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

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

Adaptation of Pseudomonas fluorescens to Low Levels of Water Activity Produced by Different Solutes

A culture of Pseudomonas fluorescens which grew at a minimal a_w of 0.97 in a glucose-salts broth adjusted to that level with NaCl, KCl, or glucose, and at a_w 0.9725 with Na₂SO₄, was adapted to grow at lower a_w levels produced by these solutes. Adaptation to growth at a low a_w produced by means of one solute did not necessarily result in growth at that a_w when another solute was employed to lower the a_w. The culture adapted to grow at a_w 0.9650 produced by addition of glucose could grow equally well in broth adjusted to that a_w with NaCl or KCl, but not Na₂SO₄. NaCl and KCl permitted growth at the lowest a_w level (0.9625), and the culture adapted in KCl did equally well in NaCl.     

Effect of water activity and protective solutes on growth and subsequent survival to air-drying of Lactobacillus and Bifidobacterium cultures

Probiotic cultures of Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were grown in media having water activities (a _w) adjusted between 0.99 and 0.94 with NaCl or with a mixture of glycerol and sucrose in order to find conditions of osmotic stress which would still allow for good growth. Cultures grown at a _w?=?0.96 or 0.99 were then recovered by centrifugation, added to a sucrose–phosphate medium and air-dried. In some assays, a 2-h osmotic stress was applied to the cell concentrate prior to air-drying. Assays were also carried out where betaine, glutamate and proline (BGP) supplements were added as protective compounds to the growth or drying media. For most strains, evidence of osmotic stress and benefits of BGP supplementation on growth occurred at a _w?=?0.96. Growing the cells in complex media adjusted at a _w?=?0.96 did not enhance their subsequent survival to air-drying, but applying the 2-h osmotic stress did. Addition of the BGP supplements to the growth medium or in the 2-h stress medium did not enhance survival to air-drying. Furthermore, addition of BGP to a sucrose–phosphate drying medium reduced survival of the cultures to air-drying. This study provides preliminary data for producers of probiotics who wish to use air-drying in replacement of freeze-drying for the stabilization of cultures.     

The effect of water activity on growth and end-product formation of two Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T

Summary The effect of water activity (a_w) on the growth and end-product formation of Lactobacillus viridescens SMRICC 174, Lactobacillus SMRICC 173 (homofermentative) and Brochothrix thermosphacta ATCC 11509^T was studied. All strains orginated from meat or meat products. The a_w was adjusted in the range 0.94–0.99 with NaCl or glycerol. A greater reduction in growth rates was found for L. viridescens and B. thermosphacta when a_w was regulated with NaCl rather than with glycerol, the opposite was true for Lactobacillus 173. L. viridescens grew at a_w >-0.94. At 0.94 a_w B. thermosphacta was totally inhibited when NaCl was the solute and Lactobacillus 173 when glycerol was the solute. Only minor variations in the end-product formation of the Lactobacillus spp. were found at different a_w values. In aerobic culture B. thermosphacta produced less l-lactic acid and more acetic acid as the a_w was decreased with NaCl, while the yields were unaffected when glycerol was used.     

Specificity of Lactococcus lactis subsp. cremoris SK11 Proteinase, Lactocepin III, in Low-Water-Activity, High-Salt-Concentration Humectant Systems and Its Stability Compared with That of Lactocepin I

Marked changes in the specificity of hydrolysis of α_s1-, β-, and κ-caseins by lactocepin III from Lactococcus lactis subsp. cremoris SK11 were found in humectant systems giving the equivalent water activity (a_w) and salt concentration of cheddar cheese. Correlations were noted between certain peptides produced by the activity of lactocepin III in the humectant systems and peptides found in cheddar cheese. The stability of lactocepin III was compared with that of lactocepin I from L. lactis subsp. cremoris HP in the humectant systems at different pHs. Significant differences between the stability of each of the lactocepin types were evident. The relationship between stability and humectant type, a_w, pH, and NaCl concentration was complex. Nevertheless, in those systems where a_w, pH, and NaCl concentration were equivalent to those in cheddar cheese, lactocepin I was generally more stable than lactocepin III. It was concluded that differences in the specificity and/or stability of various lactocepin types are likely to persist in cheese itself and therefore potentially contribute to differences in the peptide composition of ripened cheese.     

Water relations of polyol accumulation by Zygosaccharomyces rouxii in continuous culture

Summary Glycerol and arabitol were the main polyols accumulated by Zygosaccharomyces rouxii in continuous culture but the intracellular and extracellular concentrations of the polyols varied with the dilution rate and osmoticum used to adjust the water activity (a_w) to 0.960. When the a_w was adjusted with NaCl, glycerol was the main polyol accumulated intracellularly whereas glycerol and arabitol were accumulated when polyethylene glycol (PEG) 400 was used. The extracellular glycerol and arabitol concentrations at 0.960 a_w (NaCl or PEG 400) were similar or decreased relative to cultures at 0.998 a_w. Compared to steady-state cultivation at 0.998 a_w, the yeast retained at 0.960 a_w (NaCl or PEG 400) a greater proportion of the total glycerol intracellularly against an increased concentration ratio without significantly greater production of glycerol. Arabitol was only significant in osmoregulation when cultivated at 0.960 a_w (PEG 400). The intracellular glycerol concentration was insufficient to balance the a_w across the membrane, but an equilibrium could be achieved under certain conditions if arabitol was also osmotically active. Offprint requests to: P. J. van Zyl     

Survival and Filamentation of Salmonella enterica Serovar Enteritidis PT4 and Salmonella enterica Serovar Typhimurium DT104 at Low Water Activity

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a_w). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a_w for long periods, but minimum humectant concentrations of 8% NaCl (a_w, 0.95), 96% sucrose (a_w, 0.94), and 32% glycerol (a_w, 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a_w, incubation at 37°C resulted in more rapid loss of viability than incubation at 21°C. At a_w values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 μm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a_w conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a_w highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a_w (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a_w storage. If Salmonella strains form filaments in food products that have low a_w values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.     

Clostridium botulinum Type A Growth and Toxin Production in Media and Process Cheese Spread

We found that Clostridium botulinum type A grew well and produced toxin in media with a water activity (a_w) of 0.972 or 0.965 and a pH of 5.7, but no growth or toxin production was observed at or below an a_w of 0.949 during incubation at 30°C for 52 to 59 days. a_w and pH values of media were adjusted to those of cheese spreads commercially produced. Solutes used to adjust a_w included combinations of NaCl, cheese whey powder, emulsifying salt, sodium tripolyphosphate, and glycerol. In agreement with results obtained for media, toxin was produced in samples of cheese spread (a_w, 0.970; pH, 5.7) at 30 to 70 days of incubation at 30°C.     

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

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

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

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

Synthesis of Pyruvate Dehydrogenase in Staphylococcus aureus Is Stimulated by Osmotic Stress

The pyruvate dehydrogenase multienzyme complex (PDHC) was found to be upregulated by osmotic stress in the osmotolerant pathogen Staphylococcus aureus. Upregulation was detectable in the levels of both activity and protein and was judged to be about fourfold when sodium chloride was used to adjust the water activity (a_w) of the growth medium to 0.94. The upregulation of the PDHC was also found to be humectant dependent and was greatest when impermeant, nonmetabolizable humectants were used to adjust a_w. Further experiments provided evidence that in addition to osmotic upregulation, the PDHC complex is also subject to catabolite repression, thus providing a possible explanation for the observation that high concentrations of carbohydrates are generally more inhibitory to the growth of this bacterial pathogen than are high concentrations of salts.     

Osmotic Stress Leads to Decreased Intracellular pH of Listeria monocytogenes as Determined by Fluorescence Ratio-Imaging Microscopy

Intracellular pH (pH_i) of Listeria monocytogenes was determined after exposure to NaCl or sorbitol in liquid and solid media (agar). Both compounds decreased pH_i, and recovery on solid medium was impaired compared to that in liquid medium. N,N′-dicyclohexylcarbodiimide abolished pH_i recovery, and lowering a_w with glycerol showed no effect on pH_i.     

Effect of Water Activity on Enterotoxin B Production and Growth of Staphylococcus aureus

Staphylococcus aureus C-243, an enterotoxin B-producing strain, was cultured on media adjusted to various water activity (a_w) levels by means of two different solute systems. Total numbers and rate of growth were diminished at low a_w levels, and enterotoxin synthesis was extremely sensitive to reduction in a_w. A reduction of a_w from 0.99 to 0.98 in one medium and from 0.99 to 0.97 in the other medium resulted in extremely low levels of enterotoxin in spent culture media despite the attainment of high numbers of staphylococci.     

Influence of water activity on Streptococcus diacetylactis metabolism

During the cheese-making process, water activity (a_w) is one of the essential environmental parameters acting on bacterial growth and metabolic pathways. The influence of a_w on Streptococcus diacetylactis growth and lactic acid production was studied. The specific growth rate was linearly related to water availability in the milk medium. The cell behaviour was quite different above and below a_w=0.95, which can be considered a limiting value. Below this value, the lactic acid production reached 1.4–6.1 mg·g^–1, whereas the specific productivity was 2.0–2.6 mg·10^–10 cells·h^–1. Changes in the consumption of lactose and amino acids during the different growth phases was completely modified by decreasing the water availability in the medium. Correspondence to: N. Cochet     

Spore Germination and Mycelial Growth of Streptomycetes at Different Humidity Levels

This study is the first to show the ability of streptomycetes to develop at a very low humidity level. All of the streptomycetes studied produced growth at low humidity (a_w 0.86 and 0.67). This capacity was most markedly pronounced in Streptomyces odorifer, whose spores were capable of germinating, and mycelial germs increased in length, at the air humidity a_w 0.50. The formation of lateral branches (mycelium branching) at this humidity was noted only in single S. odorifer germs and only after 72 h of incubation. Study of streptomycete growth on an agarized medium with different osmotic pressures, created by various glycerol concentrations in the medium, showed that, at a_w 0.67, the spores of all the streptomycetes studied germinate, producing mycelial germs but not microcolonies. The ecological significance of mycelial prokaryotes in soil microbial communities that develop and function under conditions of extremely low humidity is discussed.     

Relation of the Heat Resistance of Salmonellae to the Water Activity of the Environment

The effect of water activity (a_w) on the heat resistance of eight strains of Salmonella was studied. Heat resistance of the organisms increased as the a_w of the heating menstruum was reduced. Sucrose afforded the cells a greater degree of protection than did fructose, glycerol, and sorbitol. A direct correlation between a_w and heat resistance could not be established over the range of a_w levels tested in this study. There was variation among the strains of salmonellae in the magnitude of the increase in heat resistance as the a_w level was reduced. All strains of Salmonella tested showed a greater increase in heat resistance than S. senftenberg 775W as the environment became drier. Washed cells had D values 25 to 75% lower than unwashed cells. Prior growth of the organisms in media with a reduced a_w increased the heat resistance of the organisms when glycerol, but not when sucrose, was the controlling substance.     

Xerotolerance in fission yeasts and the role of glycerol as compatible solute

The effect on growth of reducing the water activity (a _w) of a medium with various solutes has been investigated for 27 strains of fission yeasts (Schizosaccharomyces). The minimum-tolerated a _w (MTA) was dependent on both the nature of the solute and the species. When the strains of each species were grouped together, the lowest mean MTA values were found with glucose, fructose or glycerol as stressing solutes, being in the range 0.89–0.90 for S. pombe, S. malidevorans, S. octosporus and S. slooffiae, but in the range 0.92–0.94 for S. japonicus. With the non-metabolizable sugars sorbose and xylose and the salts NH₄Cl, KCl, and NaCl, the mean MTA values were in the range 0.96–0.985, except for (1) the single strain of S. slooffiae, which was more tolerant of NH₄Cl and KCl with values of 0.95 and 0.94, respectively, and (2) the strains of S. pombe, S. malidevorans and S. japonicus, which were less tolerant of NaCl with mean values of about 0.99. One strain of each species was examined for intracellular solutes when actively growing in the presence of near-limiting concentrations of stressing solute. With glucose, fructose or glycerol, all five strains contained substantial amounts of glycerol but no other polyol; with the other solutes no glycerol or other polyol was found, except for small amounts of glycerol in strains of S. octosporus and S. slooffiae stressed with NH₄Cl, KCl, or NaCl.Abbreviations MTA Minimum-tolerated water activity - a _w water activity - YEPG yeast extract, phosphate, glucose medium     

Influence of Reduced Water Activity on Lactose Metabolism by Lactococcus lactis subsp. cremoris at Different pH Values

The influence of reduced water activity (a_w) on lactose metabolism by Lactococcus lactis subsp. cremoris 2254 and 2272 was studied at different pH values. In control incubations (a_w, 0.99) with nongrowing cells in pH-controlled phosphate buffer, the levels of carbon recovered as l-(+)-lactate were 92% at pH 6.1 and 5.3 and 78% at pH 4.5. However, the levels of recovery decreased to ∼50% at all pH values tested when the a_w was 0.88 (with glycerol as the humectant). When growing cells in broth controlled at pH 6.3 were used, a reduction in the a_w from 0.99 to 0.96 resulted in a decrease in the level of lactose carbon recovered as l-(+)-lactate from 100 to 71%. Low levels of l-(+)-lactate carbon recovery (<50%) were also observed with cells resuspended in pH-uncontrolled reconstituted skim milk at a_w values of 0.99 and 0.87 and in young cheese curds. The missing lactose carbon could not be accounted for by acetate, ethanol, formate, acetaldehyde, or pyruvate. Attempts were made to determine where the missing lactose carbon was diverted to under the stress conditions used. Some of the missing lactose carbon was recovered as galactose (0.1 to 2.5 mM) in culture supernatants. Decreasing either the a_w or the pH resulted in increased galactose accumulation by nongrowing cells; adjusting both environmental factors together potentiated the effect. The sensitivities of the two lactococcal strains tested were different; strain 2272 was more prone to accumulate galactose under stress conditions. A methyl pentose(s) and additional galactose were found in acid-hydrolyzed supernatants from cultures containing both growing and nongrowing cells, indicating that a saccharide(s) rich in these components was formed by lactococci under low-a_w and low-pH stress conditions.Water activity (a_w) affects the growth, physiology, and metabolism of microorganisms and their resistance to inimical agents (22, 54). A number of microorganisms respond to a low-a_w environment by intracellular accumulation of low-molecular-weight compatible solutes, such as amino acids, amino acid derivatives, trehalose, and polyols (6, 12). These compatible solutes restore turgor pressure and membrane tension to levels very similar to those that occur before osmotic upshift (12), preserve enzyme activity and protein stability, and maintain the integrity and stability of membranes and nucleic acids (6).There have been several reports concerning the accumulation of compatible solutes in lactic acid bacteria. Hutkins et al. (26) found that betaine was accumulated by an osmotolerant strain of Lactobacillus acidophilus, while Molenaar et al. (38) reported that Lactococcus lactis subsp. lactis ML3 contained high levels of proline or betaine when it was grown under osmotic stress conditions in complex media. Lactobacillus plantarum, however, accumulated not only betaine and proline, but also carnitine, glutamic acid, and trehalose when it was cultured in a complex medium having a reduced a_w (29, 30, 36). The accumulation of betaine enhanced the survival of several lactic acid bacteria subjected to drying conditions (28, 30).The influence of reduced a_w on substrate metabolism and product formation by lactic acid bacteria has received little attention. Optimal production of lactic acid by some dairy lactic acid bacteria occurs at a_w values of 1.0 to 0.95, and production declines dramatically as the a_w is decreased, which is consistent with growth inhibition, whereas diacetyl production by some lactic acid bacteria increases with decreasing a_w and optimal diacetyl production occurs at a_w values of 0.95 to 0.97 (51, 52). Bassit et al. (1) studied the influence of a_w on the metabolism of Streptococcus diacetylactis (now Lactococcus lactis subsp. lactis var. diacetylactis) and found that decreasing the a_w decreased lactose consumption and lactic acid formation and inhibited growth. Blickstad (3) observed no significant variations in the formation of end products by two meat lactobacilli at different a_w values (0.99 to 0.94).Lactose is the major carbohydrate present in milk and young cheeses, and metabolism of lactose by starters during curd manufacture and early ripening is important for acid development. The cheese a_w decreases during manufacture and ripening as a result of dehydration, salting, and the production of water-soluble solutes from glycolysis, proteolysis, and lipolysis; the cheese a_w values range from 0.70 for extrahard cheeses to 0.99 for fresh, soft cheeses, such as cottage cheese, while semihard cheeses have a_w values of around 0.90 (33, 41). The cheese pH also decreases during manufacture and ripening (19). We describe here the influence of low a_w values on lactose metabolism by both growing and nongrowing cells of Lactococcus lactis subsp. cremoris at different pH values in buffer, broth, and reconstituted skim milk (RSM) with and without pH control. Cheese experiments to determine lactose utilization during ripening were also linked with our in vitro studies.     

Physiological responses of Zygosaccharomyces rouxii to osmotic stress

When cell suspensions of Zygosaccharomyces rouxii were subjected to osmotic shock with NaCl, the cell volume decreased sharply and plasmolysis was observed. The cell subsequently recovered and volumes similar to those of cells growing at the respective water activity (a_w) values were found. Cycloheximide prevented cell recovery, indicating the involvement of protein synthesis in the recovery process. The intracellular glycerol concentration of Z. rouxii incubated in the presence of [¹⁴C]glycerol increased from 13 to 96 mmol/l during the initial 20 min after an upshock from 0.998 a_w to 0.96 a_w. All the intracellular glycerol was labelled and therefore derived from the medium. Labelled glycerol was subsequently utilized and replaced by unlabelled glycerol produced by the cell within 90 min. The initial increase in glycerol concentration following the upshock was confirmed by ¹³C-nuclear magnetic resonance (NMR) spectroscopic studies of cell extracts. The combined dihydroxyacetone and dihydroxyacetone phosphate concentrations fluctuated during this period, whereas glycerol-3-phosphate initially increased and then remained constant. This indicates that the production of glycerol is regulated. Decreases in ATP and polyphosphate levels were observed following osmotic upshock and may reflect a greater demand for ATP during the period of adjustment to decreased a_w. The changes in cell volume and in ATP concentration following osmotic upshock may serve as osmoregulatory signals in Z. rouxii, as suggested previously for other microorganisms. Correspondence to: S. G. Kilian     

Regulation of glycerol metabolism in Zygosaccharomyces rouxii in response to osmotic stress

Summary Enzyme analyses indicated that the metabolism of glycerol by Zygosaccharomyces rouxii occurred via either glycerol-3-phosphate (G3P) or dihydroxyacetone (DHA). The route via DHA is significant in osmoregulation. The specific activities of glycerol dehydrogenase (GDHG) and DHA kinase, which metabolize glycerol via DHA, increased nine- and fourfold respectively during osmotic stress [0.960 water activity (a_w) adjusted with NaCl] when compared to non-stressed conditions (0.998 a_w). Both pathways are under metabolic regulation. Glycerol kinase, mitochondrial G3P dehydrogenase and DHA kinase are induced by glycerol while the latter is also repressed by glucose. Cells treated with cycloheximide prior to osmotic upshock showed significantly lower DHA kinase and GDHG levels and lower intracellular glycerol concentrations when compared to untreated control cells. Thus protein synthesis is essential for osmotic adaptation. Offprint requests to: B. A. Prior