Moisture Requirements for Growth and Metabolite Production by Lactic Acid Bacteria

The effect of water activity (a_w) reduction on growth and acid and diacetyl production by three lactic streptococci was studied. In addition, the influence of low moisture conditions on several bacteria of significance in the fermentation of sauerkraut was examined. The minimal a_w supporting growth of dairy lactics was 0.93 in a medium adjusted with glycerol. Media adjusted with sucrose generally were more inhibitory than those in which glycerol was the humectant. Titratable acidity, although not related to the type of humectant, did depend on the a_w of the medium and was directly related to the extent of growth. Diacetyl concentration increased in cultures of reduced a_w when the media were adjusted with both humectants; however, the effect was greatest with glycerol. A lactic strain associated with sauerkraut fermentation appeared to grow at a lower minimal a_w in a glycerol-adjusted medium than in a system adjusted with NaCl; however, none of the sauerkraut organisms grew at a_w levels of <0.95 when NaCl was the solute. Acid production appeared to be related to the presence and extent of growth at all of the a_w levels studied.     

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.     

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.     

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     

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     

Water relations of ethanol fermentation by Saccharomyces cerevisiae: Glycerol production under solute stress

The effect of water activity (a_w) upon glycerol production during ethanol fermentation by Saccharomyces cerevisiae was studied in continuous culture using sorbitol to adjust the a_w to 0.971. A reduction of the a_w of the medium resulted in more glycerol being produced. The glycerol yield was 3–4-fold greater at 0.971 a_w than 0.994 a_w. Ethanol yield and specific rate of ethanol production was slightly greater at 0.971 than 0.994 a_w whereas the biomass concentration was lower. Reduced a_w increased the specific rate of glucose uptake and the maintenance requirements of the organism. The results suggest that the effect of a_w on glycerol production can be reduced by controlled feeding of substrate during fermentation.     

Increasing the stability of immobilizedLactococcus lactis cultures stored at 4 °C

Summary Immobilized cell technology was used to prepare concentrated cultures ofLactococcus lactis that lost only 22% of viability over a 30-day storage period at 4°C. Concentrated cultures ofL lactis CRA-1 were immobilized in calcium alginate beads and added to glycerol, NaCl or sucrose-NaCl solutions in order to obtain a_w readings ranging from 0.91 to 0.97. The suspensions were subsequently placed at 4°C and viability (CFU g^–1 of bead) was followed during storage. Viability losses were high at a_w readings of 0.95 and 0.97 and pH dropped significantly (up to one unit) in the unbuffered solutions. Addition of 1% soytone or glycerophosphate helphed stabilize pH, and a beneficial effect on viability during storage was observed in the glycerol-soytone mix when the beads were added to the conservation solutions immediately following immobilization. When beads were added to the conservation solution immediately following immobilization, a 70% drop in cell counts occurred during the first 5 days of incubation. Dipping theL lactis-carrying beads in milk for 2h before mixing with the glycerolsoytone 0.93 a_w solution reduced this initial 5-day viability loss. Cultures grown in the alginate beads also had good stability in the 0.93 a_w glycerol-soytone solution, where 78% of the population was viable after 30 days at 4°C. The process could be used to store immobilized cells at a processing plant, or by suppliers of lactic starters who wish to ship cultures without freezing or drying.     

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     

Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Summary Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacillus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inoculum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the nitrogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.     

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     

The role of an active transport mechanism in glycerol accumulation during osmoregulation by Zygosaccharomyces rouxii

Summary At water activities (a _w) of 0.998 (no osmoticum) and 0.960 a _w(NaCl), the affinity (K _m) of glycerol transport by Zygosaccharomyces rouxii was 25.6 and 6.4 mmol/l respectively. The maximum uptake rate (V _max) was ca. 2.3 mol/g/min at both a _w's. However, at an a _wof 0.960 using polyethylene glycol (PEG) 400 the K _mand V _max for glycerol transport increased to 61.1 mmol/l and 32.2 mol/g per minute respectively. This suggests that different glycerol transport mechanisms operate during stress by the two osmotica. The addition of uncouplers (2,4-dinitrophenol or carbonylcyanide-m-chlorophenylhydrazine) resulted in the outflow of accumulated [^14C]glycerol from Z. rouxii after on osmotic upshock indicating that an active transport mechanism was operative. The transport mechanism was specific for glycerol since other polyols (mannitol, meso-erythritol and arabitol) had no effect on the uptake rate. During upshock from 0.998 to 0.960 a _w(NaCl), a transient increases in the rate of [¹⁴C]glycerol uptake was observed. However, if PEG 400 was used as osmoticum, the rate of glycerol uptake failed to increase.Offprint requests to: P. J. van Zyl     

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.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

In sterilized skim milk or sterilized 10% solution of dry skim milk at 120°C for 15 min, Lactobacillus bulgaricus, Lactobacillus helveticus and Streptococcus lactis were cultivated for 7 days at given temperature.

Both NCN (non casein type nitrogen) content and pH in each culture of lactic acid bacteria were rapidly decreased until 2 days after cultivation, But NCN content increased and the pH change got small after 3 days cultivation.

Caseins prepared from the cultures of these three kinds of lactic acid bacteria were examined electrophoretically. From the results of electrophoresis of these caseins, we have concluded that α-casein could be hydrolyzed by these lactic acid bacteria. And, it seemed that β-casein could not be hydrolyzed by these lactic acid bacteria.

Rennet easily hydrolyzed casein treated with L. bulgaricus and L. helveticus but hardly hydrolyzed that treated with S. lactis compared with control-casein. Caseins treated with L. bulgaricus and L. helveticus were hydrolyzed easier than control-casein.

Particle weights of caseins prepared from fermented milk by lactic acid bacteria, Streptococcus cremoris, Streptococcus lactis, Lactobacillus bulgaricus and Lactobacillus helveticus, and of hydrolyzed casein by rennet, trypsin or pepsin were measured according to the light scattering experiment.

Particle weights of various treated caseins were larger than that of raw native casein at both pH 7.0 and 12.0. And the heating caused the polymerization of casein to large particle.     

Metabolic changes induced during adaptation of Saccharomyces cerevisiae to a water stress

When exponentially growing Saccharomyces cerevisiae was transferred from a normal high water activity growth medium (a_w 0.997) to a medium containing 8% NaCl low water activity growth medium (a_w 0.955), glycerol accumulation during the first eight hours of the adaptation was both retarded and greatly diminished in magnitude. Investigation of the underlying reasons for the slow onset of glycerol accumulation revealed that not only was overall glycerol production reduced by salt transfer, but also the rates of ethanol production and glucose consumption were reduced. Measurement of glycolytic intermediates revealed an accumulation of glucose-6-phosphate, fructose-6-phosphate, fructose 1,6 bisphosphate and phosphoenolpyruvate in S. cerevisiae 3 to 4 h after transfer to salt, suggesting that one or more glycolytic enzymes were inhibited. Potassium ions accumulated in S. cerevisiae after salt transfer and reached a maximum about 6 h after transfer, whereas the sodium ion content increased progressively during the adaptation period. The trehalose content also increased in adapting cells. It is suggested that inhibition of glycerol production during the initial period of adaptation could be due to either the inhibition of glycerol-3-phosphate dehydrogenase by increased cation content or the inhibitin of glycolysis, glycerol being produced glycolytically in S. cerevisiae. The increased accumulation of glycerol towards the end of the 8-h period suggests that the osmoregulatory response of S. cerevisiae involves complex sets of adjustments in which inhibition of glycerol-3-phosphate dehydrogenase must be relieved before glycerol functions as a major osmoregulator.     

Modelling time to growth of Escherichia coli as a function of water activity and undissociated lactic acid

Aims: To model the effect of water activity (a_w) and concentration of undissociated lactic acid (HLac) on the time to growth (TTG) and the growth/no growth boundary of acid‐adapted generic Escherichia coli, used as model organisms for Shiga toxin‐producing E. coli (STEC). Methods and Results: For each of two E. coli strains, the TTG in brain heart infusion broth at 27°C was estimated at 30 combinations of a_w (range 0·945–0·995) and concentration of HLac (range 0–6·9 mol m^?3) by using an automated turbidity reader. Survival analysis was used to develop a model predicting the TTG and the growth/no growth boundary. Conclusions: The present model can be used to predict the TTG and to indicate the growth/no growth boundary of acid‐adapted E. coli strains as a function of a_w and concentration of HLac. Significance and Impact of the Study: Fermented food products have been implicated as sources of STEC in several outbreaks. The study results are relevant for modelling of growth of STEC in fermented food and can be used in microbiological risk assessments or in the design and validation of food‐production processes.     

Comparison of growth limits of Listeria monocytogenes in milk,broth and cheese

Aim: To determine growth initiation differences of Listeria monocytogenes between a cheesemaking context, milk and tryptic soy broth (TSB). Methods and Results: A laboratory‐scale cheese was made with a mix of two strains of L. monocytogenes at four initial pH values, five water activity (a_w) values and two contamination levels at 30°C. Counts of L. monocytogenes were determined at time 0 and after 8 h of cheese manufacture. Milk and TSB at the same pH and a_w conditions were inoculated with the L. monocytogenes mix in multi‐well plates. Growth was determined by plating each well onto Agosti & Ottaviani Listeria Agar after 8 h of incubation at 30°C. Each condition was repeated six times, and growth initiation probability was modelled with logistic regression models. Growth initiation boundaries were obtained for each matrix type. The results showed that the growth limits were matrix dependent. In the three matrix types, a_w was the most important factor affecting the probability of growth initiation. Contamination level affected growth TSB and cheesemaking conditions. Conclusions: The interface wideness and position in cheese, milk and TSB were dissimilar, indicating that the use of models evaluated in TSB or milk could not be used to predict the behaviour of L. monocytogenes under cheesemaking conditions. Significance and Impact of the Study: Predictive models generated in liquid media are not necessarily adaptable to solid food, and the generation of real food models is necessary.     

Influence of water activity and temperature on in vitro growth of surface cultures of a Phoma sp. and production of the pharmaceutical metabolites, squalestatins S1 and S2

A Phoma sp., known to produce the pharmaceutically active metabolites squalestatin 1 (S1) and squalestatin 2 (S2), was cultured on malt-extract/agar (MEA) over a range of water activities (a _w, 0.995–0.90) and temperatures (10–35 °C) to investigate the influence on growth and metabolite production. Use of the ionic solute NaCl to adjust a _w resulted in significantly lower (P < 0.01) squalestatin yields than when the Phoma sp. was grown on MEA amended with the non-ionic solute glycerol. Water activity and temperature and their interactions were highly significant factors (P < 0.001) affecting growth of the Phoma sp., with optimum conditions of 0.998–0.980 a _w and 25 °C. Squalestatin production was similarly influenced by a _w, temperature, time and their interactions (P < 0.001). S1 and S2 production occurred over a narrower a _w and temperature range than growth, with a slightly lower optimum a _w range of 0.995–0.980 a _w. The optimum temperature for squalestatin production varied from 20 °C (S1) to 25 °C (S2) and yields of S2 were up to 1000 times lower than those of S1. The ratio of S1 and S2 produced by the Phoma sp. was influenced by a _w and temperature, with highest values at 0.99–0.98 a _w, and at 15 °C. Incubation times of 28 days gave highest yields of both S1 and S2. Up to 2000-fold increases in squalestatin yields were measured at optimum environmental conditions, compared to the unmodified MEA. This indicates the need to consider such factors in screening systems used to detect biologically active lead compounds produced by fungi. Received: 2 June 1997 / Received last revision: 6 November 1997 / Accepted: 7 November 1997     

Batch propagation of Lactobacillus helveticus for production of lactic acid from lactose concentrated cheese whey with microaeration and nutrient supplementation

Continuous mix batch bioreactors were used to study the kinetic parameters of lactic acid fermentation in microaerated-nutrient supplemented, lactose concentrated cheese whey using Lactobacillus helveticus. Four initial lactose concentrations ranging from 50 to 150 g l^–1 were first used with no microaeration and no yeast extract added to establish the substrate concentration above which inhibition will occur and then the effects of microaeration and yeast extract on the process kinetic parameters were investigated. The experiments were conducted under controlled pH (5.5) and temperature (42 °C) conditions. The results indicated that higher concentrations of lactose had an inhibitory effect as they increased the lag period and the fermentation time; and decreased the specific growth rate, the maximum cell number, the lactose utilization rate, and the lactic acid production rate. The maximum lactic acid conversion efficiency (75.8%) was achieved with the 75 g l^–1 initial lactose concentration. The optimum lactose concentration for lactic acid production was 75 g l^–1 although Lactobacillus helveticus appeared to tolerate up to 100 g l^–1 lactose concentration. Since the lactic acid productivity is of a minor importance compared to lactic acid concentration when considering the economic feasibility of lactic acid production from cheese whey using Lactobacillus helveticus, a lactose concentration of up to 100 g l^–1 is recommended. Using yeast extract and/or microaeration increased the cell number, specific growth rate, cell yield, lactose consumption, lactic acid utilization rate, lactic acid concentration and lactic acid yield; and reduced the lag period, fermentation time and residual lactose. Combined yeast extract and microaeration produced better results than each one alone. From the results it appears that the energy uncoupling of anabolism and catabolism is the major bottleneck of the process. Besides lactic acid production, lactose may also be hydrolysed into glucose and galactose. The -galactosidase activity in the medium is caused by cell lysis during the exponential growth phase. The metabolic activities of Lactobacillus helveticus in the presence of these three sugars need further investigation.     

Production of the fungal biocontrol agent Epicoccum nigrum by solid substrate fermentation: effect of water activity on accumulation of compatible solutes

Epicoccum nigrum conidia were produced by solid fermentation on wheat grains (cv. Rendeveaux and Brigadier) at different water activities (a_w). Conidial production was highest at high a_w(0.996) than at reduced a_w (0.98). However, conidial production at reduced a_w was improved when the a_w of the substrate was adjusted with a mixture of glycerol and water. Maximum levels ofconidiation were 7–11 × 10⁶ conidia g⁻¹ grain. The a_w of the solid substrate affected the pattern of accumulation of compatible solutes in the conidia. Mannitol was the main polyol in all conidialtypes. However, the amounts of mannitol were higher in conidia produced at high a_w. At reduced a_w the conidia of E. nigrum accumulated moreglycerol, which is more efficient in the osmorregulation proccess than mannitol. Arabitol accumulated in low amounts, specifically in conidia produced at the lower a_w, on cv. Rendeveaux but not on cv. Brigadier. Trehalose was detected in higher amounts in cv. Rendeveaux than in cv. Brigadier, andthe amounts were higher in conidia produced at high a_w. A significant amount of endogenous solutes was detected in the washing liquid used for the separation of the conidia. This revised version was published online in June 2006 with corrections to the Cover Date.     

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