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.     

Modelling the growth rate of Escherichia coli as a function of pH and lactic acid concentration.

The growth rate responses of Escherichia coli M23 (a nonpathogenic strain) to suboptimal pH and lactic acid concentration were determined. Growth rates were measured turbidimetrically at 20 degrees C in the range of pH 2.71 to 8.45. The total concentration of lactic acid was fixed at specific values, and the pH was varied by the addition of a strong acid (hydrochloric) or base (sodium hydroxide) to enable the determination of undissociated and dissociated lactic acid concentrations under each condition. In the absence of lactic acid, E. coli grew at pH 4.0 but not at pH 3.7 and was unable to grow in the presence of > or = 8.32 mM undissociated lactic acid. Growth rate was linearly related to hydrogen ion concentration in the absence of lactic acid. In the range 0 to 100 mM lactic acid, growth rate was also linearly related to undissociated lactic acid concentration. A mathematical model to describe these observations was developed based on a Bĕlehrádek-like model for the effects of water activity and temperature. This model was expanded to describe the effects of pH and lactic acid by the inclusion of novel terms for the inhibition due to the presence of hydrogen ions, undissociated lactic acid, and dissociated lactic acid species. Preliminary data obtained for 200 and 500 mM total lactic acid concentrations show that the response to very high lactic acid concentrations was less well described by the model. However, for 0 to 100 mM lactic acid, the model described well the qualitative and quantitative features of the response.     

Expanded models for the non-thermal inactivation of Listeria monocytogenes

Previously developed four-variable response surface models for describing the effects of temperature, pH/lactic acid, sodium chloride and sodium nitrite on the time to achieve a 4-log, non-thermal inactivation ( t _4D) of Listeria monocytogenes in aerobic, acidic environments were expanded to five-variable models that distinguish the effects of pH and acidulant concentration. A total of 18 new variable combinations were evaluated and the inactivation kinetics data appended onto a consolidation of two data sets from earlier studies. The consolidated data set, which included 315 inactivation curves representing 209 unique combinations of the five variables, was analysed by response surface analysis. The quadratic model without backward elimination regression was selected for further evaluation. Three additional quadratic models were generated using the concentrations of undissociated lactic and/or nitrous acids as variables in place of percentage lactic acid and sodium nitrite concentration. Comparison of predicted t _4D values against literature values for various food systems indicated that the models provide reasonable initial estimates of the inactivation of L. monocytogenes. The models based on the concentration of undissociated lactic and nitrous acids support the hypothesis that antimicrobial activity is associated with this form of the compounds. Evaluation of several examples suggests that these models may be useful for predicting the equivalent of the compounds'minimal inhibitory concentrations'for accelerating inactivation under various conditions.     

Modelling the Growth Limits (Growth/No Growth Interface) of Escherichia coli as a Function of Temperature, pH, Lactic Acid Concentration, and Water Activity

The form of a previously developed Bělehrádek type of growth rate model was used to develop a probability model for defining the growth/no growth interface as a function of temperature (10 to 37°C), pH (pH 2.8 to 6.9), lactic acid concentration (0 to 500 mM), and water activity (0.955 to 0.999; NaCl was used as the humectant). Escherichia coli was unable to grow in broth in which the undissociated lactic acid concentration exceeded 11 mM or, with two exceptions, at a pH of 3.9 or less with no lactic acid present. Under experimental conditions at which the pH and the undissociated acid concentrations were the major growth-limiting factors, the growth/no growth interface was essentially independent of temperature at temperatures ranging from 15 to 37°C. The interface between conditions that allowed growth and conditions at which growth did not occur was abrupt. The inhibitory effect of combinations of water activity and pH varied with temperature. Predictions of the model for the growth/no growth interface were consistent with 95% of the experimental data set.     

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.     

Limitation of growth and lactic acid production in batch and continuous cultures of Lactobacillus helveticus

Summary Continuous and batch cultures of Lactobacillus helveticus operated under different conditions were studied with respect to the limitation of growth and lactic acid production by increasing undissociated lactic acid and hydrogen ion concentrations, respectively. In a single-stage continuous culture without pH control a final pH of 3.8 and 65 mm undissociated lactic acid was obtained. In two-stage continuous cultures provided with different growth media and run at different pH values, 65–70 mm free acid was obtained in the second stage. Further batch-culture experiments showed growth limitation at 60–70 mm lactic acid. After growth ceased, production of lactate continued until a lactic acid concentration of about 100 mm was reached; obviously an uncoupling of growth and acid production had occurred. Examining the effect of different concentrations of either lactic acid or hydrochloric acid, added to growing batch cultures of L. helveticus, it was shown that the undissociated lactic acid concentration was responsible for growth limitation and lactic acid production in this organism, whereas the pH value had only an indirect effect.     

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.     

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.     

Measurement and prediction of the susceptibility of lager beer to spoilage by lactic acid bacteria

J.L. FERNANDEZ AND W.J SIMPSON. 1995. The ability of 14 strains of hop-resistant lactic acid bacteria ( Lactobacillus spp., Pediococcus spp.) to grow in 17 different lager beers was assessed using a biological challenge test. All beers were spoiled by at least one strain: all strains could grow in at least one beer. The sensitivity of different beers to spoilage by lactic acid bacteria varied. Spoilage potential could be described in the form of a 'Resistance to Spoilage Value' (RSV) based on the ability of the organisms to grow in each beer. Parameters which correlated to RSV included pH, beer colour and the content of free amino nitrogen, total soluble nitrogen, a range of individual amino acids, maltotriose and the undissociated forms of SO₂ and hop bitter acids. Predictive models based on the technique of partial least squares regression and constructed using values for undissociated SO₂ content, undissociated hop bitter acids content, polyphenol content, maltotriose content, free amino nitrogen content and a colour intensity component allowed RSV to be predicted.     

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.     

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.     

Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae IGC4072 grown in lactic acid medium transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoichiometry of 1:1. The accumulation ratio measured with propionate increased with decreasing pH from ca. 24-fold at pH 6.0 to ca. 1,400-fold at pH 3.0. The symport accepted the following monocarboxylates (Km values at 25 degrees C and pH 5.5): D-lactate (0.13 mM), L-lactate (0.13 mM), pyruvate (0.34 mM), propionate (0.09 mM), and acetate (0.05 mM), whereas apparently a different proton symport accepted formate (0.13 mM). The lactate system was inducible and was subject to glucose repression. Undissociated lactic acid entered the cells by simple diffusion. The permeability of the plasma membrane for undissociated lactic acid increased exponentially with pH, and the diffusion constant increased 40-fold when the pH was increased from 3.0 to 6.0.     

Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae IGC4072 grown in lactic acid medium transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoichiometry of 1:1. The accumulation ratio measured with propionate increased with decreasing pH from ca. 24-fold at pH 6.0 to ca. 1,400-fold at pH 3.0. The symport accepted the following monocarboxylates (Km values at 25 degrees C and pH 5.5): D-lactate (0.13 mM), L-lactate (0.13 mM), pyruvate (0.34 mM), propionate (0.09 mM), and acetate (0.05 mM), whereas apparently a different proton symport accepted formate (0.13 mM). The lactate system was inducible and was subject to glucose repression. Undissociated lactic acid entered the cells by simple diffusion. The permeability of the plasma membrane for undissociated lactic acid increased exponentially with pH, and the diffusion constant increased 40-fold when the pH was increased from 3.0 to 6.0.     

Influence of pH and lactic acid concentration on Clostridium tyrobutyricum during continuous growth in a pH-auxostat

The aim of this project was to establish the minimal inhibitory concentration (MIC) of lactic acid for growth of Clostridium tyrobutyricum. A pH-auxostat was used to maintain a constant pH and to allow continuous growth at the highest possible rates at fixed, but adjustable concentrations of lactate. By raising the concentration of lactic acid and keeping the pH constant, the growth rate was shown to decrease linearly with increasing lactic acid concentration. The p K _a of lactic acid, measured in the actual growth medium at 37°C, was 3.40 (±0.03). Based on this value, the MIC_undiss values for each pH were estimated. The MIC of total lactic acid (MIC_tot) ranged from 150 mmol l⁻¹ to 1510 mmol l⁻¹ at pH 4.6–6.25, respectively. The corresponding MIC values of undissociated lactic acid (MIC_undiss) ranged from 8.9 to 2.1 mmol l⁻¹ at the same pH values. These results emphasize the importance of a rapid pH decrease and an equally rapid initial lactic acid fermentation of the ensilage, in order to sufficiently suppress clostridial growth.     

A Combined Model To Predict the Functionality of the Bacteriocin-Producing Lactobacillus sakei Strain CTC 494

The use of bacteriocin-producing lactic acid bacteria for improved food fermentation processes seems promising. However, lack of fundamental knowledge about the functionality of bacteriocin-producing strains under food fermentation conditions hampers their industrial use. Predictive microbiology or a mathematical estimation of microbial behavior in food ecosystems may help to overcome this problem. In this study, a combined model was developed that was able to estimate, from a given initial situation of temperature, pH, and nutrient availability, the growth and self-inhibition dynamics of a bacteriocin-producing Lactobacillus sakei CTC 494 culture in (modified) MRS broth. Moreover, the drop in pH induced by lactic acid production and the bacteriocin activity toward Listeria as an indicator organism were modeled. Self-inhibition was due to the depletion of nutrients as well as to the production of lactic acid. Lactic acid production resulted in a pH drop, an accumulation of toxic undissociated lactic acid molecules, and a shift in the dissociation degree of the growth-inhibiting buffer components. The model was validated experimentally.     

A combined model to predict the functionality of the bacteriocin-producing Lactobacillus sakei strain CTC 494

The use of bacteriocin-producing lactic acid bacteria for improved food fermentation processes seems promising. However, lack of fundamental knowledge about the functionality of bacteriocin-producing strains under food fermentation conditions hampers their industrial use. Predictive microbiology or a mathematical estimation of microbial behavior in food ecosystems may help to overcome this problem. In this study, a combined model was developed that was able to estimate, from a given initial situation of temperature, pH, and nutrient availability, the growth and self-inhibition dynamics of a bacteriocin-producing Lactobacillus sakei CTC 494 culture in (modified) MRS broth. Moreover, the drop in pH induced by lactic acid production and the bacteriocin activity toward Listeria as an indicator organism were modeled. Self-inhibition was due to the depletion of nutrients as well as to the production of lactic acid. Lactic acid production resulted in a pH drop, an accumulation of toxic undissociated lactic acid molecules, and a shift in the dissociation degree of the growth-inhibiting buffer components. The model was validated experimentally.     

Individual cells of Saccharomyces cerevisiae and Zygosaccharomyces bailii exhibit different short-term intracellular pH responses to acetic acid

The effects of perfusion with 2.7 and 26 mM undissociated acetic acid in the absence or presence of glucose on short-term intracellular pH (pH(i)) changes in individual Saccharormyces cerevisiae and Zygosaccharomyces bailii cells were studied using fluorescence-ratio-imaging microscopy and a perfusion system. In the S. cerevisiae cells, perfusion with acetic acid induced strong short-term pH(i) responses, which were dependent on the undissociated acetic acid concentration and the presence of glucose in the perfusion solutions. In the Z. bailii cells, perfusion with acetic acid induced only very weak short-term pH(i) responses, which were neither dependent on the undissociated acetic acid concentration nor on the presence of glucose in the perfusion solutions. These results clearly show that Z. bailii is more resistant than S. cerevisiae to short-term pH(i) changes caused by acetic acid.     

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.     

Low- and high-affinity transport systems for citric acid in the yeast Candida utilis.

Citric acid-grown cells of the yeast Candida utilis induced two transport systems for citric acid, presumably a proton symport and a facilitated diffusion system for the charged and the undissociated forms of the acid, respectively. Both systems could be observed simultaneously when the transport was measured at 25 degrees C with labelled citric acid at pH 3.5 with the following kinetic parameters: for the low-affinity system, Vmax, 1.14 nmol of undissociated citric acid s-1 mg (dry weight) of cells-1, and Km, 0.59 mM undissociated acid; for the high-affinity system, Vmax, 0.38 nmol of citrate s-1 mg (dry weight) of cells-1, and Km, 0.056 mM citrate. At high pH values (above 5.0), the low-affinity system was absent or not measurable. The two transport systems exhibited different substrate specificities. Isocitric acid was a competitive inhibitor of citric acid for the high-affinity system, suggesting that these tricarboxylic acids used the same transport system, while aconitic, tricarballylic, trimesic, and hemimellitic acids were not competitive inhibitors. With respect to the low-affinity system, isocitric acid, L-lactic acid, and L-malic acid were competitive inhibitors, suggesting that all of these mono-, di-, and tricarboxylic acids used the same low-affinity transport system. The two transport systems were repressed by glucose, and as a consequence diauxic growth was observed. Both systems were inducible, and not only citric acid but also lactic acid and malic acid may induce those transport systems. The induction of both systems was not dependent on the relative concentration of the anionic form(s) and of undissociated citric acid in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)