied models showed good agreement with the observed growth rates, Davey model proved to be the best predictor of the experimental data. The cardinal parameters as estimated by Rosso model were comparable to those reported in previous studies. Toxins were detected in the range of 0·86–0·99 a_w with optimal a_w of 0·98 and optimal temperature in the range of 25–30°C. Conclusions: The influences of a_w and temperature on the growth of A. flavus and aflatoxin production were successfully characterized, and the models developed were found to be capable of providing good, related estimates of the growth rates. Significance and Impact of the Study: The results of this study could be effectively implemented in minimizing the risk of aflatoxin contamination of the paddy at postharvest.     

Factors determining the microflora of stored barley grain

Colonisation of barley grain has been studied during storage at different water contents and with and without restriction of the air supply to simulate conditions in unsealed silos. Grain stored with a water activity >0·9 a_w (20% water content) heated spontaneously when aeration was unrestricted, the maximum temperature attained increasing with a_w to 58 °C at 1·0 a_w (39% water content). The presence of many unripe grains increased the tendency to heat at a given mean water content. Although heating was prevented by sheeting to restrict the air supply, it could occur subsequently when the sheeting was removed. Both heating and restriction of the air supply were associated with increased carbon dioxide (to >25%) and decreased oxygen concentrations (to <5%). Germination of grain after 6·9 months storage was correlated with a_w; germination levels approaching 100% were retained only at about 0·7 a_w (13·5% water content). Colonisation by Aspergillus species was correlated with a_w and temperature and similar correlations with Penicillium species were also found, with P. verrucosum var. cyclopium abundant at 0·85-0·90 a_w (17·20% water content) and P. piceum, P. funiculosum and P. capsulatum at 0·90-0·95 a_w (20–25% water content). In sealed containers P. roquefortii occurred at 1·00 a_w (39% water content) and P. hordei at 0·90-0·92 a_w (20–22% water content). Spores of fungi and actinomycetes formed during spontaneous heating of grain survived 6 months sealed storage although Absidia corymbifera and Micropolyspora faeni may have declined in numbers. Fungicides applied to the ripening grain had only limited effect on the colonisation of the grain during storage.     

Survival of alginate-entrapped cells of Azospirillum lipoferum during dehydration and storage in relation to water properties

Survival of alginate-entrapped cells of Azospirillum lipoferum was studied during dehydration using a dry air stream and during prolonged storage at various constant water activity values (a_w). During the drying operation, the viability loss remained almost constant from the initial water content to 0.35 g water/g dry weight (DW) corresponding to a 98.5% water removal, strongly increased until a water content of 0.25 g/g DW and then stopped until the end of the drying operational (final a_w 0.18). A water content of 0.25 g/g DW (a_w=0.55) corresponded to the critical point of the moisture sorption isotherm curve from which water became restricted to the dry material. A high drying rate (5 g/g DW per hour) was shown to be more detrimental for cell viability than a low drying rate (1.18 g/g DW per hour). When the product was stored in a closed chamber with a regulated a_w (0.23), the number of living cells decreased during a short period (less than 15 days) corresponding to the product a_w stabilization, and then remained constant for more than 150 days. In addition, cell survival during storage was not affected by a_w values in the range 0–0.55. Above a_w=0.55, the higher the a_w and the storage duration, the lower the residual survival percentage. The influence of the drying and storage conditions on the cell death rate are discussed with regard to both the mechanisms generally involved in viability loss and the hydration properties of water. Correspondence to: A. Pareilleux     

Impact of a Streptomyces (AS1) strain and its metabolites on control of Aspergillus flavus and aflatoxin B1 contamination in vitro and in stored peanuts

Six actinomycetes were isolated from peanuts in Egypt. Of these, a Streptomyces strain (AS1) was found in in vitro assays to inhibit directly or via secondary metabolites both germination and growth of Aspergillus flavus. Tests of the AS1 cells for direct control of A. flavus populations or aflatoxin B₁ (AFB₁) production on stored peanuts was unsuccessful over 14-day storage periods. However, crude extracts of AS1 metabolites at 50 and 100 ppm completely inhibited spore germination of conidia of A. flavus in vitro over 48 h. Comparison of solvents for extracting the metabolites showed that the ethyl acetate extract was most effective. This gave greater than 85% inhibition of mycelial growth at these concentrations at different water availabilities (water activity; a _w; 0.95, 0.92, and 0.89) and 25°C. Doses of 50, 200, and 500 ppm of AS1 metabolites significantly inhibited populations of A. flavus on stored peanuts at two water stress levels (0.90, 0.93 a _w) at 25°C over 14-day storage periods. The amounts of AFB₁ produced by A. flavus on peanuts stored at 0.90 a _w were significantly decreased by AS1 metabolites for only 7 days. However, at 0.93 a _w doses of 200 and 500 ppm significantly controlled AFB₁ accumulation in peanuts for 14 days.