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.     

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.     

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.     

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.     

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 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 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 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.     

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     

Chrysosporium species, potential spoilage organisms of chocolate

Standard methods of analysing foods for the presence of moulds are inadequate for thedetection of genera such as Chrysosporium which do not grow at the high wateractivities of most mycological media. The use of malt, yeast, 50% glucose agar (MY50G) insealed containers as an enrichment medium allowed time for germination and growth ofheat-stressed spores. Three Chrysosporium spp., C. xerophilum Pitt, C. inops (Carmichael) and C. farinicola (Burnside) Skou, were isolated fromcommercial chocolate bars with a water activity (a _w ) of approximately0·28. Chrysosporium inops was isolated from commercial milk crumb and anew Chrysosporium sp. was isolated from Ghanaian cocoa beans. In chocolates madeby coating MY50G agar (a_w = 0·89) with chocolate (a_w = 0·27) containing C. inops arthroconidia, two types of deterioration were seenafter storage. The first was fat bloom due to recrystallization of the cocoa butter on the outer andinner chocolate surface. The second was growth of C. inops which occurred on theinside chocolate surface adjacent to the MY50G agar filling and on the outside surface afterholding at 92% equilibrium relative humidity (erh) for 12 d. There was some evidence that C. inops could grow on the outside of chocolates held at 5·7% erh after 4months' storage at 25 °C. The appearance of the white fungal growth was not unlikefat bloom to the naked eye but was clearly different with the electron microscope.     

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.     

Changes in Escherichia coli O157:H7 numbers during holding on excised lean, fascia and fat beef surfaces at different temperatures

Aim:  To investigate changes in Escherichia coli O157:H7 numbers on excised beef carcass surfaces over 72 h at different temperatures.
Methods and Results:  Excised lean meat, fascia and fat were inoculated with E. coli O157:H7 and held in an environmental chamber for 72 h, at air speed 0·5 m s⁻¹, relative humidity (RH) 90%, and temperatures 4, 8 and 12°C. On lean, pathogen counts increased significantly at 12°C. On fascia, significant reductions in counts occurred at 4 and 8°C. Pathogen numbers were significantly reduced on fat at 4, 8 and 12°C (64 h). Counts on fat were significantly less at all temperatures, compared to lean or fascia and surface water activity, a_w, decreased significantly over time on fat at 4°C. Significant decreases in surface pH values were recorded on all meat substrates.
Conclusions:  The survival of E. coli O157:H7 varied in relation to the meat substrate and the holding temperature. Reductions in counts on fat surfaces appeared to be related to low surface a_w values. No relationship between pathogen survival and surface pH was established.
Significance and Impact of the Study:  The use of excised meat pieces in an environmental cabinet offers a more flexible approach to determining the use of different chilling regimes in the production of safe meat.     

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.     

The effects of two ammonium propionate formulations on growth in vitro of Aspergillus species isolated from hay

The efficacy of fully neutralized (AP) and half-neutralized ammonium propionate (H-AP) against growth of a number of Aspergillus spp. was determined in vitro on malt extract at different levels of water availability in the range 0.995–0.85 a_w (water activity) (= 40–25% water content of hay). In general, Aspergillus flavus, A. fumigatus, A. nidulans, A. niger and A. versicolor were more sensitive to the H-AP in the range 0.995–0.95 a_w than to AP. At 0.90 and 0.85 a_w growth of most species occurred in the absence of chemicals but only of a few species in 27 mmol/l AP. All three members of the A. glaucus group tested, A. amstelodami, A. repens and A. sejunctus , were similarly more sensitive to H-AP over the a_w range 0.95–0.85 than to AP. Aspergillus sejunctus was most tolerant, growing in 27 mmol/l H-AP and 54 mmol/l AP at 0.85 a_w. The number of days for growth initiation in 27 mmol/l H-AP was greater than with AP at different levels of water availability and reflected the different effects of the two chemicals on growth rate.     

The effects of temperature and water concentration on the otolith incorporation of barium and manganese in black rockfish Sebastes melanops

Observations of multiple years of geographic variation in [Ba:Ca]_otolith and [Mn:Ca]_otolith in black rockfish Sebastes melanops prompted this study to examine the effects of temperature and water concentration on the otolith incorporation of Ba and Mn in this wholly marine species. The replicated experiment design consisted of two water temperatures (7·4 and 13·0° C) and four water concentrations of Ba:Ca and Mn:Ca. A positive, linear relationship between [Ba:Ca]_water and [Ba:Ca]_otolith was observed at both temperatures. A positive temperature effect was also observed with mean partition coefficients for Ba ( D _Ba) greater in the 13° C than in the 7·4° C treatments (mean = 0·061 and 0·048, respectively). There was no relationship between [Mn:Ca]_water and [Mn:Ca]_otolith although a negative temperature effect was observed. Mean partition coefficients for Mn ( D _Mn) were lower in the 13° C than in the 7·4° C treatments (mean = 0·027 and 0·036, respectively). The data presented support the assumption of a positive, linear relationship between water and otolith Metal:Ca concentrations for Ba:Ca but not for Mn:Ca. Thus, although indicative of residence in distinct water masses, observed variation in [Metal:Ca]_otolith may not reflect variation in water concentration and can be affected by temperature. Caution should be applied in the interpretation of geographic variation of [Mn:Ca]_otolith until the mechanisms regulating its incorporation are more fully understood.     

The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava

Mucor circinelloides LU M40 produced 12·2 mU ml⁻¹ of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l⁻¹ deionized water): pectin, 10·0; (NH₄)₂SO₄,
1·0; KH₂PO₄, 2·0; Na₂HPO₄, 0·7; MgSO₄.7H₂O, 0·5; yeast extract, 1·0; Tween-80,
1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the K_m value for linamarin was 2·93 mmol l⁻¹. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.     

The influence of thermal parameters on the acclimation responses of pinfish Lagodon rhomboides exposed to static and decreasing low temperatures

Pinfish Lagodon rhomboides acclimation rates were determined by modelling changes in critical thermal minimum ( T _{crit min}, ° C) estimates at set intervals following a temperature decrease of 3–4° C. The results showed that pinfish gained a total of 3·7° C of cold tolerance over a range of acclimation temperatures ( T _acc, ° C) from (23–12° C), that cold tolerance increased with exposure time to the reduced temperature at all T _acc, but that the rate of cold tolerance accruement (mean 0·14° C day⁻¹) was independent of T _acc. A highly significant ( P < 0·001) multivariate predictive model was generated that described the acclimation rates and thermal tolerance of pinfish exposed to reduction in water temperature: log₁₀ T _{crit min}= 0·41597 − 0·01704 T _acc+ 0·04320 T _plunge− 0·08376[log₁₀ ( t + 1)], where T _plunge is plunge temperature (° C) and t is the time (days). A comparison of the present data, with acclimation rate data for other species, suggests that factors such as latitude or geographic range may play a more important role than ambient temperature in determining cold acclimation rates in fishes.     

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.     

Effect of water activity on production of β-lactam antibiotics by Streptomyces clavuligerus in submerged culture

The amount of available water in the environment of micro-organisms, defined as water activity ( a _W), has been shown to affect growth, respiration, enzyme synthesis, sporulation and other physiological functions. The aim of this study was to examine the impact of a _W on production/excretion of a secondary metabolite. For this purpose, the production of β-lactam antibiotics and biomass of Streptomyces clavuligerus was studied in relation to the a _W-depressing agents glucose, sorbitol and NaCl. These were chosen because NaCl and sorbitol are often used to depress a _W and glucose was not thought to be taken up by S. clavuligerus. The filamentous bacterium S. clavuligerus NRRL 3585 (ATCC 27064) is a prokaryotic producer of penicillin N, cephalosporins including cephamycin C and clavulanic acid. Under water stress conditions, a greater effect upon antibiotic biosynthesis than upon growth was consistently observed. When a _W was decreased to below 0·997, antibiotic production began to decrease. For growth, inhibition was much more gradual and did not become intensive until an a_W of 0·990 was reached.