Impact of some environmental factors on growth and production of ochratoxin A of/by <Emphasis Type="Italic">Aspergillus tubingensis,A. niger</Emphasis>, and <Emphasis Type="Italic">A. carbonarius</Emphasis> isolated from moroccan grapes

The effects of temperature, water activity (a_w), incubation time, and their combinations on radial growth and ochratoxin A (OTA) production of/by eight Aspergillus niger aggregate strains (six A. tubingensis and two A. niger) and four A. carbonarius isolated from Moroccan grapes were studied. Optimal conditions for the growth of most studied strains were shown to be at 25°C and 0.95 a_w. No growth was observed at 10°C regardless of the water activity and isolates. The optimal temperature for OTA production was in the range of 25°C∼30°C for A. carbonarius and 30°C∼37°C for A. niger aggregate. The optimal a_w for toxin production was 0.95∼0.99 for A. carbonarius and 0.90∼0.95 for A. niger aggregate. Mean OTA concentration produced by all the isolates of A. niger aggregate tested at all sampling times shows that maximum amount of OTA (0.24 μg/g) was produced at 37°C and 0.90 a_w. However, for A. carbonarius, mean maximum amounts of OTA (0.22 μg/g) were observed at 25°C and 0.99 a_w. Analysis of variance showed that the effects of all single factors (a_w, isolate, temperature and incubation time) and their interactions on growth and OTA production were highly significant.     

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     

The production of cyclopiazonic acid by Penicillium commune and cyclopiazonic acid and aflatoxins by Aspergillus flavus as affected by water activity and temperature on maize grains

The combined effects of water activity (a_w) and temperature on mycotoxin production by Penicilium commune (cyclopiazonic acid — CPA) and Aspergillus flavus (CPA and aflatoxins — AF) were studied on maize over a 14-day period using a statistical experimental design. Analysis of variance showed a highly significant interaction (P 0.001) between these factors and mycotoxin production. The minimum a_w/temperature for CPA production (2264 ng g^–1 P. commune, 709 ng g^–1 A. flavus) was 0.90 a_w/30 °C while greatest production (7678 ng g^–1 P. commune, 1876 ng g^–1 A. flavus) was produced at 0.98 a_w/20 °C. Least AF (411 ng g^–1) was produced at 0.90 a_w/20 °C and most (3096 ng g^–1) at 0.98 a_w/30 °C.     

Effect of water activity adjusted with different solutes on growth and lactic acid production byLactobacillus helveticus

Effect of water activity (a _w) adjusted with NaCl or glycerol on the growth and metabolism ofLactobacillus helveticus var.pragensis at 40°C was demonstrated by growth curves (generation time) and changes in the degree of acidity of a milk medium expressed as lactic acid content. NaCl-regulateda _w of 0.970 inhibited growth and acid production completely. The same, glycerol-regulateda _w of 0.970 increased the generation time only from 33 min (a _w=0.994) to 67 min which was less than the generation time at the highera _w of 0.982 adjusted with NaCl (103 min). Glycerol-regulateda _w of 0.970 did not decrease the acid production (2.6% lactic acid). Ata _w of 0.951, the acid production was decreased by 39% compared with the values found in milk media with the original, unadjusteda _w of 0.994, after the same time of incubation. Media witha _w adjusted to the same values with NaCl or glycerol do not influence the growth and acid production ofL. helveticus in the same way. In contrast to glycerol, NaCl has a strong effect.     

Effect of water activity on the growth and the production of cAMP phosphodiesterase inhibitor with Bacillus subtilis

Summary Bacillus subtilis C-756, a producer of cyclic adenosine 3,5-monophosphate (cAMP) phosphodiesterase inhibitor, was cultured in media adjusted to various water activity (a_w) levels by addition of three different solutes, sodium chloride, ethylene glycol and polyethylene glycol 1540 (PEG). B. subtilis C-756 can grow, however weakly, at a_w levels of 0.94 and 0.93.The presence of all three solutes in the medium inhibited growth, cell mass as well as inhibitor production. PEG was found to be most inhibitory, but the effect can not be explained in terms of a decreased water activity in the medium. It is rather the increased viscosity of the medium, which results in a decreased oxygen transfer rate.Comparing ethylene glycol and sodium chloride, the presence of ethylene glycol appears to favour inhibitor production, whereas sodium chloride favours cell mass production.     

Effect of environmental factors on in vitro and in situ interactions between atoxigenic and toxigenic A. flavus strains and control of aflatoxin contamination of maize

Abstract

The potential of three atoxigenic strains from different geographical origins in Africa were examined for in vitro and in situ competitiveness against two toxigenic strains of Aspergillus flavus and subsequent inhibition of aflatoxin B₁ (AFB₁) production under different environmental conditions. Temperature, water activity (a _w) and substrate influenced the types of interaction between the three AFL^? and two AFL⁺ strains. The competitiveness and AFB₁ reduction ability of the three atoxigenic strains when interacting with the two toxigenic strains were evaluated by inoculation of 100, 25:75, 50:50, 75:25 and 100% ratios of mixed spore suspensions in vitro on malt extract and milled maize agars over 28 days and in situ on stored maize grain for 14 days, respectively at 0.99, 0.96 and 0.90 a _w. For all the treatments, the effect of a _w and inoculum ratio and their interaction was highly significant. Toxin inhibition was >80% in vitro at both 0.99 and 0.96 a _w. In situ AFB₁ reduction was influenced by the toxigenic strain assayed, a _w and the inoculum ratio. Where control was achieved, it was more variable at 0.96 a _w, while with more stringent water stress conditions (0.90 a _w) the percentage inhibition was up to 77.2%. The study shows the importance of including environmental factors in screening and identifying effective atoxigenic strains for control of AFs (aflatoxins).     

Changes in protein secretion of Aspergillus niger caused by the reduction of the water activity by potassium chloride

The effect of the rapid reduction of the water activity (a_w) on the extracellular protein and amylolytic activity of Aspergillus niger was studied. An a_w value gradient from 0.90 to 0.99 in KCl solutions was applied for the mycelium treatment. It was found that the a_w reduction considerably influenced the protein secretion. This phenomenon was dependent on the age of the treated mycelium and the range of the a_w gradient. The highest protein and enzyme secretion yields were obtained at a_w = 0.98 using a 72-h old mycelium. In comparison with the non-treated mycelium, the increase in the secretion amounted to about 60% for the amylolytic activity and 37% for the soluble protein, respectively. It was shown that the mycelium incubated in KCl solutions of an a_w value from 0.90 to 0.99 had the ability for regeneration in fresh CZAPEK-DOX medium. The effect of the osmotic shock on the protein secretion was limited only for the treated cell population and declined in the mycelium which was regenerated after the transfer into the culture medium.     

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.     

Influence of water activity on the enzyme biosynthesis and enzyme activities produced by Trichoderma viride TS in solid-state fermentation

Influence of water activity (a_w) on biosynthesis of polygalacturonase, d-xylanase and β-glucosidase in solid culture system of Trichoderma viride TS was studied. It was found that the production of enzymes was strongly affected by water activity of substrate and nature of a_w depressor used. The polygalacturonase and d-xylanase production were maximized at a_w = 0.995 whereas β-glucosidase formation was favored at a_w = 0.96–0.98. The influence of water activity on catalytic effect of enzymes using sodium chloride, glycerol and sorbitol as a_w depressor was also investigated. It was observed that sorbitol improved the thermal stability of polygalacturonase and d-xylanase.     

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.     

Effect of the sugar-beet pulp water activity on the solid-state culture of Trichoderma viride TS

Summary The growth and the sporulation of Trichoderma viride TS in relation to water activity (a _w) of sugar-beet pulp medium was studied. It was found that the maximum growth, monitored by protein production, substrate utilization and pH alteration, appeared at a _w=0.990–0.992. Optimal water activity of the medium for sporogenesis was 0.980. It was observed that both physiological phenomena appeared in narrow ranges of water activity which caused the rigorous a _w control in solid-state fermentation to be postulated.     

Influence of water activity and temperature on in vitro growth of surface cultures of a Phoma sp. and production of the pharmaceutical metabolites, squalestatins S1 and S2

A Phoma sp., known to produce the pharmaceutically active metabolites squalestatin 1 (S1) and squalestatin 2 (S2), was cultured on malt-extract/agar (MEA) over a range of water activities (a _w, 0.995–0.90) and temperatures (10–35 °C) to investigate the influence on growth and metabolite production. Use of the ionic solute NaCl to adjust a _w resulted in significantly lower (P < 0.01) squalestatin yields than when the Phoma sp. was grown on MEA amended with the non-ionic solute glycerol. Water activity and temperature and their interactions were highly significant factors (P < 0.001) affecting growth of the Phoma sp., with optimum conditions of 0.998–0.980 a _w and 25 °C. Squalestatin production was similarly influenced by a _w, temperature, time and their interactions (P < 0.001). S1 and S2 production occurred over a narrower a _w and temperature range than growth, with a slightly lower optimum a _w range of 0.995–0.980 a _w. The optimum temperature for squalestatin production varied from 20 °C (S1) to 25 °C (S2) and yields of S2 were up to 1000 times lower than those of S1. The ratio of S1 and S2 produced by the Phoma sp. was influenced by a _w and temperature, with highest values at 0.99–0.98 a _w, and at 15 °C. Incubation times of 28 days gave highest yields of both S1 and S2. Up to 2000-fold increases in squalestatin yields were measured at optimum environmental conditions, compared to the unmodified MEA. This indicates the need to consider such factors in screening systems used to detect biologically active lead compounds produced by fungi. Received: 2 June 1997 / Received last revision: 6 November 1997 / Accepted: 7 November 1997     

Effect of Challenge Temperature and Solute Type on Heat Tolerance of Salmonella Serovars at Low Water Activity

Salmonella spp. are reported to have an increased heat tolerance at low water activity (a_w; measured by relative vapor pressure [rvp]), achieved either by drying or by incorporating solutes. Much of the published data, however, cover only a narrow treatment range and have been analyzed by assuming first-order death kinetics. In this study, the death of Salmonella enterica serovar Typhimurium DT104 when exposed to 54 combinations of temperature (55 to 80°C) and a_w (rvp 0.65 to 0.90, reduced using glucose-fructose) was investigated. The Weibull model (LogS = −btⁿ) was used to describe microbial inactivation, and surface response models were developed to predict death rates for serovar Typhimurium at all points within the design surface. The models were evaluated with data generated by using six different Salmonella strains in place of serovar Typhimurium DT104 strain 30, two different solutes in place of glucose-fructose to reduce a_w, or six low-a_w foods artificially contaminated with Salmonella in place of the sugar broths. The data demonstrate that, at temperatures of ≥70°C, Salmonella cells at low a_w were more heat tolerant than those at a higher a_w but below 65°C the reverse was true. The same patterns were generated when sucrose (rvp 0.80 compared with 0.90) or NaCl (0.75 compared with 0.90) was used to reduce a_w, but the extent of the protection afforded varied with solute type. The predictions of thermal death rates in the low-a_w foods were usually fail-safe, but the few exceptions highlight the importance of validating models with specific foods that may have additional factors affecting survival.     

Influence of Water Potential on γ-Decalactone Production by the Yeast Sporidiobolus salmonicolor

The influence of water potential on γ-decalactone production by the yeast Sporidiobolus salmonicolor cultivated in a liquid medium was evaluated by gas-chromatographic analysis. Modifications in water potential led to a number of variations in the aroma production. Maximum extracellular production occurred at water activity (a_w) with a value of 0.99. Further analyses revealed an important phenomenon of cellular accumulation of aroma for a_w values between 0.97 and 0.99.     

Effects of water activity on Vmax and KM of lipase catalyzed transesterification in organic media

Summary The V_max and K_M of various forms of lipase from Pseudomonas cepacia (powder, adsorbed onto Celite or covalently linked to polyethylene glycol) were determined in organic solvents preequilibrated to water activities (a _w) from <0.1 to 0.84. The model reaction was the transesterification between n-octanol and vinyl butyrate. It was found that K_M for the nucleophile increased with increasing a _w for all three lipase forms. V_max increased with increasing a _w for polyethylene glycol-lipase, whereas there was an optimum at intermediate a _w values (0.11 – 0.38) for lipase powder and Celite-immobilized lipase.     

Potential of ochratoxin A production by Aspergillus carbonarius strains isolated from grapes at different ecological factors

Aspergillus carbonarius is known to colonize and produce ochratoxin A (OTA) on grapes and its derived products which is harmful to humans. We screened and tested A. carbonarius strains which isolated from grapes for production of OTA and selected three high OTA producing strains (ACSP1, ACSP2, ACSP3) for this study. These strains were further tested for their ability to produce OTA at different ecological factors [temperature 15, 25, 30, 35°C; water activity (a_w) 0.98, 0.95, 0.90, 0.88; and pH 4.0, 7.0, 9.0, 10.0]. Out of the three strains tested, A. carbonarius ACSP3 produced high levels of OTA than ACSP2 and ACSP1 in all the ecological factors. At 30°C A. carbonarius strains produced the highest OTA compared with other temperature regimes. With reference to water activity, a_w 0.98 favoured mycelial growth and accumulation of more OTA with all the three A. carbonarius strains. Further, pH 4.0 was encouraged the greatest production of OTA in all the strains. No growth was observed at a_w 0.88 and pH 10.0 in all the three strains except the strain ACSP3 at high pH. Our work demonstrated that temperature 30°C, a_w 0.98 and pH 4.0 is optimum for growth and production of OTA by A. carbonarius strains. Maximum amounts of OTA were found at earlier growth stages (7–9 days of incubation) in all the strains of A. carbonarius. The present study revealed that different ecological factors had great impact on OTA production by A. carbonarius which is useful for understanding OTA contamination and to develop proper management practices in future research programmes.     

Effect of water activity on production and activity of Rhizopus oligosporus polysaccharidases

Summary During tempeh fermentation, Rhizopus oligosporus produced polysaccharidases to degrade soya bean cell walls; the maximum activity for all polysaccharidases tested occurred 20–30 h after inoculation. R. oligosporus was also grown in a soya bean extract model medium to which glycerol was added to control water activity (a _w). The overall activities of the major enzymes produced by the fungus, polygalacturonase, endocellulase and xylanase, appeared to be strongly influenced by a _w. The production of enzymes as well as their specific activities were affected by a _w. The optimum a _w for polygalacturonase and xylanase activity coincided with that for mycelial growth, namely 0.99–1.00. In contrast, the optimum a _w for (endo)cellulase was 0.98, at which mycelial growth was significantly reduced. Correspondence to: M. Sarrette     

Effect of temperature and water activity on spore germination and mycelial growth of three fungal biocontrol agents against water hyacinth (Eichhornia crassipes)

Aims: To determine the effect of water activity (a_w = 0·880–0·960) and temperature (15–35°C) on the percentage of viable conidia and mycelial growth of three biocontrol agents effective against water hyacinth in Mali: Alternaria sp. isolate Mlb684, Fusarium sacchari isolate Mln799 and Cadophora malorum isolate Mln715. Methods and Results: The fungi were grown in vitro on plates containing potato dextrose agar medium at different a_w values (glycerol being added to adjust the a_w). The percentage of viable conidia and radial growth rate decreased with decreasing water activity. Statistical analysis showed a significant effect of a_w, temperature and the a_w × temperature interaction on mycelial growth (P < 0·0001). Water activity emerged as the factor exerting the greatest influence. Differences were observed between the fungi tested, the C. malorum appearing more tolerant to low a_w and the F. sacchari more tolerant to high temperature (35°C). Growth models predicting the combined effect of a_w and temperature were developed and response surfaces generated, showing fairly good agreement with the experimental values. Conclusions: Our results confirm the previous finding that a_w has a greater influence than temperature on fungal growth. Under most conditions, variation of environmental factors has a detrimental influence on the percentage of viable conidia and mycelial growth rate of fungal isolates. Significance and Impact of the Study: The developed models may contribute to predicting the best environmental conditions for use of these fungi as effective biocontrol agents against water hyacinth.     

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     

Effects of reduction in beef surface water activity on the survival of Salmonella Typhimurium DT104 during heating

Aim: To investigate the influence of reducing beef surface water activity (a_w) on the survival of Salmonella Typhimurium DT104 during heating. Methods and Results: Beef discs were surface inoculated with S. Typhimurium DT104 and either untreated or dried to achieve surface a_w values of 0·95, 0·85 and 0·70. The samples were vacuum packed, heat‐treated at 60°C and removed at predetermined times. The inactivation curves were influenced by a_w and treatment time. Biphasic inactivation curves were observed for S. Typhimurium DT104 heat‐treated on beef samples with altered a_w values, which were characterized by an initial decline in cell numbers at commencement of heating followed by a much slower rate of inactivation during the remaining treatment period. Point estimates of the heating time required to achieve a 1 log reduction on beef surfaces with a_w of 0·99, 0·95, 0·85 and 0·70 were 0·5, 1·55, 11·25 and 17·79 min, respectively. Conclusions: A decrease in beef surface a_w can substantially enhance the survival of S. Typhimurium DT104 after heating. Significance and Impact of the Study: Caution needs to be taken using dry air as a decontamination method as this may rapidly decrease product surface and pathogen a_w values resulting in enhanced survival.