Interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii

The interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii were investigated. Growth rates in media supplemented with glucose, sucrose or NaCl to a _w 0.93 were more rapid than in unsupplemented media ( a _w 0.99). Although growth in unsupplemented medium was lower at 35°C, incubation at 21°C or 35°C had little effect on growth in media supplemented with glucose and sucrose. The addition of 300 μg potassium sorbate/ml to media resulted in reduced growth rates, particularly at 35°C. Heat resistance of Z. rouxii was substantially greater in cultures previously incubated at 35°C than in cultures incubated at 21° in media both with and without 300 μg potassium sorbate/ml. Zygosaccharomyces rouxii was tolerant to freezing at - 18°C for up to 120 d in all test media supplemented with glucose, sucrose or NaCl. The addition of 300 μg potassium sorbate/ml to sucrose-supplemented media resulted in increased resistance to freezing in cultures previously incubated at 21°C. Sensitivity to freezing increased when cultures were incubated at 21°C in media not supplemented with solutes. Glucose and sucrose provided the best protection against inactivation by heating and freezing, regardless of the presence of potassium sorbate in growth media.     

Effects of potassium sorbate on growth patterns, morphology, and heat resistance of Zygosaccharomyces rouxii at reduced water activity.

A study was made of the effects of potassium sorbate on growth, morphology, and heat sensitivity of an osmotolerant yeast, Zygosaccharomyces rouxii, grown in media (water activity (aw) 0.93) supplemented with glucose and sucrose. Growth patterns of Z. rouxii in YM broth supplemented with glucose (YMBG) and sucrose (YMBS) were similar, although increased potassium sorbate concentration in both media resulted in decreased growth rates. Growth in YMBS containing potassium sorbate was not as prolific as that in YMBG containing potassium sorbate. Inhibition of growth was indicated by decreased absorbance (at 600 nm) of cells grown in YMBS and in YMBG and YMBS supplemented with potassium sorbate at 600 or 1000 micrograms/mL. Slight decreases in cell size and alteration of cellular morphology were associated with increased potassium sorbate concentration. Plasmolysis increased as potassium sorbate concentration was elevated in YMBS but not in YMBG. Tolerance of Z. rouxii to potassium sorbate was enhanced by previous adaptation of cells in media with elevated potassium sorbate concentrations. Heat resistance of cells unadapted to potassium sorbate showed little or no increase regardless of culture age, but increased substantially in cells grown in media containing potassium sorbate, particularly YMBS.     

Combined effects of pH and sugar on growth rate of Zygosaccharomyces rouxii, a bakery product spoilage yeast.

The effects of citric acid-modified pH (pH 2.5, 2.75, 3, 3.5, 4, 4.5, 5, and 5.5) and a 30% glucose-70% sucrose mixture (300, 400, 500, 600, 700, 800, 875, and 900 g/liter) on an osmophilic yeast, Zygosaccharomyces rouxii, were determined by using synthetic medium. One hundred experiments were carried out; 50-ml culture flasks were inoculated with 10(3) CFU ml(-1) by using a collection strain and a wild-type strain cocktail. The biomass was measured by counting cell colonies, and growth curves were fitted by using a Baranyi equation. The growth rate decreased linearly with sugar concentration, while the effect of pH was nonlinear. Indeed, the optimal pH range was found to be pH 3.5 to 5, and pH 2.5 resulted in a 30% reduction in the growth rate. Finally, we evaluated the performance of two nonlinear predictive models developed previously to describe bacterial contamination. Equations derived from the Rosso and Ratkowsky models gave similar results; however, the model that included dimensionless terms based on the Ratkowsky equation was preferred because it contained fewer estimated parameters and also because biological interpretation of the results was easier.     

Salt tolerance and glycerol accumulation of a respiration-deficient mutant isolated from the petite-negative, salt-tolerant yeast Zygosaccharomyces rouxii

A respiration-deficient (RD) mutant was isolated from the petite-negative, salt-tolerant yeast Zygosaccharomyces rouxii. One strain among sixteen glycerol-non-utilizing mutants exhibited vigorous liberation of CO2 but no uptake of O2. Furthermore, this strain lacked cytochrome aa3 and had a reduced level of cytochrome b. The few mitochondria found in cells of this strain contained few or no cristae. Salt tolerance and intracellular accumulation of glycerol by the RD strain were almost equal to that of the wild-type strain in media containing NaCl up to 2.5 M. In media with more than 3 M NaCl, the growth of the RD mutant was retarded and the intracellular accumulation of glycerol was depressed in spite of ample production.     

Effect of pH, temperature, and potassium sorbate on amino acid uptake in Salmonella typhimurium 7136.

The effect of sorbate on L-serine and L-histidine uptake in Salmonella typhimurium was studied at various pH levels, temperatures, and amino acid and sorbate concentrations. Low pH had an apparent synergistic effect on amino acid uptake inhibition caused by sorbate. The relationship between sorbate concentration and the amount of amino acid uptake inhibition was not linear. Compared with L-histidine, L-serine uptake was more sensitive to changes in pH, temperature, and sorbate concentration. Various degrees of amino acid uptake inhibition by sorbate may be related to differences between amino acid transport systems. The results of this study suggest that sorbate acts as a noncompetitive inhibitor of amino acid uptake in S. typhimurium.     

Inhibition of Bacillus spores by combinations of heat,potassium sorbate,NaCl and pH

Viability of spores of Bacillus cereus was totally inhibited at 85°C over 30 min by adding 0.4% (w/v) potassium sorbate with 6% (w/v) NaCl at pH 4.5. Viability of B. stearothermophilus spores was totally inhibited at 95°C for 45 min in a buffer at pH 4.2 containing 0.8% (w/v) potassium sorbate and 8% (w/v) NaCl. A synergistic inhibitory effect was demonstrated in some of the combinations. The inhibition may be due to interference with the heat-resistance apparatus of the spores.O.B. Oloyede was and J. Scholefield is with the Department of Bioscience & Biotechnology, Food Science Division, University of Strathclyde, Glasgow G1 1SD, UK. O.B. Oloyede is now with the Department of Biochemistry, University of Ilorin, Ilorin, Nigeria.     

Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii.

A novel ketoreductase isolated from Zygosaccharomyces rouxii catalyzes the asymmetric reduction of selected ketone substrates of commercial importance. The 37.8-kDa ketoreductase was purified more than 300-fold to > 95% homogeneity from whole cells with a 30% activity yield. The ketoreductase functions as a monomer with an apparent Km for 3,4-methylenedioxyphenyl acetone of 2.9 mM and a Km for NADPH of 23.5 microM. The enzyme is able to effectively reduce alpha-ketolactones, alpha-ketolactams, and diketones. Inhibition is observed in the presence of diethyl pyrocarbonate, suggesting that a histidine is crucial for catalysis. The 1.0-kb ketoreductase gene was cloned and sequenced from a Z. rouxii cDNA library using a degenerate primer to the N-terminal sequence of the purified protein. Furthermore, it was expressed in both Escherichia coli and Pichia pastoris and shown to be active. Substrate specificity, lack of a catalytic metal, and extent of protein sequence identity to known reductases suggests that the enzyme falls into the carbonyl reductase enzyme class.     

Effect of growth temperature,surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectants

The goal of this study was to investigate the effect of the environmental conditions such as the temperature change, incubation time and surface type on the resistance of Staphylococcus aureus biofilms to disinfectants. The antibiofilm assays were performed against biofilms grown at 20 °C, 30 °C and 37 °C, on the stainless steel and polycarbonate, during 24 and 48 h. The involvement of the biofilm matrix and the bacterial membrane fluidity in the resistance of sessile cells were investigated. Our results show that the efficiency of disinfectants was dependent on the growth temperature, the surface type and the disinfectant product. The increase of growth temperature from 20 °C to 37 °C, with an incubation time of 24 h, increased the resistance of biofilms to cationic antimicrobials. This change of growth temperature did not affect the major content of the biofilm matrix, but it decreased the membrane fluidity of sessile cells through the increase of the anteiso-C19 relative amount. The increase of the biofilm resistance to disinfectants, with the rise of the incubation time, was dependent on both growth temperature and disinfectant product. The increase of the biofilm age also promoted increases in the matrix production and the membrane fluidity of sessile cells. The resistance of S. aureus biofilm seems to depend on the environment of the biofilm formation and involves both extracellular matrix and membrane fluidity of sessile cells. Our study represents the first report describing the impact of environmental conditions on the matrix production, sessile cells membrane fluidity and resistance of S. aureus biofilms to disinfectants.     

Prevention of surface mold growth on Italian dry sausage by natamycin and potassium sorbate.

Inhibition of uncontrolled mold growth on three types of raw cured Italian dry salami was studied under commercial production conditions. Salami were dipped or sprayed with natamycin (pimaricin) or were given a combined organic acid-plus-potassium sorbate treatment. Acetic and citric acids potentiated the inhibitory effects of potassium sorbate significantly, but lactic and succinic acids showed little or no effect. Treatment of salami by dipping in 2.5% (wt/vol) potassium sorbate or 2,000 ppm (mg/liter) of pimaricin did not successfully prevent the growth of surface molds. At 10% potassium sorbate on all types of salami and at 2.5% sorbate on Casalingo salami, visual inhibition of mold growth was observed, but numbers of viable fungi on all salami types treated with 2.5% sorbate were not significantly (95% confidence) different from numbers found in the untreated controls. Pimaricin spray (2 X 1,000 ppm) was as good as or slightly better than 2.5% potassium sorbate, but greater concentrations of each were required to satisfactorily inhibit surface mold growth during the 25- to 50-day ripening period.     

Interactive effects of cryosolvents, ionic and macromolecular solutes on protein structures and functions

Surprisingly, cryosolvents may mimic the effects of ionic solutes on the structures and functions of macromolecular assemblages, showing additive or opposite effects depending on the respective concentrations. These interactive effects are hard to analyze precisely because they may result from so many possible contributions. However, studies on model systems clearly show the nature of the interactive effects and bring about useful information concerning the mechanism of action of cryosolvents and ions and the response of enzyme systems. Such results suggest new studies on the interaction of biopolymers and water and their possible impact on the cryobehavior of highly organized and living systems.     

Effects of natamycin and potassium sorbate on growth and aflatoxin production in olives

Aspergillus flavus NRRL 6555 was inoculated onto whole olives and olive paste samples containing variable amounts of either natamycin or potassium sorbate and incubated at 15 degrees, 25 degrees, and 35 degrees C for 7, 14 and 21 days for whole olives and at 15 degrees and 25 degrees C for 8 and 16 days for olive pastes. The initiation time of growth was parallel to the concentrations of either preservatives applied. However, at 15 degrees C, natamycin at 160 and 320 micrograms/g (ppm) completely inhibited the growth of mold on whole olives for 21 days and olive paste for 7 and 15 days, respectively. All levels of potassium sorbate inhibited mold growth at 15 degrees C, but at 25 degrees C, 6000 micrograms/g (ppm) only, delayed growth for 15 days. The extent of growth at the end of the incubation periods was parallel to the temperatures of incubation. The analyses for aflatoxin B1 production in all samples at all levels of preservatives and control were negative.     

Combining heat treatment and subsequent incubation temperature to prevent growth from spores of non-proteolytic Clostridium botulinum

Refrigerated processed foods of extended durability rely on a mild heat treatment combined with refrigerated storage to ensure microbiological safety and quality. The principal microbiological safety risk in foods of this type is non-proteolytic Clostridium botulinum. In this article the combined effect of mild heat treatment and refrigerated storage on the time to growth and probability of growth from spores of non-proteolytic Cl. botulinum is described. Spores of non-proteolytic Cl. botulinum (two strains each of type B, E and F) were heated at 90°C for between 0 and 60 min and subsequently incubated at 5°, 10° or 30°C in PYGS broth in the presence or absence of lysozyme. The number of spores that resulted in turbidity depended on the combination of heat treatment, incubation time and incubation temperature they received. Heating at 90°C for 1 or more min ensured a 10⁶ reduction when spores were subsequently incubated at 5°C for up to 23 weeks. Heating at 90°C for 60 min ensured a 10⁶ reduction over 23 weeks when subsequent incubation was at 10°C in the presence of added lysozyme. The same treatment did not reduce the spore population by 10⁶ when subsequent incubation was at 30°C.     

Prevention of surface mold growth on Italian dry sausage by natamycin and potassium sorbate

Inhibition of uncontrolled mold growth on three types of raw cured Italian dry salami was studied under commercial production conditions. Salami were dipped or sprayed with natamycin (pimaricin) or were given a combined organic acid-plus-potassium sorbate treatment. Acetic and citric acids potentiated the inhibitory effects of potassium sorbate significantly, but lactic and succinic acids showed little or no effect. Treatment of salami by dipping in 2.5% (wt/vol) potassium sorbate or 2,000 ppm (mg/liter) of pimaricin did not successfully prevent the growth of surface molds. At 10% potassium sorbate on all types of salami and at 2.5% sorbate on Casalingo salami, visual inhibition of mold growth was observed, but numbers of viable fungi on all salami types treated with 2.5% sorbate were not significantly (95% confidence) different from numbers found in the untreated controls. Pimaricin spray (2 X 1,000 ppm) was as good as or slightly better than 2.5% potassium sorbate, but greater concentrations of each were required to satisfactorily inhibit surface mold growth during the 25- to 50-day ripening period.     

Latent effects of egg incubation temperature on growth in the lizard Anolis carolinensis

Varied egg incubation temperatures can result in immediate effects on the phenotype of reptiles, and also latent effects that can augment or contradict effects evident at egg hatching. I examined the effects of incubation temperature on embryonic development, hatching morphology, and subsequent growth in multiple populations of the lizard Anolis carolinensis. Eggs from wild-caught females in four populations were incubated at up to three temperatures, 23.5, 27, and 30 degrees C. Measures of body size were collected immediately after hatching and weekly thereafter, while juveniles were maintained in a common laboratory environment for 8 weeks. Cooler incubation temperatures resulted in longer incubation periods but did not affect conversion of egg mass to hatchling mass. Incubation temperature did not affect hatchling mass or snout vent length (SVL), but did affect subsequent growth in both mass and SVL, which varied by population. Cooler incubation temperatures generally resulted in greater overall growth over 8 weeks of housing all juveniles in a common environment. In A. carolinensis, egg incubation temperature had latent effects on juvenile growth despite the absence of any detected immediate effects on hatchling phenotype. Therefore, the total impact and evolutionary importance of developmental environment should not be assessed or assumed based solely on the phenotype of reptiles at birth or hatching.     

A note on the effect of dilution and temperature on the bactericidal activity of potassium sorbate

O ragui , J.I. M ara , D.D.1984. A note on a modified membrane-Bovis agar for the enumeration of Streptococcus bovis by membrane filtration. Journal of Applied Bacteriology 56 , 179–181.
A modified membrane-Bovis agar, containing a reduced quantity of sodium azide, for the isolation and enumeration of Streptococcus bovis is described and evaluated. Higher counts, with larger colonies, were obtained from water and sewage samples with this medium than with the original formulation.     

Effect of threonine, cystathionine, and the branched-chain amino acids on the metabolism of Zygosaccharomyces rouxii*

Zygosaccharomyces rouxii is an important yeast in the formation of flavor in soy sauce. In this study, we investigated the separate effects of exogenous threonine, cystathionine, and the branched-chain amino acids on the metabolism of Z. rouxii. The addition of these amino acids had significant effects on both Z. rouxii growth and glycerol and higher alcohol production. It also seemed that Z. rouxii displayed the Crabtree effect, which was independent of the added amino acids. Furthermore, we investigated the regulation of the metabolism of alpha-ketobutyrate, which is a key-intermediate in Z. rouxii amino acid metabolism. Threonine and cystathionine were introduced separately to stimulate the formation rate of alpha-ketobutyrate and the branched-chain amino acids to inhibit its conversion rate. Enzyme activities showed that these amino acids had a significant effect on the formation and conversion rate of alpha-ketobutyrate but that the alpha-ketobutyrate pool size in Z. rouxii was in balance all the time. The latter was confirmed by the absence of alpha-ketobutyrate accumulation.     

In vitro effects of water activity, temperature and solutes on the growth rate of P. italicum Wehmer and P. digitatum Sacc

AIMS: To evaluate the effect of water activity (a(w) 0.98-0.89, adjusted with glycerol, sorbitol, glucose, or NaCl) and temperature (5-25 degrees C) on the lag phase and radial growth rate (mm day(-1)) of the important citrus spoilage fungi, such as Penicillium italicum and Penicillium digitatum grown in potato dextrose agar (PDA) medium. To select, among models based on the use of different solutes, a model fitting accurately the growth of these species in relation to a(w) and temperature. METHODS AND RESULTS: Extensive data analyses showed for both Penicillium species a highly significant effect of a(w), temperature, solutes and their interactions on radial growth rate (P < 0.0001). Radial growth rate was inhibited and the lag phase (i.e. the time required for growth) lengthened as the a(w) of the medium decreased. NaCl appeared to causes the greatest stress on growth when compared with other nonionic solutes. Penicillium italicum stopped growing at 0.96 a(w) and P. digitatum at 0.93 a(w). Under the dry conditions where growth was observed, P. italicum grew faster than P. digitatum at low temperature and P. digitatum remained more active at ambient temperature. Multiple regression analysis applied to the square roots of the growth rates observed in the presence of each solute showed that both the 'glycerol model' and the 'sorbitol model' yielded a good prediction of P. italicum growth and the 'sorbitol model' gave an accurate fit for P. digitatum growth, offering high-quality prediction within the experimental limits described. CONCLUSIONS: Mathematical models describing and predicting, as a function of a(w) and temperature, the square root of the radial growth rate of the agents responsible for blue and green decays are important tools for understanding the behaviour of these fungi under natural conditions and for predicting citrus fruit spoilage. SIGNIFICANCE AND IMPACT OF THE STUDY: Implementation of these results should contribute towards a more rational control strategy against citrus spoilage fungi.     

The effects of solutes on the freezing properties of and hydration forces in lipid lamellar phases.

Quantitative deuterium nuclear magnetic resonance is used to study the freezing behavior of the water in phosphatidylcholine lamellar phases, and the effect upon it of dimethylsulfoxide (DMSO), sorbitol, sucrose, and trehalose. When sufficient solute is present, an isotropic phase of concentrated aqueous solution may coexist with the lamellar phase at freezing temperatures. We determine the composition of both unfrozen phases as a function of temperature by using the intensity of the calibrated free induction decay signal (FID). The presence of DMSO or sorbitol increases the hydration of the lamellar phase at all freezing temperatures studied, and the size of the increase in hydration is comparable to that expected from their purely osmotic effect. Sucrose and trehalose increase the hydration of the lamellar phase, but, at concentrations of several molal, the increase is less than that which their purely osmotic effect would be expected to produce. A possible explanation is that very high volume fractions of sucrose and trehalose disrupt the water structure and thus reduce the repulsive hydration interaction between membranes. Because of their osmotic effect, all of the solutes studied reduced the intramembrane mechanical stresses produced in lamellar phases by freezing. Sucrose and trehalose at high concentrations produce a greater reduction than do the other solutes.