Quantitative Analysis of the Modes of Growth Inhibition by Weak Organic Acids in Saccharomyces cerevisiae

Weak organic acids are naturally occurring compounds that are commercially used as preservatives in the food and beverage industries. They extend the shelf life of food products by inhibiting microbial growth. There are a number of theories that explain the antifungal properties of these weak acids, but the exact mechanism is still unknown. We set out to quantitatively determine the contributions of various mechanisms of antifungal activity of these weak acids, as well as the mechanisms that yeast uses to counteract their effects. We analyzed the effects of four weak organic acids differing in lipophilicity (sorbic, benzoic, propionic, and acetic acids) on growth and intracellular pH (pH_i) in Saccharomyces cerevisiae. Although lipophilicity of the acids correlated with the rate of acidification of the cytosol, our data confirmed that not initial acidification, but rather the cell''s ability to restore pH_i, was a determinant for growth inhibition. This pH_i recovery in turn depended on the nature of the organic anion. We identified long-term acidification as the major cause of growth inhibition under acetic acid stress. Restoration of pH_i, and consequently growth rate, in the presence of this weak acid required the full activity of the plasma membrane ATPase Pma1p. Surprisingly, the proposed anion export pump Pdr12p was shown to play an important role in the ability of yeast cells to restore the pH_i upon lipophilic (sorbic and benzoic) acid stress, probably through a charge interaction of anion and proton transport.     

Evidence that sorbic acid does not inhibit yeast as a classic 'weak acid preservative'

Weak-acid preservatives are widely used to maintain microbial stability in foods and beverages. Classical weak-acid theory proposes that undissociated acid molecules pass through the plasma membrane, dissociate in the neutral pH of the cytoplasm, release protons and inhibit growth through acidification of the cytoplasm. Inhibitory concentrations of sorbic acid are shown to liberate fewer protons than other weak-acid preservatives. Sorbic acid shows similar inhibition to other six-carbon acids, alcohols and aldehydes, the latter being unable to act as weak acids. A survey of 22 yeasts showed high correlation between sorbate resistance and ethanol tolerance. Inhibition by short-chain acids or alcohols showed strong correlation with lipophilicity. It is proposed that sorbic acid acts as a membrane-active substance rather than as a weak-acid preservative.     

Caffeine induces macroautophagy and confers a cytocidal effect on food spoilage yeast in combination with benzoic acid

Weak organic acids are an important class of food preservatives that are particularly efficacious towards yeast and fungal spoilage. While acids with small aliphatic chains appear to function by acidification of the cytosol and are required at high concentrations to inhibit growth, more hydrophobic organic acids such as sorbic and benzoic acid have been suggested to function by perturbing membrane dynamics and are growth-inhibitory at much lower concentrations. We previously demonstrated that benzoic acid has selective effects on membrane trafficking in Saccharomyces cerevisiae. Benzoic acid selectively blocks macroautophagy in S. cerevisiae while acetic acid does not, and sorbic acid does so to a lesser extent. Indeed, while both benzoic acid and nitrogen starvation are cytostatic when assayed separately, the combination of these treatments is cytocidal, because macroautophagy is essential for survival during nitrogen starvation. In this report, we demonstrate that Zygosaccharomyces bailii, a food spoilage yeast with relatively high resistance to weak acid stress, also exhibits a cytocidal response to the combination of benzoic acid and nitrogen starvation. In addition, we show that nitrogen starvation can be replaced by caffeine supplementation. Caffeine induces a starvation response that includes the induction of macroautophagy, and the combination of caffeine and benzoic acid is cytocidal, as predicted from the nitrogen starvation data.     

Multiple modes of inhibition of spore germination and outgrowth by reduced pH and sorbate

Germination and outgrowth of three strains of Clostridium botulinum in PYEG medium were measured by phase contrast microscopy. Reduction in pH from 7 to 5·5 completely inhibited germination of strain 12885A, reduced the extent of germination of strain 62A and had no effect on the extent of germination of strain 53B. At pH 5·5, 225 mg/1 of undissociated sorbic acid had no effect on the germination of strain 53B, while at pH 6·5, 225 mg/1 of undissociated sorbic acid completely inhibited germination of strains 62A and 12885A. Outgrowth of germinated spores of strains 62A and 53B was not inhibited at pH 5·5, but the addition of sorbate (225 mg/1 undissociated sorbic acid) completely inhibited outgrowth. Sorbate inhibited germination of Cl. botulinum and Bacillus cereus spores triggered to germinate by amino acids. Inhibition occurred after germinant binding, as measured by commitment to germinate.     

Individual and Combined Effects of pH and Lactic Acid Concentration on Listeria innocua Inactivation: Development of a Predictive Model and Assessment of Experimental Variability

In food technology, organic acids (e.g., lactic acid, acetic acid, and citric acid) are popular preservatives. The purpose of this study was to separate the individual effects of the influencing factors pH and undissociated lactic acid on Listeria innocua inactivation. Therefore, the inactivation process was investigated under controlled, initial conditions of pH (pH₀) and undissociated lactic acid ([LaH]₀). The resulting inactivation curves consisted of a (sometimes negligible) shoulder period followed by a descent phase. In a few cases, a tailing phase was observed. Depending on the conditions, the descent phase contained one or two log-linear parts or had a convex or concave shape. In addition, the inactivation process was characterized by a certain variability, dependent on the severity of the conditions. Furthermore, in the neighborhood of the growth/no growth interface sometimes contradictory observations occurred. Overall, the individual effects of the influencing factors pH and undissociated lactic acid could clearly be distinguished and were also apparent based on fluorescence microscopy. Appropriate model types were developed and enabled prediction of which conditions of pH₀ and [LaH]₀ are necessary to obtain a predetermined inactivation (number of decimal reductions) within a predetermined time range.     

Influence of Medium Buffering Capacity on Inhibition of Saccharomyces cerevisiae Growth by Acetic and Lactic Acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (ΔpH), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

Effect of external pH on acidification and excretion of ethanol intermediates byCandida utilis

External pH affects the acidification induced by ethanol. The apparent specific rate of acidification depends on the dissociation properties and production of carbon dioxide, acetic and lactic acid which are intermediates of ethanol oxidation. The organic acids are transported by Nernst — Einstein diffusion. We designed and identified a new and simple mathematical model that allows us to describe the effect of external pH on nonstationary transport of dissociated intermediates of ethanol oxidation.     

Unstructured model for free and immobilized cell culture without pH control of Bifidobacterium animalis subsp. lactis Bb 12—Inhibitory effect of the undissociated organic acids

A model was developed to describe growth and organic acids production of Bifidobacterium animalis growing without pH control in free and immobilized cell culture. The Verlhust model was considered for growth, and to account for the inhibition observed at acidic pH, the Luedeking–Piret production model was modified by introducing an additional term involving the undissociated form of the organic acids, acetic and lactic acids, the main inhibitory species. To describe the relationship between pH and both the dissociated and the undissociated forms of organic acids, the Henderson–Hasselbach equation was considered. The model was found to satisfactory describe experimental growth and production data recorded during free and immobilized cell cultures. The part of each acid produced can be deduced from the calculated production data, since a constant lactic to acetic acid mass ratio was found, 1.29 and 1.66 during free and immobilized cell cultures. Owing to the acidic pH values recorded, 4.43 at lowest, higher amounts of undissociated acetic acid were produced, leading to a higher inhibitory effect of this acid if compared to lactic acid.     

Multiple modes of inhibition of spore germination and outgrowth by reduced pH and sorbate

Germination and outgrowth of three strains of Clostridium botulinum in PYEG medium were measured by phase contrast microscopy. Reduction in pH from 7 to 5.5 completely inhibited germination of strain 12885A, reduced the extent of germination of strain 62A and had no effect on the extent of germination of strain 53B. At pH 5.5, 225 mg/l of undissociated sorbic acid had no effect on the germination of strain 53B, while at pH 6.5, 225 mg/l of undissociated sorbic acid completely inhibited germination of strains 62A and 12885A. Outgrowth of germinated spores of strains 62A and 53B was not inhibited at pH 5.5, but the addition of sorbate (225 mg/l undissociated sorbic acid) completely inhibited outgrowth. Sorbate inhibited germination of Cl. botulinum and Bacillus cereus spores triggered to germinate by amino acids. Inhibition occurred after germinant binding, as measured by commitment to germinate.     

Influence of medium buffering capacity on inhibition of Saccharomyces cerevisiae growth by acetic and lactic acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (Delta(pH)), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

Inhibition of type A and type B (proteolytic) Clostridium botulinum by sorbic acid.

The effect of sorbic acid in the pH range 4.9 to 7.0 on the probability P of growth of a single vegetative bacterium of proteolytic strains of Clostridium botulinum has been determined by comparison of the most probable number count of the bacteria in media at pH 4.9 to 7.0 containing a series of concentrations of potassium sorbate and in a nutrient medium at pH 6.8 to 7.0. The media were maintained under strictly anaerobic conditions at a redox potential equivalent to lower than -350 mV at pH 7. In medium adjusted to the required pH with HCl, P for strain ZK3 (type A) at pH 5.1 or 5.5 after 2 days at 30 degrees C was similar to that at pH 6.8 to 7.0 but was slightly lower at pH 4.9. Potassium sorbate inhibited growth, the inhibition being a function of the concentration of undissociated sorbic acid. A calculated undissociated sorbic acid concentration of 156 mg/liter delayed growth of strain ZK3 (type A) but did not result in a significant decrease in P after an incubation time of 14 days. Higher concentrations of undissociated sorbic acid caused longer delays before maximum most probable number counts developed, and a calculated undissociated sorbic acid concentration of 282 mg/liter decreased log P for strain ZK3 after an incubation time of 14 days by a factor of 5.5 to 7.5. Four additional type A strains and five type B strains were inhibited to an extent comparable to inhibition of strain ZK3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of type A and type B (proteolytic) Clostridium botulinum by sorbic acid

The effect of sorbic acid in the pH range 4.9 to 7.0 on the probability P of growth of a single vegetative bacterium of proteolytic strains of Clostridium botulinum has been determined by comparison of the most probable number count of the bacteria in media at pH 4.9 to 7.0 containing a series of concentrations of potassium sorbate and in a nutrient medium at pH 6.8 to 7.0. The media were maintained under strictly anaerobic conditions at a redox potential equivalent to lower than -350 mV at pH 7. In medium adjusted to the required pH with HCl, P for strain ZK3 (type A) at pH 5.1 or 5.5 after 2 days at 30 degrees C was similar to that at pH 6.8 to 7.0 but was slightly lower at pH 4.9. Potassium sorbate inhibited growth, the inhibition being a function of the concentration of undissociated sorbic acid. A calculated undissociated sorbic acid concentration of 156 mg/liter delayed growth of strain ZK3 (type A) but did not result in a significant decrease in P after an incubation time of 14 days. Higher concentrations of undissociated sorbic acid caused longer delays before maximum most probable number counts developed, and a calculated undissociated sorbic acid concentration of 282 mg/liter decreased log P for strain ZK3 after an incubation time of 14 days by a factor of 5.5 to 7.5. Four additional type A strains and five type B strains were inhibited to an extent comparable to inhibition of strain ZK3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat resistance of Paenibacillus polymyxa in relation to pH and acidulants

The efficacy of different organic acids in decreasing the heat resistance of Paenibacillus polymyxa spores was assessed. The relationship between concentration of the undissociated form of different organic acids and decrease in heat resistance was also investigated. The heat resistance of P. polymyxa spores was tested in distilled water at 85, 90 and 95 degrees C, at pH4 and in the presence of 50, 100 and 200 mmol l(-1) of the undissociated form of lactic, citric or acetic acid and sodium citrate or acetate. The undissociated form of organic acids was responsible for increasing the heat sensitivity of spores. The most effective acid was lactic acid. The D values of the spores decreased rapidly (between 74 and 43%) in the presence of 50 mmol l(-1) of the undissociated form of organic acid, and increasing concentrations of these forms affected the heat resistance of spores less than proportionally. The heat resistance of the spores in milk was approximately threefold lower than in distilled water. This work has shown that the undissociated fraction of organic acids increases, albeit non-linearly, the sensitivity of spores to heat, even in complex substrates such as milk. By knowing the amount of organic acids added to a given substrate, their dissociation constants and the final pH, it could be possible to estimate the concentration of undissociated forms and the corresponding increase in lethality of heat treatments. This would help the food industry to maximize the lethality achieved by heat processes and/or safely reduce the heat treatments already in use.     

Inhibition of enterobacteria and Listeria growth by lactic, acetic and formic acids

Minimum inhibitory concentrations (MIC) of undissociated lactic, acetic and formic acids were evaluated for 23 strains of enterobacteria and two of Listeria monocytogenes. The evaluation was performed aerobically and anaerobically in a liquid test system at pH intervals of between 4.2 and 5.4. Growth of the enterobacteria was inhibited at 2–11 mmol 1⁻¹, 0.5–14 mmol 1⁻¹ and 0.1–1.5 mmol 1⁻¹ of undissociated lactic, acetic and formic acids, respectively. The MIC value was slightly lower with anaerobic conditions compared with aerobic conditions. The influence of protons on the inhibition was observed for acetic acid at the low pH values. Undissociated lactic acid was 2 to 5 times more efficient in inhibiting L. monocytogenes than enterobacteria. Acetic acid had a similar inhibitory action on L. monocytogenes compared with enterobacteria. Inorganic acid (HCl) inhibited most enterobacteria at pH 4.0; some strains, however, were able to initiate growth to pH 3.8. The results indicate that the values of undissociated acid which occur in a silage of pH 4.1–4.5 are about 10–100 times higher than required in order to protect the forage from the growth of enterobacteria and L. monocytogenes.     

Electrophysiological studies of salty taste modification by organic acids in the labellar taste cell of the blowfly.

Using the labellar salt receptor cells of the blowfly, Phormia regina, we electrophysiologically showed that the response to NaCl and KCl aqueous solutions was enhanced and depressed by acetic, succinic and citric acids. The organic acid concentrations at which the most enhanced salt response (MESR) was obtained were found to be different: 0.05-1 mM citric acid, 0.5-2 mM succinic acid and 5-50 mM acetic acid. Moreover, the degree of the salt response was not always dependent on the pH values of the stimulating solutions. The salt response was also enhanced by HCl (pH 3.5-3.0) only when the NaCl concentration was greater than the threshold, indicating that the salty taste would be enhanced by the comparatively lower concentrations of hydrogen ions. Another explanation for the enhancement is that the salty taste may also be enhanced by undissociated molecules of the organic acids, because the MESRs were obtained at the pH values lower than the pKa(1) or pKa(2) values of these organic acids. On the other hand, the salty taste could be depressed by both the lower pH range (pH 2.5-2.0) and the dissociated organic anions from organic acid molecules with at least two carboxyl groups.     

The effect of food preservatives on pH homeostasis in Escherichia coli

The effects of cinnamic, propionic, benzoic and sorbic acids on the growth and intracellular pH of Escherichia coli were investigated. The data suggest that the potency of weak acids as food preservatives is related to their capacity to reduce specifically the intracellular pH. The data also suggest that although both the undissociated forms of the acid cause the intracellular pH to fall, growth inhibition is due predominantly to the undissociated acid.     

Changes in Membrane Lipids of Roots Associated with Changes in Permeability: I. EFFECTS OF UNDISSOCIATED ORGANIC ACIDS

Previous work has shown that undissociated forms of organic acids, such as formic, acetic, and propionic acids, increase the permeability of barley roots to ions. The work here was undertaken to test whether these undissociated acids affect the lipids from the root membranes in such a way as to account for the permeability increase. Relative amounts of the principal fatty acids from barley root membranes were measured as a function of organic acid concentration, pH, and time of treatment of barley roots under conditions similar to those of the previous studies.     

Listeria contamination at a poultry processing plant

Escherichia coli grown in broth initially at pH 5.0 (pH 5.0-grown organisms) survived exposure to inorganic acid or to acid pH plus organic acid which prevented subsequent growth by pH 7.0-grown organisms. This resistance of pH 5.0-grown organisms to organic acids was observed at acid pH with lactic, propionic, benzoic, sorbic, trans-cinnamic and acetic acids. Such resistance might allow acid-habituated organisms to survive in acid foods or at body sites such as the urinary tract where organic acids are present at acid pH.     

Resistance of some wine spoilage yeasts to combinations of ethanol and acids present in wine

The most important factors affecting microbial growth in an alcoholic beverage are the ethanol content and the low pH. The effectiveness of different organic acids in conjunction with ethanol concentration in controlling growth of yeasts was determined for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Brettanomyces lambicus, Pichia anomala, Zygosaccharomyces bailii, Saccharomycodes ludwigii and Kluyveromyces thermotolerans in malt extract broth (MEB). The results are summarized as undissociated concentrations of different acids required to inhibit growth of yeasts in MEB containing 10% (v/v) ethanol. About half the amount of undissociated malic or tartaric acid is necessary for inhibition of the yeasts, compared with acetic and lactic acid but the concentrations of acid necessary to inhibit growth were generally very high and unrealistic in wines for controlling growth of most of the yeasts tested. All the yeasts tested were able to grow in acidified non- or low alcoholic beverages but, at higher ethanol concentrations, Sacch. cerevisiae and Zygosacch. bailii have the greatest spoilage potential.     

Measurement of the Effects of Acetic Acid and Extracellular pH on Intracellular pH of Nonfermenting, Individual Saccharomyces cerevisiae Cells by Fluorescence Microscopy

The effects of acetic acid and extracellular pH (pH_ex) on the intracellular pH (pH_i) of nonfermenting, individual Saccharomyces cerevisiae cells were studied by using a new experimental setup comprising a fluorescence microscope and a perfusion system. S. cerevisiae cells grown in brewer’s wort to the stationary phase were stained with fluorescein diacetate and transferred to a perfusion chamber. The extracellular concentration of undissociated acetic acid at various pH_ex values was controlled by perfusion with 2 g of total acetic acid per liter at pH_ex 3.5, 4.5, 5.6, and 6.5 through the chamber by using a high-precision pump. The pH_i of individual S. cerevisiae cells during perfusion was measured by fluorescence microscopy and ratio imaging. Potential artifacts, such as fading and efflux of fluorescein, could be neglected within the experimental time used. At pH_ex 6.5, the pH_i of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.035 g/liter, whereas at pH_ex 3.5, 4.5, and 5.6, the pH_i of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.10 g/liter. At concentrations of undissociated acetic acid of more than 0.10 g/liter, the pH_i remained constant. The decreases in pH_i were dependent on the pH_ex; i.e., the decreases in pH_i at pH_ex 5.6 and 6.5 were significantly smaller than the decreases in pH_i at pH_ex 3.5 and 4.5.