Combined Effects of Water Activity, pH and Temperature on the Growth and Spoilage Potential of Fungi

S ummary . An arbitrary parameter 'rejection time', i.e. the time required for a fungal inoculum to form a 2 mm diam. colony, was used to express the shelf life of jam after unsealing and exposure of the contents to airborne contamination. Individual and combined effects of water activity ( a_w ), pH value and temperature on rejection time of low sugar jam were estimated from the radial growth on agar of colonies of 9 fungi. The decreases in a_w (0·94–0·90) and temperature (25–15°) practicable for low sugar jam were more effective in increasing rejection time than the feasible decrease in pH value (3·7–2·9). The interaction between a_w and temperature was significant. The effect of the changes in a_w , temperature and pH on rejection time was broadly similar for media adjusted by either sucrose or glycerol. At a given a_w , moulds were slightly more tolerant of glycerol than sucrose but yeasts, except for the osmophile Saccharomyces rouxii , were markedly more tolerant of glycerol than sucrose.     

Czapek Casein 50% Glucose (CZC50G): a new medium for the identification of foodborne Chrysosporium spp.

A new medium Czapek Casein 50% Glucose agar (CZC50G) has been developed, on which the four foodborne Chrysosporium spp., C. xerophilum, C. inops, C. farinicola and C. fastidium can be distinguished by differences in growth rates and colony morphology. Chrysosporium xerophilum and C. inops both produced dense white colonies, but C. xerophilum grew faster than C. inops , 22 mm in 14 d compared to 9–12 mm in 14 d at 25°C. Some isolates produced a yellow or red reverse due to the reaction of ferric ammonium citrate incorporated in the medium with a fungal metabolite. Chrysosporium farinicola and C. fastidium both grew poorly on this medium and produced sparse colonies: C. farinicola grew faster. Electron micrographs of arthroconidia with a cryo-scanning electron microscope showed thickening of the spore walls in C. inops but not in C. xerophilum . The aleurioconidia of C. farinicola and C. fastidium were different in shape. The differences in colony morphology and growth rate on CZC50G reflected these differences and demonstrated that these four species could be distinguished easily on CZC50G.     

Effect of oscillatory high hydrostatic pressure treatments on Byssochlamys nivea ascospores suspended in fruit juice concentrates

The effect of continuous (689 MPa with holding times of 5, 15 or 25 min) and oscillatory (one, three or five cycles at 689 MPa with holding times of 1 s) high hydrostatic pressure treatments on the viability of Byssochlamys nivea ascospores suspended in apple and cranberry juice concentrates adjusted by dilution to water activities (a_w) of 0·98 and 0·94 was evaluated at 21 and 60 °C. Inactivation of the initial spore inocula was achieved after three or five cycles of oscillatory pressurization at 60 °C when the a_w was 0·98 in both fruit juices. With a_w 0·94, the initial inocula were reduced by less than 1 log-cycle after five pressure cycles. Inactivation was not observed within 25 min with continuous pressurization at 60 °C. In treatments at 21 °C, no effect on spore viability was observed with continuous or oscillatory treatments.     

The Effect of Water Activity and aw-Controlling Solute on Sporulation of Bacillus cereus

The influence of water activity (aw) on the formation of phase bright, heat stable, and dipicoiir.ic acid-containing spores of Bacillus cereus T from stage III to stage IV forespores has beer. investigated. Decreasing a_w levels reduced the rate of sporulation and the number of forespores which lysed was determined by the a_w-controlling solute used. The limiting a_w value for ir.e formation of mature spores was about 0·95 for glucose, sorbitol and NaCl whereas it was about 0·91 for glycerol. The development of refractility. the synthesis of dipicolinic acid, and acquisition of heat stability were affected equally by decrease in a_w during sporulation. With the range of a_w value where spores could be formed NaCl and glycerol had no signifcant: influence on the D value of the resulting spores whereas at all a_w levels, when sorbitol was use: as the a_w-controlling solute, the heat resistance was greater than in the basal medium. It Is suggested that the a_w of the sporulation medium determines the quantity of spores rather than. the spore properties.     

Effect of Water Activity on Heat Survival of Staphylococcus aureus, Salmonella typhimurium and Salm. senftenberg

The viability of Staphylococcus aureus heated at 55° in phosphate buffer was determined on recovery media of different water activity ( a_w ) levels. The basal recovery medium, tryptone–soya agar ( a_w 0.997) was adjusted to lower a_w levels by the addition of NaCl, glycerol or sucrose. Maximum survival occurred at a_w 0.997. Viability was reduced to 1/10 of the maximum at a_w 0.98 when a_w , was controlled by sucrose or NaCl but not until a_w 0.93 with glycerol. To eliminate effects such as incomplete mixing or post-heating dilution and in order to use conditions comparable to those occurring in foods, a solid medium heating/recovery method was also used. This involved heating, by immersion, of surface-inoculated agar plates and recovery of survivors in situ . Heat resistance studies could thus only be carried out at a_w levels permitting growth on the heating/recovery medium. Staphylococcus aureus, Salmonella typhimurium and Salm. senftenberg 775W were heated at 55° and recovered in situ on tryptone-soya agar adjusted to lower a_w levels as above. Maximum survival of Staph. aureus occurred at a higher a_w with glycerol ( a_w 0.965) than with NaCl (0.92) or sucrose (0.90). The maximum survival of both Salm. typhimurium and Staph. aureus heated at 55° occurred at the same a_w (0.965) with glycerol. This maximum was not affected by the duration of heating. As a contrast, heat resistance of Salm. senftenberg 775W was virtually unaffected by reduction in the a_w of the heating/recovery medium.     

Water relations and metabolism of propionate in two yeasts from hay

Many yeasts and bacteria were isolated from moist hays (>30% water content) treated with up to 3% propionic acid-based preservatives. Predominant yeasts were Candida guilliermondii var. guilliermondii and Hyphopichia burtonii. Growth of both species was decreased more than 50% in liquid medium containing 54 mmol/l ammonium propionate but some still occurred in 135 mmol/l propionate. Both metabolized between 80 and 85% of 27 mmol/l ammonium propionate in 1% malt broth within four weeks at 25°C. Growth on solid malt extract agar containing ammonium propionate was decreased by decreasing the water availability (water activity, a_w) in the medium. Growth rates were slightly greater when glycerol rather than NaCl was used to alter a_w in the range 0.995 to 0.93. At both 0.995 and 0.95 a_w optimum growth was at pH 6. The significance of these findings with regard to the preservation of moist hay is discussed.     

The effect of water activity and pH on the production of mycotoxins by fungi growing on a bread analogue

Mycotoxin production by various toxigenic fungi, growing on a bread analogue, was investigated at various water activities (a_w) and pH combinations. Citrinin, ochratoxin A and sterigmatocystin could be detected at a_w > 0·80, while patulin was only observed at a_w= 0·95. These results show that some toxins may be produced at lower water activities than have been reported on synthetic media and suggest that, where possible, natural substrates should be used to investigate factors affect-ing mycotoxin production in foodstuffs.     

Comparison of combinations of diluents and media for enumerating Zygosaccharomyces rouxii in intermediate water activity foods

Combinations of five diluents (0.1% peptone, 40 and 50% glucose, and 18 and 26% glycerol) and three enumeration media (tryptone glucose yeast extract, dichloran 18% glycerol and malt extract yeast extract 50% glucose (MY50G) agars) were evaluated for recovering a xerotolerant yeast, Zygosaccharomyces rouxii , from foods with intermediate water activity ( a _w). Combinations of 40% ( a _w, 0.936) or 50% ( a _w 0.898) glucose diluent and MY50G agar ( a _w 0.890) were superior in recovering highest populations. The type of solute in the diluent, as well as a reduced a _w, influences efficiency of recovering viable cells.     

Interactions among xerophilic fungi associated with dried salted fish

Interactions were investigated among five xerophilic fungi, Polypaecilum pisce, Basipetospora halophila, Eurotium rubrum, Aspergillus wentii and A. penicillioides, isolated from Indonesian dried salted fish. A range of water activities ( a _w) (0·98, 0·95, 0·90 and 0·84) and temperatures (15°, 25° and 30°C) were studied on agar media in Petri dishes, and with dried fish as a substrate at 0·90 and 0·84 a _w at 30°C. Generally, the fungi exhibited one of two interaction types: mutual inhibition on contact, or inhibition of one or both species on contact, with the inhibited species continuing to grow at a significantly reduced rate. On glucose-based agar media A. wentii and E. rubrum were most competitive at all a _w values and temperatures studied, while on NaCl media P. pisce and B. halophila were usually most competitive. The Petri dish system was a useful model, but did not completely simulate the interactions observed on dried fish. Polypaecilum pisce and B. halophila were able to compete more strongly on fish than on agar media, especially at 0·90 a _w. This study provides some evidence that each species examined had a niche in which it was dominant, and that species interactions as well as environmental factors are important in determining the dominant fungal species on dried salted fish.     

Influence of water activity and nutrients on growth and production of squalestatin S1 by a Phoma sp

D. ALDRED, N. MAGAN and B.S. LANE.1999.This study investigated the effects of temperature, nutrient status and water activity (a_W) on the production of squalestatin S1 by a Phoma sp. The fungus was grown on malt extract (MEA), wheat extract (WEA), oat extract (OEA) and oil seed rape extract (OSREA) agars at 15, 20 and 25 °C and 0·998, 0·995, 0·990, 0·980 and 0·960 a_W levels. The growth rate and secondary metabolite formation were followed over a total of 30 d. The maximum growth rate was observed at 25 °C and 0·998–0·990 a_W for all media types, which was significantly reduced ( P = 0·05) for most media at 0·96 a_w. The growth rate was greatest for WEA and OEA but the growth form was an effuse exploitative type compared with the dense assimilative type on the richer MEA. The lipid-based OSREA appeared to be a poor growth substrate for this fungus. In contrast to the growth rate data, squalestatin S1 production was maximal for all media types at slightly reduced a_w in the range 0·990–0·980. There was greater production of the secondary metabolite under significant water stress (0·960 a_W) compared with that with freely available water (0·998 a_W). Maximum production was observed in WEA. Production began earlier in WEA and OEA compared with MEA. Squalestatin S1 production was not significantly affected by incubation temperature ( P = 0·05). This study has shown that nutritionally depleted substrates may be usefully employed in the production of squalestatin S1 and perhaps also for other secondary metabolites.     

Ecophysiological characterization of common food-borne fungi in relation to pH and water activity under various atmospheric compositions

The combined effects of pH, water activity (a_w), oxygen (O₂) and carbon dioxide (CO₂) levels on growth and sporulation of 10common food-borne fungi were studied. The use of a multivariate statistical method (PLS) for the analysis of data showed that the fungi could be grouped according to their physiological response to changes in the four tested factors. Carbon dioxide, a_w and pH were found to be the most significant factors describing differences and similarities among the fungi. Maximal inhibitory effect of elevated levels of CO₂ (5–25%) and decreased a_w (0·99–0·95) varied among the 10 species from 6 to 77% and from 52 to 100%, respectively. Sporulation of the fungi was sensitive to all tested factors. Furthermore, interaction of CO₂ and a_w displayed a significant effect on sporulation. It was shown that different fungal species associated with the same ecosystem responded similarly to changes in the tested factors. Thus, fungi which are not phylogenetically related may be physiologically related or show a common strategy of life.     

Reproductive investment in the Silurus meridionalis

A comparison of pre- and postspawning Silurus meridionalis showed that 20·7% of body stored energy was utilized during spawning for a standard male (74·5 cm) and 23·8% for a standard female (85·3 cm). About one-third of the loss of the stored energy was released as eggs by females, and almost all of the energy loss for males and about two-thirds for females were expended in metabolism. Stored lipid as fuel for metabolism supplied 90·0% of energy in males and 95·2% in females, and protein supplied the rest of the energy. Models for predicting energy in released gametes ( G _g), deposited in the body as somatic growth ( G _s), utilized in spawning activity ( S _a), expended in maintenance ( M , including metabolism, faeces and excretion), and food energy ( C ) were developed, and annual energy budgets were compiled. The balanced budget for a male aged 4 was: 100 C =0·06 G _g+11·17 S _a+19·5 G _s+69·2 M , and for a female aged 5: 100 C =5·48 G _g+8·51 S _a+15·8 G _s+70·2 M .     

The effect of age on the thermal resistance of arthroconidia from Chrysosporium inops

J. L. KINDERLERER. 1996. Food-borne members of the genus Chrysosporium have been isolated relatively infrequently. The heat resistance of arthroconidia of the xerophilic fungus, Chrysosporium inops Carmichael, was determined in 0.1% peptone at 66C. The survival curve was sigmoid in shape. The initial lag period was due to the chains of arthroconidia. Thermal inactivation occurred when one viable conidium was left per chain. The presence of chains of arthroconidia was confirmed with the cryo scanning electron microscope. The decimal reduction times were obtained from the regression line of the linear death phase for the heat-sensitive spores. The decimal reduction time (D₆₆) increased with increasing spore age. It was 1.67 min for 3-week-old spores, 1.95 min for 4-week-old spores and 5.49 min for 6-week-old spores. The older spores could recover from thermal death if they were given sufficient time. There was a significant increase in D₆₆ value for 6-week-old spores from 3.97 min to 5.49 when the counts were obtained after 14 d incubation (compared to counts after incubation for 10 d). This effect was not seen for the 3- and 4-week-old spores. There was a small population of heat-resistant spores. The initial population of arthroconidia was greater than log 7 cfu ml^-1. After heating for 1 h at 66C approximately log 2.2 cfu ml^-1 survived. These survivors represented approximately 0.001% of the original population.     

Resistance of food spoilage yeasts to sorbic acid

Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus were grown on Sabouraud Dextrose Agar (SDA) modified with KCl to give a range of water activity (a_w) from 0.938 to 0.998. Growth of all three species was optimal at 0.983 a_w and growth occurred over the a_w range tested. Acyclic sugar alcohol (polyol) and trehalose content of conidia was determined by HPLC and found to vary with species and a_w. Conidia of B. bassiana and P. farinosus were found to contain totals of 1.5% and 2.3% polyols respectively at 0.998 a_w, and double these amounts at <0.950 a_w. Conidia of M. anisopliae contained from 5.7% to 6.8% polyols at each a_w tested. In conidia of all three species the predominant polyol was mannitol. The lower molecular weight polyols, arabitol and erythritol, were found to accumulate at reduced a_w. Small amounts of glycerol were present in conidia of each species; <15% total polyols. Conidia of B. bassiana and M. anisopliae contained about 0.5% trehalose from 0.970 to 0.998 a_w, but only trace amounts below 0.950 a_w. Conidia of P. farinosus contained 2.1% trehalose at 0.998 a_w and this decreased to <0.1% below 0.950 a_w. Potential to manipulate the endogenous reserves of conidia of these biological control agents to enhance viability and desiccation toierance is discussed     

Determination of optimal growth parameters for the bioincising fungus Physisporinus vitreus by means of response surface methodology

Aim:  To evaluate the influence of water activity ( a _w), temperature and pH on the radial growth and lag phase of Physisporinus vitreus (E-642), a basidiomycete was used in the biotechnological process of bioincising.
Methods and Results:  Radial growth was monitored for 20 days on malt extract agar medium. Five levels of a _w (0·998, 0·982, 0·955, 0·928, 0·892) were combined with three incubation temperatures (10, 15, 20°C) and three pH values (4, 5, 6). Data analyses showed a highly significant effect of a _w and temperature ( P <  0·0001) and a significant effect of pH ( P <  0·05). The radial growth rate and lag phase of P. vitreus were very sensitive to a _w reduction. Although P. vitreus was able to grow at all the selected temperatures and pH values, the lag phase increased with decreasing a _w and growth became inhibited at a _w = 0·955. Optimal conditions for growth of P. vitreus were a _w = 0·998, 20°C and pH 5. The response surface model provided reliable estimates of these growth parameters and confirmed a greater dependence on a _w than on temperature or pH under in vitro conditions.
Conclusions:  Low levels of a _w can prevent growth of P. vitreus , so wood moisture content should be adjusted accordingly.
Significance and Impact of the Study:  Implementation of these results should contribute towards the optimization and efficiency of bioincising.     

Seasonal variability in growth of larval Japanese anchovy Engraulis japonicus driven by fluctuations in sea temperature in the Kii Channel, Japan

Seasonal variability in the growth of larval Japanese anchovy Engraulis japonicus was examined through otolith microstructure analysis based on the samples collected from the northern side (inner area, IA) and the southern side (outer area, OA) of the Kii Channel from April 2006 to March 2007. Growth trajectories (otolith backcalculated mean standard length of 5 day intervals from 5 days after hatch to 24 days) as well as the most recent 5 day mean growth rate of larvae before capture ( G ₅) differed among months. Growth trajectories showed the same pattern as G ₅. In IA, mean ± s.d. G ₅ ranged from 0·31 ± 0·04 mm day⁻¹ (January) to 0·73 ± 0·06 mm day⁻¹ (October). In OA, mean ± s.d. G ₅ ranged from 0·36 ± 0·05 mm day⁻¹ (January) to 0·79 ± 0·11 mm day⁻¹ (August). G ₅ values declined from November to January and then started to increase. In general, the seasonal patterns of growth were similar between IA and OA, and a clear seasonal pattern in growth was identified. When the relationships among larval growth rate, sea temperature, zooplankton density and larval density were examined, growth rate was positively related with sea temperature in both areas and not related with the other factors. The similar pattern in growth observed between IA and OA was probably due to the low spatial variability in sea temperature compared to its seasonal variability.     

Water relations of solute accumulation in Pseudomonas fluorescens

When Pseudomonas fluorescens was grown in a glucose salts medium adjusted with NaCl to a water activity (a_w) value of 0.980, the intracellular glutamic acid concentration increased 23-fold and comprised 90% of the total amino acid pool. This increase was not observed when the a_w of the medium was reduced to 0.980 with sorbitol. Sorbitol was taken up rapidly over a 30 min period and accumulated intracellularly to a level approximately two-fold greater than the concentration in the growth medium. In continuous culture, the specific rate of glutamic acid production and glucose uptake was greater at 0.980 (NaCl) than at 0.997 a_w. The maintenance coefficients for glucose uptake were similar at both a_w values but were 2.4-fold greater for glutamic acid production at 0.980 (NaCl) than at 0.997 a_w.     

Growth and physiological changes during metamorphosis of Senegal sole reared in the laboratory

The metamorphosis of Solea senegalensis was studied in larvae reared at 20° C and fed four different feeding regimes. A, Artemia (4 nauplii ml⁻¹); B, Artemia (2 nauplii ml⁻¹); C, mixed diet (2 nauplii ml⁻¹ and 3 mg ml⁻¹ microencapsulated diet); and D, microencapsulated diet (3·7 mg ml⁻¹). Rotifers were also supplied in all cases during the first days of feeding. These feeding regimes supported different growth rates during the pre-metamorphosis period (regime A, G=0·376 day⁻¹; regime B, G=0·253 day⁻¹; regime C, G=0·254 day⁻¹; regime D, G=0·162 day⁻¹). Larvae started metamorphosis 9 days after hatching (DAH) when fed the regime A, 13 DAH with regime B, 11 DAH with regime C and 15 DAH with regime D. A minimum 5·6–5·9 mm L_T was required under all feeding regimes to initiate the metamorphosis. Eye translocation was completed when the larvae reached 8·6–8·7 mm L_T (regimes A, B and C), but only 7·3 mm L_T with regime D. 4·4–6·2 days were required to complete eye migration under the regimes A, B and C, and 18·3 days under the regime D. This transformation is concomitant with changes in body reserves, and with the pattern of some digestive enzymes.     

Survival of Listeria monocytogenes in sea water and effect of exposure on thermal resistance

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes , Scott A and an environmental strain KM, on exposure to sea water at 12·8 or 20·8 °C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T₉₀ values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0·6% yeast extract) at 12·8 and 20·8 °C were 61·7 and 69·2 h for L. monocytogenes Scott A, and 103·0 and 67·0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T₉₀ values at 12·8 and 20·8 °C were 60·6 and 56·9 h for L. monocytogenes Scott A, and 83·0 and 65·9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12·8 and 20·8 °C was 1·7 and 17·7%, respectively, while for KM the values were 19·0 and 1·6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 °C showed an apparent increase in heat resistance after exposure to sea water at 20·8 °C for 7 d ( D ₅₈= 2·64 min) compared with before exposure ( D ₅₈= 1·24). This increase in thermal resistance was not apparent at temperatures greater than 63 °C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different ( P > 0·05) over the temperature range tested (58–62 °C).     

A blood-free enrichment medium for growing Campylobacter spp. under aerobic conditions

Detection limits for Campylobacter jejuni strains JH93 and ATCC 29428 in a new blood-free enrichment broth (BFEB) were investigated under aerobic conditions. Cultures of Camp. jejuni were inoculated into 50 ml BFEB containing 10% food homogenate in 50 ml screw-cap tubes. After 24 h enrichment under aerobic conditions, Camp. jejuni were isolated on four selective agar media. The least squares means of the detection limit 50% endpoint (DL₅₀) values were 0·4 (plain BFEB), 0·9 (crabmeat), 1·7 (mushroom), 1·7 (raw milk) and 2·1 (oyster) colony forming units (cfu) 5 g⁻¹ food. The efficiency of the BFEB was significantly affected ( P < 0·05) by food type and bacterial strain. Overall, the BFEB enrichment compared favourably with the existing US Food and Drug Administration method under modified atmosphere. In addition, the BFEB method did not require the use of blood, special equipment or Oxyrase^® to reduce oxygen tensions.