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 water activity of common liquid bacteriological media

The water activities ( a_w ) of 27 (21 different) bacteriological media were determined. It was found that a_w values of most media were above the range reported in the literature as optimal for rates of growth of many bacteria.     

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.     

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     

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.     

Growth of Fusarium moniliforme and its biosynthesis of fumonisin B1 on maize grain as a function of different water activities

The production of fumonisin by Fusarium moniliforme during its growth on maize depends on extrinsic factors. In particular, experiments on maize grain at different water activities ( a _w)(1, 0.95, 0.90, 0.85) have demonstrated the influence of a _w on fumonisin biosynthesis, and on fungal growth defined by measurement of ergosterol levels. Fumonisin levels dropped threefold when a _w was lowered by 5%, but growth rate was unchanged. A 10% reduction in a _w from 1 to 0.90 resulted in a 20-fold drop in fungal growth, and fumonisin production was reduced 300-fold. At a threshold a _w of 0.85–0.86, F. moniliforme exhibited virtually no measurable metabolic activity, and hence no fumonisin production.     

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.     

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.     

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.     

Physiological response of Pseudomonas putida S12 subjected to reduced water activity

Abstract The effect of osmotic stress, given as decreased water activity (a_w), on growth and the accumulation of potassium and the compatible solute betaine by Pseudomonas putida S12 was investigated. Reduced a_w was imposed by addition of sodium chloride, sucrose, glycerol or polyethylene glycol to the growth medium. Accumulation of potassium and betaine was established when sodium chloride and sucrose were used to cause osmotic stress. No accumulation of these solutes was found in the presence of glycerol. Addition of polyethylene glycol to the medium strongly decreased the growth rate in comparison with the other osmolytes tested at the corresponding a_w. Although polyethylene glycol did decrease the a_w, neither potassium nor betaine was accumulated by the cells.     

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.     

The effect of water activity on Legionella spp. growth and survival

Growth and survival of Legionella spp. at various water activity (a_w) levels were determined. Compared with Escherichia coli , the growth of Legionella spp. was limited to a high a_w environment (≥0.98).     

Effect of Some Environmental Factors on Psychrophilic Microbacteria

S ummary . The growth of three psychrophilic strains of Microbacterium isolated from meat was studied at 10 temperatures between 0° and 35° and at 6 water activity (a_w) levels between 0.99 and 0.94. The temperature and water relations of the three strains were similar. For all strains the rate of growth was fastest at 25° and a_w 0.99, and growth did not occur at 35°.
Further experiments on one strain showed that under aerobic conditions the range of water activities permitting growth was independent of temperature, but under anaerobic conditions the minimum water activity increased from about 0.94—0.96 as the temperature was reduced from 25° to 0°. Growth was inhibited by low concentrations of undissociated nitrous acid, and inhibition was greater at 0° than at 10° or 25°.     

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.     

Determination of dissolved carbon dioxide by coulometric titration in modified atmosphere systems

The solubility of carbon dioxide (CO₂) in microbiological media at different pH values, water activities ( a_w ), temperatures, buffering capacities and ratios of headspace to media volumes was determined by using a coulometer. Buffering capacity and ratio of headspace to media volume were shown to be the major factors influencing the solubility of CO₂ in modified atmosphere model systems. The growth inhibitory effects of different dissolved CO₂ concentrations (0–50 μmol ml^-1) were determined for Pseudomonas fragi at 8°C and 22 C. Pseudomonas fragi was shown to be strongly affected by the CO₂ concentration in the media. A carbon dioxide concentration of 40 μmol ml^-1 was needed to inhibit Ps. fragi at 8°C. The importance of measuring dissolved CO₂ concentrations in modified atmosphere packaging applications was shown and the coulometer proved to be an excellent tool for this purpose.     

Secretion of the Hormoconis resinae glucoamylase P enzyme from Trichoderma reesei directed by the natural and the cbh1 gene secretion signal

Abstract We have isolated two alkaline phosphatases (H-AP and L-AP, for high and low molecular mass, respectively) from Pseudomonas aeruginosa PA01. These two enzymes were found to differ in mobility on sodium dodecyl sulphate polyacrylamide gels (H-AP, M _r = 51 000 and L-AP, M _r = 39 500), amino-terminal amino acid sequence and did not cross-react. Both enzymes were active as phosphomonoesterases while only L-AP demonstrated any phosphodiesterase activity. Both enzymes were purified from P. aeruginosa grown in phosphate limiting conditions using the same protocol and were identified in both periplasmic and extracellular locations. A low level of H-AP was produced constitutively whereas L-AP was produced only after induction by reduced phosphate concentration in the growth medium. An L-AP-like enzyme has been previously described, however, this is the first report of a second P. aeruginosa alkaline phosphatase.     

Seasonal changes in some blood chemistry parameters and in relative liver and gonad weights of pike (Esox lucius L.) from the River Danube

Samples from a natural population of pike (Esox lucius L.) from the River Danube were used in a 12-month study to determine seasonal variations in biochemical parameters of pike blood sera, hepatosomatic index (HSI) and gonadosomatic index (GSI). The ranges of enzyme activities for sample means were: aspartate aminotransferase (ASAT) 252.0–583.8 U 1⁻¹, alanine aminotransferase (ALAT) 4.9–11 -2 U1 and alkaline phosphatase (AP) 39.5–91.8 U1⁻¹. The ranges of other parameters analysed in serum were total protein 27.7–40.1 g 1 ¹, urea 0.57–l.52 mmol 1 ¹ and creatinine 21.2–118.6 μmol 1 ¹. The range of sample means for HSI and GSI were 1.28–4.16 and 0.07–20.2 respectively. Temperature ranged from 4.5 to 23.5°C. The activity of serum AP was positively correlated to water temperature in males only, while urea and creatinine showed a positive correlation to water temperature in individuals of both sexes. GSI was correlated significantly with HSI in females. Total protein reached the lowest values during the spawning period, while creatinine levels depended on both the sex and season.     

The Effect of Sugars and Polyols on the Heat Resistance of Salmonellae

S ummary . The heat resistance at 65° of 3 strains of salmonellae in solutions of sugars or polyols was enhanced as the concentration of the solutes increased. There was no linear relationship between heat resistance and water activity ( a_w ), but for all solutes except glycerol there was a linear relationship between log D ₆₅ and concentration (% w/w) of solute. Comparison of D ₆₅ at a particular a_w or percentage (w/w) solute concentration showed that the value decreased in the order: sucrose > glucose > sorbitol > fructose > glycerol. In glycerol, D ₆₅ values were always very much lower than in any other solute. With sucrose–glycerol or sucrose–glucose mixtures, heat resistance depended both on the total concentration (% w/w) of solutes present and also on the a_w of the solution.     

Accumulation of carbohydrate by Escherichia coli B/r/1 during growth at low water activity

The carbohydrate content of Escherichia coli B/r/1, grown in a glucose or arabinose-limited salts medium in a chemostat, increased by a factor of 2–4 when the water activity (a_w) of the medium was reduced to 0.986 by addition of NaCl, KCl or sucrose. The biomass decreased by 30–45%. The sucrose system resulted in the lowest biomass and carbohydrate content. The monosaccharide part of the accumulated carbohydrate consisted of glucose or glucose and arabinose in the cultures fed glucose and arabinose, respectively, and accounted for 50% or more of the total carbohydrate in the NaCl and KCl systems and 16.79% in the sucrose system. In addition, the K⁺ content depended on the solute and related inversely to the monosaccharide content, being highest in the sucrose system. The combined molarity of the monosaccharide and K⁺ was deduced to be far in excess of that required for osmotic equilibration of the cultures, especially in the sucrose system. These observations are discussed in the context of osmoregulation, the effects of solutes on glucose metabolism and the morphological changes that occur in cultures at low a_w.     

Population differences in water-use efficiency of Eucalyptus microtheca seedlings under different watering regimes

Gas exchange, water-use efficiency (WUE), carbon isotope composition ( Δ ¹³C) and growth traits were compared among 5 populations of Eucalyptus microtheca F. Muell. Seedlings grown from seed collected across the natural distribution of the species were maintained under water-stressed and well-watered conditions. Gas exchange was measured in terms of net photosynthesis (A) and transpiration (E); WUE was measured in terms of instantaneous water-use efficiency (WUE_i) and transpiration efficiency (WUE_T); growth traits were measured in terms of total biomass (TB), root/shoot ratio (RS), and specific leaf area density (DEN). Significant differences in all traits were detected among the populations. Overall population variation was 1.68–2.50 and 1.48–2.26 μmol CO₂ uptake per mmol H₂O transpired (WUE_i), 1.97–3.04 and 1.64–2.36 g dry matter accumulation per kg water transpired (WUE_T), and Δ ¹³C was −28.81 to −26.75‰ and −30.56 to −30.04‰ under the water-stressed and well-watered conditions, respectively. In addition, WUE_i, WUE_T and Δ ¹³C were significantly correlated with A, E, RS, DEN and TB. The study indicated that measurement of WUE may be a useful trait for selecting genotypes with improved drought adaptation and biomass productivity under different environmental conditions.