Survival of Staphylococcus aureus and enterotoxin A in glassy and rubbery states of gelatin

About 1% of Staphylococcus aureus cells survived the production of gelatin sheets containing nutrient broth. Those cells which survived showed no evidence of injury. Growth occurred in rubbery state gelatin with a _w values of 0.98 and 0.93; viability decreased during storage at a _w values of 0.89, 0.62 and 0.36 but there was little loss of viability in gelatin at an a _w of 0.25 over 27 d storage at 26°C. Assays for enterotoxin A detected no synthesis of new toxin but no loss in pre-formed toxin. The results suggest that high levels of Staph. aureus and its toxins should be excluded from glassy and rubbery state food products in order to prevent illness.     

The survival of Salmonella enterica serovar Typhimurium DT104 and total viable counts on beef surfaces at different relative humidities and temperatures

Aims:  The aim of this study was to investigate changes in Salmonella and total viable count (TVC) survival on beef carcass surfaces stored for 72 h under different combinations of relative humidity (i.e. RH 75% or 96%) and temperature (5°C or 10°C).
Methods and Results:  The influence of low water activity ( a _w) and temperature on the survival and growth of Salmonella enterica serovar Typhimurium DT104 and the aerobic mesophilic flora on meat pieces from different sites on beef carcasses was investigated, under controlled conditions (75% or 96% RH; 5 or 10°C) in an environmental cabinet. Salmonella counts declined during storage at low a _w (75% RH) conditions at 5°C or 10°C. Salmonella counts increased during storage at high a _w (96% RH) at 10°C only. At 5°C, TVCs increased during storage at high a _w, but not at low a _w. TVCs increased on all samples from carcasses stored at high or low a _w at 10°C, except those samples taken from areas of surface fat.
Conclusions:  This suggests that substrate composition dictates growth rates under low a _w conditions. The results are discussed in terms of the possible protective effects of substrate osmolyte accumulation in bacterial survival and/or growth.
Significance and Impact of the Study:  The data obtained in this study provides useful insights on the influence of a _w and temperature on pathogen survival on meat surfaces at chill temperature.     

Effect of temperature and water activity on in vitro germination of Monilinia spp.

Aims:  This study evaluated the effect of temperature (0–38°C) and water activity ( a _w: 0·87–0·99) on the lag phase prior to germination and the percentage of germination over time for Monilinia laxa , Monilinia fructicola and Monilinia fructigena .
Methods and Results:  More than 80% of viable conidia germinated at 25°C and 0·99 a _w within 2 h for M. fructicola and M. fructigena and 4 h for M. laxa . There was no germination at 38°C, and all three Monilinia spp. germinated at 0°C. At the lowest a _w (0·87), none of the Monilinia spp. was able to germinate at any of the incubation temperatures studied. Whereas at 0·90 a _w, conidia were only able to germinate at 15, 25 and 30°C for the three species studied, except for M. fructicola at 15°C. In contrast, at 0·95, 0·97 and 0·99 a _w, germination occurred at all studied temperatures less 38°C. Generally, the lag phase was longer at low levels of a _w (0·90–095), and differences were more evident as temperatures were far from the optimum (0–5°C).
Conclusions:  Germination and lag phase period were markedly influenced by temperature and a _w, and in general when conditions of temperature and a _w were suboptimal, the lag phase was longer and the percentage of germination was lower.
Significance and Impact of the Study:  Knowledge of the germination requirements of this fungus is important in order to understand their behaviour in natural situations and to provide baseline data required for the construction of new prediction models. Our study might be used to develop a predictive model to understand and control the disease caused by Monilinia spp.     

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.     

The Effect of Water Activity on the Growth and Respiration of Pseudomonas fluorescens

The growth rate of Pseudomonas fluorescens was greater and continued at lower water activity ( a _w) values when glycerol controlled the a _w of glucose minimal medium than when the a _w was controlled by NaCl and sucrose. Growth was not observed below 0·945, 0·970 and 0·964 a _w when glycerol, sucrose and NaCl respectively controlled the a _w. The catabolism of glucose, Na lactate and DL-arginine as measured by respirometry was completely inhibited at a _w values greater than the minimum for growth when the a _w was controlled with NaCl. When the a _w was controlled with glycerol, catabolism of the three substrates continued at a _w values significantly below the a _w for growth on glucose. Catabolism of glucose in the presence of sucrose occurred at a level below the minimum growth a _w but catabolism of the other two substrates ceased at a _w values greater than the minimum growth a _w. Arrhenius plots between 10° and 34°C of the growth rate in glucose minimal medium at 0·98 a _w showed that the order of inhibition was sucrose > NaCl > glycerol. The order of inhibition differed when Arrhenius plots of catabolism of glucose was examined between 10° and 34 °C, namely NaCl > sucrose > glycerol. The mechanism of action of solutes controlling a _w is discussed.     

Modelling the growth of Weissella cibaria as a function of fermentation conditions

Aims:  To investigate the effect of pH, water activity ( a _w) and temperature on the growth of Weissella cibaria DBPZ1006, a lactic acid bacterium isolated from sourdoughs.
Methods and Results:  The kinetics of growth of W. cibaria DBPZ1006 was investigated during batch fermentations as a function of pH (4·0–8·0), a _w (0·935–0·994) and temperature (10–45°C) in a rich medium. The growth curve parameters (lag time, growth rate and asymptote) were estimated using the dynamic model of Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294). The effect of pH, a _w and temperature on maximum specific growth rate (μ_max) were estimated by fitting a cardinal model. μ_max under optimal conditions (pH = 6·6, a _w = 0·994, T  = 36·3°C) was estimated to be 0·93 h⁻¹. Minimum and maximum estimated pH and temperature for growth were 3·6 and 8·15, and 9·0°C and 47·8°C, respectively, while minimum a _w was 0·918 (equivalent to 12·2% w/v NaCl).
Conclusions:  Weissella cibaria DBPZ1006 is a fast-growing heterofermentative strain, which could be used in a mixed starter culture for making bread.
Significance and Impact of the Study:  This is the first study reporting the modelling of the growth of W. cibaria , a species that is increasingly being used as a starter in sourdough and vegetable fermentations.     

Effect of water activity and temperature on growth and fumonisin B1 and B2 production by Fusarium proliferatum and F. moniliforme on maize grain

S. MARIN, V. SANCHIS, I. VINAS, R. CANELA AND N. MAGAN. 1995. The effect of different water activities ( a _w, 0.968, 0.956, 0.944, 0.925) and temperature (25°C and 30°C) on colonization and production of fumonisin B₁ (FB₁) and B₂ (FB₂) on sterile layers of maize by Fusarium proliferatum and F. moniliforme isolates was determined over periods of 6 weeks. Generally, both F. moniliforme and F. proliferatum grew faster with increasing a _w and best at 30°C. All three isolates produced more FB 1 than FB₂ regardless of a _w or temperature. Very little FB₁ and FB₂ were produced at 0.925 a _w, with maximum produced at 0.956 and 0.968 a _w at both temperatures tested. Most FB₁ and FB₂ were produced by F. moniliforme (25N), followed by F. proliferatum isolates (73N and 131N). At all a _w levels and both temperatures there was an increase in FB₁ and FB₂ concentration with time. Statistical analyses of a _w, temperature, time, two- and three-way interactions showed some significant differences between isolates and FB₁ and FB₂ production.     

Water Relations of Glucose-catabolizing Enzymes in Pseudomonas fluorescens

Examination of the catabolism of glucose via the Entner-Doudoroff pathway by standard enzyme assays showed that the activity of glucose-6-phosphate dehydrogenase, glucokinase and 2-ketoglu-conokinase plus phosphoketogluconate reductase was completely inhibited at a _w values less than 0.965, 0.98 and 0.96 respectively when NaCl was used to adjust the a _w. The other glucose-catabolizing enzymes were inhibited to a lesser degree. When sucrose was used to control a _w, glucokinase and glucose-6-phosphate dehydrogenase were inhibited at 0.92 a _w but the other enzymes remained active below 0.86 a _w. Enzymes were relatively active at reduced a _w when adjusted with glycerol and most remained active even at 0.80 a _w. When a _w was controlled by potassium glutamate, the activity of glucokinase and glucose-6-phosphate dehydrogenase was markedly less inhibited than by NaCl at similar a _w. Possible reasons for the variation in activity by glucose-catabolizing enzymes in response to a _w controlled by various solutes could be location of the enzyme in the cell, ability of the solute to penetrate the cell and ability to withstand high salt and sucrose concentrations. When the a _w of the growth medium was reduced to 0.98 by glycerol, NaCl and polyethylene glycol 400, levels of glucokinase were significantly reduced while higher levels of glucose dehydrogenase and gluconate dehydrogenase were induced. This suggests that reduction in a _w could regulate the routes of catabolism in the Entner-Doudoroff pathway. When sucrose was used to control a _w of the growth medium high levels of most enzymes were induced, suggesting catabolism of the sucrose by the organism.     

Predictive assessment of ochratoxin A accumulation in grape juice based-medium by Aspergillus carbonarius using neural networks

Aims:  To study the ability of multi-layer perceptron artificial neural networks (MLP-ANN) and radial-basis function networks (RBFNs) to predict ochratoxin A (OTA) concentration over time in grape-based cultures of Aspergillus carbonarius under different conditions of temperature, water activity ( a _w) and sub-inhibitory doses of the fungicide carbendazim.
Methods and Results:  A strain of A. carbonarius was cultured in a red grape juice-based medium. The input variables to the network were temperature (20–28°C), a _w (0·94–0·98), carbendazim level (0–450 ng ml⁻¹) and time (3–15 days after the lag phase). The output of the ANNs was OTA level determined by liquid chromatography. Three algorithms were comparatively tested for MLP. The lowest error was obtained by MLP without validation. Performance decreased when hold-out validation was accomplished but the risk of over-fitting is also lower. The best MLP architecture was determined. RBFNs provided similar performances but a substantially higher number of hidden nodes were needed.
Conclusions:  ANNs are useful to predict OTA level in grape juice cultures of A. carbonarius over a range of a _w, temperature and carbendazim doses.
Significance and Impact of the Study:  This is a pioneering study on the application of ANNs to forecast OTA accumulation in food based substrates. These models can be similarly applied to other mycotoxins and fungal species.     

Chemostat adaptation of Escherichia coli B/r/1 to low water activity

Chemostat cultures of Escherichia coli B/r/1 under conditions of glucose limitation in a salts medium at high water activity ( a _w of 0.999) and at an a _w of 0.987, controlled by NaCl, have been compared. The system was run at dilution rates above 0.035 h^-1. The results suggested that the organism adapted to the lower a _w of the medium as no significant change was observed in the energy requirement for maintenance, although the maximum molar growth yield for glucose decreased by 23.2%. Such cultures showed also a shorter lag and a higher growth rate in batch at an a _w of 0.987, as compared with cultures initiated with an unadapted ( a _w of 0.999) inoculum.     

A novel method for floristic quality assessment using the vegetation of the Jiuding Mountain, Sichuan, China as an example

Floristic Quality Assessment (FQA) is proposed as a new method to assess floristic integrity. In the present study, we use both the conventional method broadly adopted in the USA and a new one proposed herein to assess the floristic quality of the Jiuding Mountain. For the application of the FQA, each plant species was assigned an integer from 0 to 10 termed coefficient of conservatism (C) based on its global distribution area. The coefficient shows the degree of fidelity of each plant species to the flora of the west slope of the Jiuding Mountain and represents a rank based on its distribution area. Those species receiving a C value of 0–1 are distributed all over the world, those receiving a C value of 2–3 mostly in tropical areas, and those receiving a C value of 4–6 broadly in temperate areas. Species with C values of 7–8 are endemic to China and those with coefficients 9–10 are restricted to the Hengduan Mountains or even endemic to the Jiuding Mountain. Based on the fact that different plant species show different relative importance values (IV), the new method estimates the weighted averages for the coefficient of conservatism called the weighted mean coefficient of conservatism (     _w). The floristic quality index (FQI) was then derived from     or     _w, native species richness (N) and non-native species (A). As expected when using the new method, the dominant species of the communities obtained different Cs, which resulted in different     _ws and FQI_ws as compared to the conventional method.     

Microbial growth at reduced water activities: some physicochemical properties of compatible solutes

C hirife , J., F avetto , G. & F erro F ontán, c. 1984. Microbial growth at reduced water activities: some physicochemical properties of compatible solutes. Journal of Applied Bacteriology 56 , 259–268.
The water activities ( a _w) of solutions of D-arabitol (a major compatible solute in yeasts grown at reduced a _w) and also of related polyols (L-arabitol, xylitol and ribitol) were determined at 25C. The a _w of saturated solutions of L-proline and monopotassium L-glutamate (solutes which accumulate in bacterial cells at reduced a _w) were also determined. The viscosities of L-proline, monopotassium L-glutamate and D-arabitol solutions were measured at 25, 30, 35 and 40C for various concentrations up to high molalities. The relative viscosity curves were described using a theoretical model for the concentration dependence of viscosity. No relationship of viscosity of the electrolytes to effectiveness as 'compatible solutes' were evident, neither were the compatible solutes particularly effective 'structure makers' or 'structure breakers.'     

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.     

Growth-induced water potentials may mobilize internal water for growth

Abstract. Wphen there is no external source of water, plants can grow by mobilizing internal water from nongrowing tissues. We investigated how this internal water moves by measuring continuously and simultaneously the water potential (ψ_w) of soybean ( Glycine max L. Merr.) seedlings in the upper, growing stem tissues and the lower, non-growing stem tissues. When external water was available to the roots, the stems grew rapidly and the ψ_w of the growing tissue was continually below that of the nongrowing tissue and the medium around the roots. This indicated that a growth-induced gradient in ψ_w favoured water movement from the external source to the growing cells. When the external source was removed, the ψ_w of the growing tissue remained constant for a time and the ψ_w of the nongrowing tissue decreased somewhat. Growth took place slowly as water was withdrawn from the nongrowing tissue but ψ_w gradients continued to favour water transport to the growing cells. On the other hand, if this internal source was removed by excision, growth ceased abruptly. In this case, the cell walls relaxed and the ψ_w of the growing tissue decreased by about 0.1 MPa instead of remaining constant. The ψ_w of the detached nongrowing tissues remained constant instead of decreasing. This indicates not only that water mobilization required attached nongrowing or slowly growing tissues but also that mobilization affected wall relaxation. Thus, ψ_w differences may mobilize internal water, may explain the continued growth of plants and plant parts removed from external sources of water, and may account for discrepancies in measurements of cell wall properties in growing tissues.     

Water potential and plant metabolism: comments on Dr P. J. Kramer's article 'Changing concepts regarding plant water relations', Volume 11, Number 7, pp. 565–568, and Dr J. B. Passioura's Response, pp. 569–571

Abstract. Kramer (1988) and Passioura (1988) highlight an important principle in plant physiology, namely that roots affect the activity of shoots. It is safe to say that anyone who has observed plants could cite many examples. What is in contention, however, is the interpretation of the water potential (Ψ_w) after some investigators (Bates & Hall, 1981; Blackman & Da vies, 1985; Eavis & Taylor, 1979; Gollan, Passioura & Munns, 1986; Termaat, Passioura & Munns, 1985; Turner, Schulze & Gollan, 1985) observed that roots in water deficient media can affect shoot physiology in ways not involving the Ψ_w of the shoot. As a result, some investigators (Sinclair & Ludlow, 1985; Passioura, 1988) question the Ψ_w concept on the grounds indicated by Passioura (1988): if roots affect shoot activity by means other than by changing shoot Ψ_w, the Ψ_w concept must be wrong. This is unfortunate. It has been known for years that metabolic reactions generally do not respond directly to the Ψ_w or its components. Nevertheless, the shoot Ψ_w, and its components play important roles in the physiology and metabolism of plants. Often these roles are interactive with roots. In what follows, I will review some of the evidence for these principles.     

Inositol 1,4,5-trisphosphate formation in leaves of winter oilseed rape plants in response to freezing, tissue water potential aed abscisic acid

Time courses of formation of inositol 1,4,5-trisphosphate (IP₃) were followed in the leaves of non-acclimated and cold (2°C)-acclimated winter oilseed rape ( Brassica napus L. var. oleifera ) plants, subjected to different freezing temperatures or to polyethylene glycol 8000 (PEG) and abscisic acid (ABA) treatments. Changes in water potential (Ψ_w) and in ABA level in the frost- and PEG-treated tissues were also determined. Results obtained indicate that temperatures sligthly higher than LT₅₀ induced a transient and substantial increase in IP₃ level, both in non-acclimated and cold-acclimated tissues. At comparable freezing temperature (–5°C) the response of cold-acclimated leaves was lower than that of non-acclimated ones. The PEG-depedent decrease in Ψ_w to –0.9 MPa or ABA (0.1 m M ) treatment gave rise to a transient increase in IP₃ content in non-acclimated tissues only. Collectively, the data indicate that cold acclimation of plants may lead to lower cell responsiveness to the factors studied in terms of induction of IP₃ formation. Changes in the IP₃ content, observed in the present experiments, support our previous suggestion that non-killing freezing temperatures may induce the phosphoinositide pathway, both in non-acclimated and cold-acclimated tissues. Lowering of tissue water potential to some threshold value or a high exogenous ABA supply may mimic the freezing-dependent reaction in the non-acclimated leaves.     

Microbial growth at reduced water activities: studies of aw prediction in solutions of compatible solutes

Studies were made of the prediction of intracellular water activity ( a _w) of microorganisms grown at reduced a _w. These included the determination and prediction of a _w of potassium L-glutamate solutions and the prediction of a _w in mixed solutions of various compatible solutes and macromolecules. Compatible solutes studied were L-proline, glycerol, potassium chloride and D-arabitol. The results indicate that a _w predictions of such mixtures may be done with acceptable accuracy by neglecting solute-solute interactions.     

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.     

Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativd) plants

Susceptibility of alfalfa ( Medicago saliva L. cv. Aragón) nodules and leaves to water stress has been investigated. Nodule acetylene reduction activity (ARA), leaf CO₂ exchange rate (CER) as well as soluble protein, proline and total soluble sugar (TSS) contents were determined during drought. Water status was estimated as water potential (Ψ_w) and Relative water content (RWC) of the respective tissues. Maximum rates of ARA required higher Ψ_w than CER. Nodules had lower RWC for a given Ψ_w than leaves. Water stress reduced soluble protein content in both tissues; however, the decline in soluble protein content was detected at greater Ψ_w in nodules than in leaves. Proline and TSS increased in leaves and nodules, and again the threshold Ψ_w triggering such accumulation was higher in nodule tissues. Oior results suggest that alfalfa nodules are more susceptible to water shortage than leaves. Effects of accumulated TSS and proline upon leaf and nodule physiology are discussed in relation to protein stability (proline), pH control (proline) and osmotic adjustment (proiine and TSS). The TSS accumulation induced by water stress suggests that substrate shortage would not be the primary effect of drought on nodule activity.     

Growth studies with fish—overcoming the problems of size variation

The relationship between growth rate and fish size is described by the equation log_e G _w= a —0·4 log_e W, where G_w is the specific growth rate and W is fish weight. Since the intercept (a) represents the log_e G_w of a fish unit size, the relationship presents a method allowing comparison of data from experiments involving fish of different sizes. The application of the method is demonstrated by examining the effects of environmental temperature on growth rates of cod, Gadus morhua , and it is suggested that the optimum temperature for growth of cod is 13–15° C.